Does inter-organellar proteostasis impact yeast quality and performance during beer fermentation?
HHYPOTHESIS AND THEORY MANUSCRIPT
Does inter-organellar proteostasis impact yeast quality andperformance during beer fermentation?
Bianca de Paula Telini, Marcelo Menoncin and Diego Bonatto*
Brewing Yeast Research Group, Centro de Biotecnologia da UFRGS, Departamento de BiologiaMolecular e Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
Running title:
Brewing yeast proteostasis *Corresponding author:
Diego BonattoCentro de Biotecnologia da UFRGS - Sala 107Departamento de Biologia Molecular e BiotecnologiaUniversidade Federal do Rio Grande do Sul – UFRGSAvenida Bento Gonçalves 9500 - Prédio 43421Caixa Postal 15005Porto Alegre – Rio Grande do SulBRAZIL91509-900Phone: (+55 51) 3308-7765E-mail: [email protected]/grant sponsor: CNPq bstract During beer production, yeast generate ethanol that is exported to the extracellularenvironment where it accumulates. Depending on the initial carbohydrate concentration in the wort,the amount of yeast biomass inoculated, the fermentation temperature, and the yeast attenuationcapacity, a high concentration of ethanol can be achieved in beer. The increase in ethanolconcentration as a consequence of the fermentation of high gravity (HG) or very high gravity(VHG) worts promotes deleterious pleiotropic effects on the yeast cells. Moderate concentrations ofethanol (5% v/v) change the enzymatic kinetics of proteins and affect biological processes, such asthe cell cycle and metabolism, impacting the reuse of yeast for subsequent fermentation. However,high concentrations of ethanol (>5% v/v) dramatically alter protein structure, leading to unfoldedproteins as well as amorphous protein aggregates. It is noteworthy that the effects of elevatedethanol concentrations generated during beer fermentation resemble those of heat shock stress, withsimilar responses observed in both situations, such as the activation of proteostasis and proteinquality control mechanisms in different cell compartments, including endoplasmic reticulum (ER),mitochondria, and cytosol. Despite the extensive published molecular and biochemical dataregarding the roles of proteostasis in different organelles of yeast cells, little is known about howthis mechanism impacts beer fermentation and how different proteostasis mechanisms found in ER,mitochondria, and cytosol communicate with each other during ethanol/fermentative stress.Supporting this integrative view, transcriptome data analysis was applied using publicly availableinformation for a lager yeast strain grown under in beer production conditions. The transcriptomedata indicated upregulation of genes that encode chaperones, co-chaperones, unfolded proteinresponse elements in ER and mitochondria, ubiquitin ligases, proteasome components, N- glycosylation quality control pathway proteins, and components of processing bodies (p-bodies) andstress granules (SGs) during lager beer fermentation. Thus, the main purpose of this hypothesis andtheory manuscript is to provide a concise picture of how inter-organellar proteostasis mechanismsare connected with one another and with biological processes that may modulate the viability and/orvitality of yeast populations during HG/VHG beer fermentation and serial repitching. 2 eywords: Proteostasis; Brewing Yeasts; Ethanol Stress; Beer Fermentation; Inter-organellar Communication; Transcriptome 3 ntroduction
During beer production, ethanol generated as a by-product of fermentation is exported to theextracellular environment, where it accumulates. Depending on the initial mono-, di-, andtrisaccharide concentrations present in the wort, the amount of yeast cell biomass inoculated,fermentation temperature, and the attenuative capability of yeast strains employed by the brewer, ahigh concentration of ethanol can be achieved in beer (Puligundla et al., 2011). At present, the brewing industry is trying to implement the use of very high gravity (VHG)worts (24 °P or approximately 1.101 kg.L -1 dissolved solids) to produce beer, which can saveenergy, time, labor, and capital costs, and improve plant efficiency (Silva et al., 2008; Puligundla etal., 2011). Beer produced from VHG worts contains high quantities of ethanol and other volatiles,which are dissolved in oxygen-free water to produce regular beers with 5% (v/v) ethanol (Stewart,2010). However, the use of VHG worts imposes challenges for serial repitching due to the osmoticand oxidative stresses that yeast cells experience in the first hours of fermentation, which arefollowed by ethanol, nutritional, and thermal (cold shock) stresses in the later phases offermentation and the beginning of cold maturation (Gibson et al., 2007). These stress conditions canlead to yeast sluries that display sluggish fermentation and poor viability, which precludes their usein subsequent fermentations (Huuskonen et al., 2010).The increase in ethanol concentrations, as a consequence of VHG wort fermentation, canhave pleiotropic effects in yeast. Ethanol is a chaotropic substance that affects cell macromolecularstructures by reducing hydration (Hallsworth, 1998; Cray et al., 2015). Moderate concentrations ofethanol (around 5% v/v) can alter the enzymatic kinetics of proteins associated with primarymetabolism (e.g., glycolysis), and affect different biological processes, such as the cell cycle(Hallsworth, 1998). In comparison, high concentrations of ethanol (>5% v/v) can cause substantialchanges in the structure and composition of hydrophobic molecules within the cell (Hallsworth,1998). Thus, by reducing the water activity in the cell, ethanol promotes a water stress condition(Hallsworth, 1998). In a general sense, the effects of high concentrations of ethanol resemble thoseobserved during heat shock conditions (defined as exposure to temperatures >35 °C), and similar4esponses are observed in response to both stress situations, such as changes in membranecomposition and synthesis of small protective osmolytes (e.g., glycerol and trehalose) (Piper, 1995).Interestingly, it was recently demonstrated by transcriptome analysis using RNA-seq data thatethanol tolerance in different Saccharomyces cerevisiae strains also depends on a series ofenvironmental conditions (e.g., the presence or absence of dissolved oxygen), pointing to a strain-by-oxygen-by-alcohol interactions that lead to ethanol tolerance (Sardi et al., 2018).Protein folding and activity, key features of “proteostasis”, are strongly affected by ethanol.In this review, proteostasis mechanisms are defined as all steps required for a protein to exert itsfunction(s), from protein biogenesis to degradation, including all post-translational changes that theprotein experiences in between. In mammalian models, it has been shown that post-translational modifications of proteins,like mannosylation and galactosylation, are substantially changed in ER and Golgi after ethanolshock (Ghosh et al., 1995; Esteban-Pretel et al., 2011). In yeast, there is limited available data onhow ethanol affects post-translational modification of proteins, but it is clear that protein structureand activity change in the presence of ethanol (Hallsworth, 1998). It has been observed that ethanolcan induce heat shock proteins like Hsp104p, Hsp70p, and Hsp26p, and oxidative stress-responseproteins, like Ctt1p, Sod1, and Sod2p under moderate concentrations of ethanol (6% v/v) (Stanleyet al., 2010). DNA microarray data supports the idea of a fermentative stress response associatedwith ethanol toxicity in industrial lager fermentations (Gibson et al., 2007).Despite the paucity of data regarding the effects of ethanol toxicity in the modulation ofyeast proteostasis mechanisms during VHG beer fermentation and serial repitching by usingpublicly available DNA microarray data (Table S1 and Figure S1), we observed the upregulation ofgenes linked to lager beer fermentation (Figures S2A and B), including differentially expressedgenes (DEGs) associated to organellar proteostasis mechanisms in DNA microarray single analysis(Figures S3A and B to S4A and B) and DNA microarray meta-analysis (Figures S5A and B to S6Aand B). The Pan-DEGs resulting from both DNA microarray analyses (Figures S1 and S9A and B)include genes linked to ER-associated unfolded protein response (UPR), endoplasmic reticulum-5ssociated protein degradation (ERAD) responses (Figures 1A and B), and mitochondria-associatedproteostasis (Figures 2A and B), suggesting cellular cross-talk among organellar proteostasismechanisms. It is important to note that all three gene expression datasets (GSEs) analyzed in thiswork (Table S1) employed the Affymetrix Yeast Genome 2.0 Array for transcript detection of both
S. cerevisiae and
Schizosaccharomyces pombe
Saccharomyces pastorianus are evaluated due to the hybrid genome of thisspecies (Okuno et al., 2016). In order to evaluate if the parental genomes of
S. pastorianus displaysome specific expression pattern, Horinouchi et al. (2010) designated a custom DNA microarrayplatform for
S. pastorianus transcriptome analysis containing probes for both
S. cerevisiae and
Saccharomyces bayanus genomes. This custom DNA microarray was employed to evaluate geneexpression pattern in the lager brewing strain Weihenstephan 34/7 during a pilot-scale fermentationcondition. The transcriptome data gathered by the authors indicated a strong correlation between theexpression levels of
S. cerevisiae and
S. bayanus orthologous genes during fermentation, allowingdiscriminate only a small set of
S. cerevisiae or S . bayanus
DEGs (Horinouchi et al., 2010). On theother hand, the use of RNA sequencing technologies for evaluation of gene expression in
S.pastorianus strains during beer production is virtually absent, making it difficult to understand thecontribution of parental genomes of
S. pastorianus in ethanol tolerance and proteostasis. Thus,considering the importance of
S. pastorianus for brewing industry in general and for hybrid yeastspecies research (Gorter de Vries et al., 2019; Gorter de Vries et al., 2019), it is imperative to designnew experimental procedures for the analysis of the influence of hybrid genomes in proteostasis andethanol tolerance.
Yeast ER proteostasis and ethanol tolerance
The endoplasmic reticulum (ER) consists of an extensive network of membranes thatoriginates at the nuclear envelope and flows through the cytoplasm (English and Voeltz, 2013). It isthe site of secretory, membrane, lysosomal, and vacuolar protein synthesis. Besides proteins, the ER6s also fundamental for the synthesis of lipids and the assembly of lipid bilayers (van Meer et al.,2008). In the ER, proteins are structurally modified, which involves cleavage of signal sequences, N- linked glycosylation, disulfide bond formation, folding of monomers and oligomerization(Braakman and Hebert, 2013). Correct protein folding is facilitated by different molecularchaperones and folding enzymes present in the ER, such as protein disulfide isomerases (PDIs).When a protein is unable to fold correctly, an ER quality control (ERQC) system is activated,comprised of both UPR and ERAD mechanisms (Brodsky and Wojcikiewicz, 2009). Considering that many proteins found in the ER contain N- linked glycans, it is logical toconsider that proteostasis mechanisms are largely associated with N- glycan synthesis in the ER. Infact, N- glycan modification by different glycanases found in ER defines the final destination ofpolypeptides, and the trimming of glucose residues recruit lectin chaperones that facilitate proteinfolding (Molinari, 2007; Ferris et al., 2014). Until now, data regarding N- glycan processing in yeastduring VHG beer fermentation or yeast reuse has been extremely limited. However, ourtranscriptome data single- and meta-analysis (Figure S1) of the proprietary lager yeast CB11 strain(Coors Brewing Limited (Burton on Trent, UK) (Lawrence et al., 2012) under fermentationconditions, when compared to propagation conditions, point to upregulation of genes related to N- glycan processing, like PDI1 and
PMT1 , which are also important components of the ERADresponse (Figures 1A to B). ERAD components export unfolded proteins to the cytosol, which areubiquitinated and degraded by the 26S proteasome (Brodsky and Wojcikiewicz, 2009; Hetz et al.,2015). The recognition step of unfolded protein can occur either on the luminal side (ERAD-L), thecytosolic side (ERAD-C), or inside of the ER membrane (ERAD-M) (Thibault and Ng, 2012).Protein disulfide isomerase 1, or Pdi1p, is essential for cell viability and is highly abundant in theER (Mizunaga et al., 1990; Pfeiffer et al., 2016). Pdi1p is also involved in the removal of aberrantdisulfide bridges (Gilbert, 1997; Pfeiffer et al., 2016). Interestingly, Pdi1p has chaperone activity,even with proteins that do not form disulfide bridges (Pfeiffer et al., 2016), assisting in theunfolding and the export of ERAD-client proteins from the ER (Weissman and Kimt, 1993). Finally,Pmt1p is an O- mannosyltransferase that, together with Pmt2p, exerts proteostasis control of ER7roteins. Pmt1p interacts with Pdi1p in order to promote the correct folding of ER-resident proteinsor to target misfolded proteins to Hrd1p, a major ERAD-associated E3 ubiquitin-protein ligase(Goder and Melero, 2011). It is worth noting that Pdi1p interacts with Htm1p/Mnl1p, an alpha-1,2-specific exomannosidase that generates Man7GlcNac2, an oligosaccharide structure onglycoproteins target for ERAD (Clerc et al., 2009). Moreover, Htm1p/Mnl1p is required for Yos9pactivity (Clerc et al., 2009), a 75 kDa soluble ER glycoprotein (Friedmann et al., 2002) that hasbeen shown to have an important role in glycoprotein degradation (Szathmary et al., 2005). Itshould be point that YOS9 gene was found overexpressed in DNA microarray single analysis only(Figures S4 and B). The roles of Htm1p/Mnl1p in yeast cells subjected to VHG beer fermentationand/or ethanol stress are poorly understood, but it has been demonstrated that ethanol can impair thebiosynthesis of N- glycans in liver cell models in vitro (Welti and Hülsmeier, 2014). This indicatesthat N- glycan biosynthesis and processing may be negatively affected by ethanol/fermentationstress during VHG or even high gravity (HG) beer production.In addition to N- glycan structural alterations promoted by ethanol, the presence of unfoldedproteins in ER reduces or even stops the translation of new proteins, and also exposes stickyhydrophobic amino acids in unfolded proteins, promoting so-called proteotoxicity (Ron, 2002;Mori, 2015), which is sensed by the transmembrane protein Ire1. Ire1p undergoes oligomerizationand autophosphorylation and activates the endoribonuclease domain on the cytosolic side of themembrane that removes a regulatory intron in the HAC1 mRNA (Chapman and Walter, 1997;Sidrauski and Walter, 1997), leading to the translation of active Hac1p, a bZip transcription factorassociated with ER proteostasis (Liu and Chang, 2008). Noteworthy, Navarro-Tapia et al. (2017)showed that low to high concentrations of ethanol (≤ 8% v/v) did not promote protein unfolding inyeast cells, but did trigger UPR through an unknown mechanism in laboratory yeast strains culturedin synthetic medium. However, Miyagawa et al. (2014) showed that an increase in ethanolconcentration, from 8 to 16% (v/v) in a synthetic culture medium, promoted the constant expressionof
HAC1 spliced form mRNA, which demonstrated that UPR can become chronically activated. Inaddition, the same authors verified that Kar2p associated with unfolded protein aggregates in the8R when yeast cells were challenged with ethanol at a concentration of 16% (v/v), supporting theidea that very high concentrations of ethanol potentially induce protein aggregates in the ER andtrigger ERQC (Miyagawa et al., 2014). Our transcriptome data indicated that Pan-DEGs related to the classical UPR pathway, like
KAR2, PTC2, and
YPT1 , are upregulated in lager beer fermentation compared to the yeastpropagation step (Figures 1A and B). Ptc2p is a type 2C serine/threonine phosphatase thatdownregulates the UPR mechanism by dephosphorylating Ire2p (Welihinda et al., 1998), whileYpt1p is a yeast Rab1 homolog that interacts with unspliced
HAC1 mRNA and regulates the UPRby promoting the decay of
HAC1 mRNA (Tsvetanova et al., 2012). Ypt1p has been linked to themaintenance of Golgi morphology and protein composition, participates in ER to Golgianterograde/retrograde transport, and is necessary for intra Golgi transport (Kamena et al., 2008).While anterograde/retrograde ER to Golgi responses have been extensively studied in yeast andother model organisms, and the functions of a number of different protein complexes involved inthese processes have been discerned (Lee et al., 2004), the influence of anterograde/retrograde ERto Golgi transport in brewing yeast vitality or beer fermentation is unknown. However, wehypothesize that this mechanism may be negatively modulated by high ethanol concentrationsduring VHG beer fermentation or yeast reuse. In support of this idea, it was previously shown thatthe rat PC12 cell line, when subjected in vitro to a low alcohol concentration (30 mM), exhibiteddelayed anterograde ER to Golgi transport, fragmented Golgi morphology, and a decreased numberof secretory vesicles (Tomás et al., 2012). Interestingly, 5% of all eukaryotic proteins (referred to astail-anchored (TA) proteins) possess a unique carboxy-terminal transmembrane region that targetsthem to the ER membrane (Stefanovic and Hegde, 2007). Considering that these proteins contain ahydrophobic domain that makes them prone to aggregation in the aqueous environment of the ERlumen, they should be targeted to the ER membrane to avoid the formation of protein aggregates.Thus, in order to guide the entry of TA proteins into the ER membrane, the guided entry of TAproteins (GET) pathway mediates the process, also acting in vesicle fusion and retrograde Golgi toER responses (Denic et al., 2013). Moreover, the GET pathway is necessary for the retrieval of the9rd2p HDEL receptor from the Golgi to the ER (Schuldiner et al., 2005). Erd2p is an importantcomponent that retain proteins bearing a C-terminal tetrapeptide HDEL sequence in the ER, like theER chaperone Kar2p (Semenza et al., 1990), invertase, and many other secreted proteins. In ourtranscriptome data analysis, we found that during lager beer fermentation, the Pan-DEGs
GET1 , GET2 , GET3 , GET4 , and
SGT2 are significantly upregulated (Figures 1A and B). GET proteins arecore components of GET pathway that promote the transfer of TA proteins from ribosomes to theGet4p/Get5p/Sgt2p complex and to the chaperone Get3p (Chartron et al., 2012). Then, Get1p andGet2p, which comprise a transmembrane complex, drive a conformational change that enables therelease of TA proteins from Get3p and, as a consequence, insertion into the ER membrane (Wang etal., 2014). In the context of beer fermentation and ethanol stress, we speculate that ethanolgenerated during fermentation induces conformational changes in N- glycans and secreted proteinsthat potentially leads to the formation of aggregates in the ER, followed by modification of thestructure and function of Golgi. This may result in the activation of ERQC mechanisms andpromote the retrograde response of Golgi to ER by stimulating the function of the GET pathway(Figure 4). Finally, the induction of ERQC due to ethanol generated during beer fermentation mayalso occur in cytoplasm and mitochondria, especially due to the activity of multi-organellarubiquitin ligases and chaperones. Cytosol proteostasis in brewing yeast and the impact on beer fermentation
In the cytosol, misfolded proteins that have exposed hydrophobic amino acid residues arerecognized by protein quality control mechanisms (Buchberger et al., 2010). The cytoplasmicproteostasis mechanism in yeast comprises the heat shock response (HSR) (Mager and Ferreira,1993), which promotes the expression of molecular chaperones and the proteasome system (Parsellet al., 1993). Similar to UPR, the HSR is induced by different stress conditions that lead toproteotoxicity. In
S. cerevisiae, the HSR is regulated by the heat shock factor 1 (Hsf1p)transcription factor, encoded by the
HSF1 gene (Weindling and Bar-Nun, 2015). Hsf1p promotes anadaptive response to different stressor agents, including ethanol (Weindling and Bar-Nun, 2015).10east cells treated with 6% (v/v) ethanol show induction of Hsf1p activity (Lee et al., 2000), whileHsf1p mutants were defective in ethanol stress-induced target gene expression (Takemori et al.,2006). Interestingly, the ER oxidoreductin, which is encoded by
ERO1 and induces protein disulfidebonds, was upregulated by Hsf1p in yeast cells exposed to ethanol (Takemori et al., 2006), pointingto a crosstalk between HSR and ERQC mechanisms. Unfortunately, the activity of HSR and ERQCin conditions of VHG beer fermentation or yeast serial repitching is not well understood, but wespeculate that modulation of the crosstalk between HSR and ERQC mechanisms may promoteethanol tolerance and cell adaptability during beer fermentation. In line with this hypothesis,ubiquitin ligases, which function by transferring ubiquitin to misfolded/unfolded proteins thustargeting them to the 26S proteasome complex, are key components that regulate both HSR andERQC (Szoradi et al., 2018). It is well known that different organelles have their own specificubiquitin ligases, such as Hrd1p and Doa10p in the ER (Ruggiano et al., 2014), San1p in thenucleus (Gardner et al., 2005), and Ubr1p, Ubr2p, Hul5p, and Rsp5p in the cytosol (Prasad et al.,2018). However, different ubiquitin ligases have overlapping functions, such as Doa10p in nucleusand cytosol, San1p in cytoplasm, and Ubr1p in the ER (Szoradi et al., 2018). This ubiquitin ligasenetwork is an essential component of inter-organellar proteostasis, yet very little is known abouthow this communication is mediated. For example, the overexpression of cytosolic Rsp5p, aNEDD4 family E3 ubiquitin ligase, improve thermoresistance and stress tolerance in yeast strainsused for bioethanol production (Hiraishi et al., 2006; Shahsavarani et al., 2012). Disruption of
RSP5 increase the production of isoamyl alcohol and isoamyl acetate in laboratory yeast strains (Abe andHorikoshi, 2005). Rsp5p is part of the so-called “Rsp5-ART ubiquitin ligase adaptor network”,which acts to promote the endocytosis and degradation of misfolded integral membrane proteinsfound in the ER, Golgi, and plasma membrane (Zhao et al., 2013). Additionally, Rsp5p interactswith another important cytosolic E3 ubiquitin ligase named Ubr1p, which is a component of thestress-induced homeostatically-regulated protein degradation (SHRED) pathway (Szoradi et al.,2018). 11he SHRED pathway is initially activated by transcription of the hydrophilin-coding gene
ROQ1 by different stress conditions due to the presence of Msn2p/4p and Hsf1p-associated stressresponse elements in the
ROQ1 promoter (Yamamoto et al., 2005; Verghese et al., 2012; Szoradi etal., 2018). Once translated, Roq1p is cleaved by the endopeptidase Ynm3p, and cleaved Roq1pbinds to Ubr1p changing its substrate specificity and promoting the degradation of misfoldedproteins at the ER membrane and in the cytosol by the proteasome (Szoradi et al., 2018). Ubr1pinteracts with the chaperone Hsp70p and with Sse1p, the ATPase component of the heat shockprotein Hsp90 chaperone complex (Nillegoda et al., 2010). Moreover, it has been demonstrated thatUbr1p is a fundamental component of ERAD when yeast cells are exposed to heat or ethanol stress,bypassing the functions of the canonical Hrd1p/Der3p and Doa10p (Stolz et al., 2013). Thus,considering the importance of Rsp5p and Ubr1p in heat and ethanol stress response, we hypothesizethat under conditions of VHG/HG beer fermentation, the Rsp5-ART ubiquitin ligase adaptornetwork and SHRED pathway actively target protein aggregates present in the ER and cytosol toERAD (Figure 4). Besides ubiquitin ligases, many chaperones are essential to repair and/or prevent misfoldedproteins even before they can be targeted to ERAD. In yeast, chaperones are classified in eightdistinct families, which are the small heat-shock proteins (SMALL), the AAA+ family, theCCT/TRiC complex, the prefoldin/GimC (PFD) complex, Hsp40, Hsp60, Hsp70, and Hsp90families (Gong et al., 2009). From transcriptome data analysis, we observed the upregulation of 54Pan-DEGs linked to chaperone activity (Figures 2A and B) in the lager yeast strain during beerfermentationas compared to the propagation step. Of these 54 Pan-DEGs linked to chaperoneactivity, 21 Pan-DEGs encode for chaperone proteins that are found in the cytoplasm andmitochondria (Figure 3A) and belong to the HSP70, HSP40, SMALL, AAA+, HSP60, and HSP90families (Figure 3B). Considering the chaperones found in cytoplasm that belong to the Hsp70 family, we foundthat the Pan-DEGs
SSA1-4, SSZ1, and
SSB2 were upregulated during beer fermentation incomparison to propagation (Figures 2A and B). The roles of Hsp70s proteins in yeast subjected to12thanol stress are extensively documented, including in beer production. For example, it wasreported that
FES1, SSA2, SSA3, SSA4 , and
SSE1 are upregulated in a synthetic wort that mimickeda VHG beer fermentation (Qing et al., 2012). Other studies based on proteomics and quantitativeRT-qPCR also confirmed the expression of cytosolic Hsp70p during the early phases of beerfermentation in different lager strains (Brejning et al., 2005; Smart, 2007), and it was clearlydemonstrated that moderate concentrations of ethanol (>4% v/v) induce the expression of Hsp70proteins (Piper et al., 1994). In fact, proteins of the Hsp70 family display important functions notonly as chaperones, but also in targeting misfolded proteins for proteasome degradation (Kettern etal., 2010; Kim et al., 2013). In addition, Hsp70 proteins form a bi-chaperone system with Hsp104p,a heat shock protein belonging to the AAA+ family (Zolkiewski et al., 2012), and promote thedisaggregation and resolubilization of misfolded proteins (Weibezahn et al., 2005). Thetranscriptome data also indicated that
HSP104 is upregulated during beer fermentation compared topropagation (Figures 2A and B), supporting our hypothesis that ethanol may promote the formationof misfolded protein aggregates in lager yeast strains during beer fermentation, which likely triggersthe activity of Hsp70p and Hsp104p to refold and resolubilize the protein aggregates or target themto the proteasome. Corroborating the importance of
HSP104 for VHG beer fermentation, Rautio et al. (2007)showed that
HSP104 is induced in the first 10-30 hours of fermentation together with
TPS1 , whichencodes trehalose phosphate synthase, a key enzyme involved in trehalose biosynthesis and ethanolstress protection (Alexandre et al., 2001) during beer fermentation. The roles of trehalose as amolecular chaperone in protecting yeast cells against protein aggregation are well understood(Singer and Lindquist, 1998) and a synergistic effect of Hsp104p on trehalose accumulation anddegradation has been observed (Iwahashi et al., 1998). However, trehalose and Hsp104p are bothrequired when protein aggregation can be reversible in yeast cells (Sethi et al., 2018). It will beinteresting to determine if Hsp104p and trehalose act synergistically in VHG beer protecting yeastcells in the early phases of the fermentation process. 13n addition to Hsp70 and AAA+ families, we also observed two additional HSP memberswith high expression in the cytosol of lager yeast cell during beer fermentation compared to cellpropagation, which were the SMALL and Hsp40 proteins (Figure 3B). The SMALL or small heatshock proteins/α-crystallin (sHSP) family is comprised of Hsp26p and Hsp42p in
S. cerevisiae, twoproteins important for preventing unfolded protein aggregation that have overlapping functions innon-stressed and stressed yeast cells (Haslbeck et al., 2004). It was previously demonstrated thatHsp26p co-assembles with misfolded proteins and allows the Hsp104p/Hsp70p/Hsp40p complex todisaggregate them (Cashikar et al., 2005). Interestingly,
HSP26 and other HSP-coding genes werefound to be upregulated in yeast strains isolated from sherry wines (Aranda et al., 2002), as well asin lager yeast cells in 16 ºP and 24 ºP wort after 24 hours of fermentation (Odumeru et al., 1992). Inaddition, it was shown that Hsp26P is a key HSP for ethanol production (Sharma, 2001). Another interesting target of our transcriptome analysis was the
HSP82
Pan-DEG, whichwas found to be upregulated in lager yeast during beer fermentation (Figures 2A and B),corroborating the previous data of Gibson et al. (2008). Additionally, in brewing yeast, it has beendemonstrated by proteomics and transcriptomics that ethanol stress induces the expression ofHsp82p in wine yeasts (Aranda et al., 2002; Navarro-Tapia et al., 2016) and bioethanol yeast strains(Li et al., 2010). The Hsp82 protein, which belongs to the HSP90 family, is an abundant andessential dimeric ATP-dependent chaperone (Borkovich et al., 1989; Richter et al., 2001). It isrequired to reactivate proteins damaged by heat without participating in de novo folding of mostproteins (Nathan et al., 1997). Hsp82 target proteins include steroid hormone receptors and kinases(Mayr et al., 2000). It has been demonstrated that Hsp82p is regulated by several co-chaperones,including Aha1p and Hch1p, both of which activate the ATPase function of Hsp82p (Panaretou etal., 2002) and whose Pan-DEGs were found upregulated in lager yeast during beer fermentation(Figures 2A and B). A third co-chaperone named Cpr6p, a peptidyl-prolyl cis-trans isomerase(cyclophilin) that interacts with Hsp82p, and together with Cpr7p, is required for normal yeastgrowth (Zuehlke and Johnson, 2012).
CPR6 was found to be upregulated in our transcriptomeanalysis during beer fermentation (Figures 2A and B), but little is known about its roles during beer14ermentation. However, protein-protein interaction data (Figure S10) indicate that Cpr6p interactswith Pbp1p, a component of processing bodies (p-bodies) and stress granules (SGs), which may beinduced by severe ethanol stress, heat shock, or glucose deprivation (Kato et al., 2011). Induction ofp-bodies and SGs by UPR, which has been observed in mammalian cells (Harding et al., 2000;Anderson and Kedersha, 2008) may also occur in yeast cells. In fact, it would be interesting todetermine whether p-bodies/SGs are formed during beer fermentation and if they are associatedwith proteostasis in cytosol and/or the ER. Cpr6p also interacts with Rpd3p (Figure S10), aconserved histone deacetylase that together with Sin3p and Ume1p comprise the Sin3 complex, aglobal regulator of transcription that is linked to a series of physiological conditions in yeast andother organisms (Silverstein and Ekwall, 2005), such as ethanol stress (Ma and Liu, 2012). Thus,Cpr6p could be an important co-chaperone that together with Hsp82 may serve as a hub for p-bodies/SGs and epigenetic regulation of genes linked to beer fermentation and proteostasis.
Mitochondrial proteostasis in brewing yeast
During beer production, yeast mitochondria exert important functions despite the catabolicrepression of nuclear genes encoding mitochondrial proteins linked to respiration (O’Connor-Cox etal., 1996). In fact, mitochondria are not only the primary site of lipid and ergosterol synthesis, butthey also provide a series of metabolites originating from central carbon and proline-argininemetabolism (Kitagaki and Takagi, 2014). A large proportion of cellular radical molecules areproduced as a result of mitochondrial metabolism, which can strongly affect yeast physiology(Kitagaki and Takagi, 2014). Despite the metabolic and physiological importance of mitochondria,mutations linked to the mitochondrial genome that result in petite phenotypes can result in theproduction of off-flavors (related to synthesis of esters and fusel alcohols) in beer fermentation(Ernandes et al., 1993). Finally, in lager yeasts and possibly in ale strains, mitotype can have astrong influence on temperature tolerance (Baker et al., 2019). Proteostasis in mitochondria includes different chaperones and proteases, as well as proteinsthat participate in inter-organellar communication, where defects in mitochondrial proteostasis15mpacts health and aging (Moehle et al., 2019). Similar to ER, mitochondria have a so-called“mitochondrial unfolded protein response” or mtUPR, which was initially characterized inmammalian cells (Zhao, 2002). Considering that mitochondria have distinct subcompartments within the organelle (e.g.,matrix, outer membrane, and intermembrane space), protein import and sorting processes are verycomplex (Neupert and Herrmann, 2007). Most mitochondrial proteins are imported as unfoldedprecursors by means of the translocase of outer membrane (TOM) and translocase of innermembrane (TIM) complexes. Upon translocation into the mitochondria, the proteins undergochaperone-assisted folding (Neupert and Herrmann, 2007). The transcriptome analysis of lager yeast cells during beer fermentation revealed that TIM-related Pan-DEGs including
TIM8 , TIM9 , TIM12 , TIM17 , and TIM54 are upregulated (Figures 2Aand B). Tim8p and Tim9p belong to the mitochondrial intermembrane space protein transportercomplex, which together with Tim10p, Tim12p, and Tim13p, mediates the transit of proteinsdestined for the inner membrane across the mitochondria intermembrane space (Davis et al., 2007).Tim9p/Tim10p and Tim9p/Tim10p/Tim12p interact with Tim22p, comprising a multioligomericcomplex with Tim54p, Tim22p, Tim18p, and Sdh3p (Gebert et al., 2011). The Tim22 complexmediates the insertion of large hydrophobic proteins, like carrier proteins with multipletransmembrane segments, as well as Tim23p, Tim17p, and Tim22p into the inner membrane(Mokranjac and Neupert, 2009). Tim17p is a component of the Tim23 complex, which promotes thetranslocation and insertion of proteins into the inner mitochondrial membrane (Mokranjac andNeupert, 2009). The Tim23 complex is composed of a membrane-embedded part, which forms theimport motor. This component is formed by Tim14p (Pam18p), Tim16 (Pam16p), Tim44p, Mge1p,and mitochondrial Hsp70p (Mokranjac and Neupert, 2009). The Pan-DEGs encoding Pam16p andPam18p were found to be upregulated in our transcriptome analysis (Figures 2A and B). Despite thelarge amount of data collected so far about the roles of Tim22 and Tim23 complexes in yeastmitochondria, considerably less is known about their roles in yeast fermentation/ethanol stress.However, Short et al. (2012) showed that yeast temperature sensitive mutant strains for
PAM16 have16efects in fermentation linked to lipid metabolism. Moreover, an upregulated Pan-DEG in ourtranscriptome analysis,
MDJ2, encode a chaperone belonging to the HSP40 family that regulatesHsp70 chaperone activity and interacts with Pam18p (Mokranjac et al., 2005). In addition, thetranscriptome analysis of lager yeast cells revealed upregulation of
TOM6 and
TOM7
Pan-DEGs(Figure 2A and B), both encoding small protein components of the TOM complex (Dekker et al.,1998). At present, the roles of Tom6p and Tom7p in yeast fermentation/ethanol stress remainunknown.Two important Pan-DEGs found to be upregulated in our transcriptome analysis,
PHB1 and
PHB2 (Figures 2A and B), encode the proteins prohibitin 1 (Phb1p) and 2 (Phb2p), which are partof a large chaperone complex that stabilizes protein structures and is involved in the regulation ofyeast replicative life span and mtUPR (Coates et al., 1997; Nijtmans, 2000). In the context of agingand replicative life span, the impact of Phb1p/2p expression during VHG/HG beer fermentationand/or yeast reuse is unknown, despite the fact that a mixed aged yeast population is commonlyobserved in mostly ale/lager fermentations (Smart et al., 2000; Powell et al., 2003). Moreover, yeast phb1 and phb2 mutants are defective in mitochondrial segregation from mother cells to daughtercells, resulting in delayed segregation of mitochondria (Piper et al., 2002). Interestingly, loss of theorthologous prohibitin in
Caenorhabditis elegans affected the morphology of mitochondria,resulting in fragmented and disorganized structures (Sanz et al., 2003), a phenotype previouslyobserved in yeast strains used for sake (Kitagaki and Shimoi, 2007) and cider (Lloyd et al., 1996)after prolonged anaerobiosis under high concentration of ethanol (>10% v/v). In animal cells,mitochondrial fragmentation is a feature of mitochondrial proteostasis that is activated in responseto a high number of misfolded proteins, but that is also observed during mitophagy andprogrammed cell death (Moehle et al., 2019). Similarly, Fis1p, a protein involved in mitochondriaand peroxisome maintenance in yeast, is upregulated when cells are subjected to high ethanolconcentrations, thereby promoting mitochondrial fragmentation and inhibition of apoptosis(Kitagaki et al., 2007). 17aking into account mitochondria structure,
YME1 and
AFG3 were also found to beupregulated in lager yeast cells (Figures 2A and B). These Pan-DEGs encode the mitochondrialATP-dependent metallopeptidase (AAA protease) Yme1p and Afg3p, respectively, which arenecessary for degradation of unfolded or misfolded proteins associated with the mitochondrial innermembrane (Arlt et al., 1996; Schreiner et al., 2012). Despite the fact that the specific roles ofYme1p and Afg3p in VHG/HG beer fermentation or yeast reuse are currently unknown, dataregarding the modulation of mitochondria activity upon ethanol exposure indicates that ethanolincreases oxidative stress and induces the formation of mitochondrial permeability transition(MPT). MPT is a protein structure that forms a pore across the inner and outer membranes ofmitochondria, leading to the depolarization of membrane potential, uncoupling of oxidativephosphorylation and ATP depletion, rupture of the outer mitochondrial membrane, and apoptosisinduction (Pastorino et al., 1999; Hoek et al., 2002). Interestingly, AAA proteases seem to beessential to coordinate many functions within mitochondria, including mitochondrial genomestability, respiratory chain complexes synthesis, and the mitochondrial membrane metabolism(Patron et al., 2018). Moreover, AAA proteases are essential to modulate the activity of themitochondrial Ca uniporter (MCU) complex. Mutations in mammalian mitochondrial AAAproteases induce constitutive MCU activity and deregulated mitochondrial Ca influx, leading tocell death (König et al., 2016). This suggests that yeast AAA proteases may have essential roles inmaintaining mitochondrial structure and function during beer fermentation and ethanol stress, anddysfunctions in mitochondrial AAA proteases are likely to affect brewing yeast viability andvitality.Besides the protein complexes linked to mitochondrial structure and function, ourtranscriptome analysis revealed an additional eight upregulated genes during beer fermentation(Figure 3A) that encode for mitochondrial molecular chaperones. These included three upregulatedPan-DEGs belonging to the HSP40 family ( MDJ1 , MDJ2 , and JAC1 ), one to the HSP60 family(
HSP60 ), one to the HSP70 family (
ECM10 ) and one to the AAA+ family (
HSP78 ) (Figures 2A andB). The interaction between mitochondrial Hsp40 and Hsp70 proteins has been extensively18ocumented, being involved in the translocation of proteins to the matrix and folding (Liu et al.,2001). The chaperone Hsp60p is a fundamental protein required to assist the folding and import ofdifferent target proteins to the mitochondrial matrix (Reading et al., 1989), also being important forthe replication of mitochondrial DNA in yeast (Kaufman et al., 2003). Finally, Hsp78p is achaperone that displays similar functions with those of the mitochondrial Hsp70 system (Schmitt etal., 1995). Biochemical studies have indicated cooperation between the Hsp70 system and Hsp78p,forming a bichaperone Hsp70-Hsp78 system that assists in protein refolding after stress induction(Krzewska et al., 2001). Similar to Hsp60p, available evidence suggests that Hsp78p is required forthe maintenance of mitochondrial genome integrity (Schmitt, 1996). Thus, it is clear thatproteostasis mechanisms in mitochondria play a central role in the maintenance of both proteins andmitochondria nucleoid structure and function, the latter of which profoundly affects beerfermentation (Smart, 2007) and hybrid brewing yeast strain adaptability to temperature (Baker etal., 2019).
Discussion
Different organelles such as ER, cytosol, and mitochondria display a set ofmolecules/proteins that are essential for proteostasis under environmental conditions that are proneto induce protein misfolding/unfolding and amorphous aggregate formation, both potentiallyleading to proteotoxicity. One such condition is beer fermentation, where brewing yeast strainsrequire protection from the toxic and pleiotropic effects of ethanol. In order to deal with ethanol andmaintain proteostasis during beer fermentation, the major cellular compartments (e.g.,mitochondria, ER, and cytosol) must communicate with one another to mount a systemic cellresponse (Figure 4).Multiple lines of evidence indicate that organellar proteostasis is a concerted process that isdirectly connected with different biological processes, such as metabolism and aging (Raimundoand Kriško, 2018). This so-called “inter-organellar/cross-organellar communication/response” orCORE is dependent on a series of signaling-associated and/or protein networks that include HSPs19nd their target molecules (Raimundo and Kriško, 2018). Interestingly, one hallmark of the COREis the upregulation of multiple genes and proteins linked to proteostasis, including
PDI1 , HSP26 ,and
HSP90 (Perić et al., 2016). However, we speculate that other protein and small moleculenetworks, such as those composed of E3 ubiquitin ligases, the SHRED pathway, trehalosebiosynthesis, ERAD, and the prohibitin complex, could be essential components of a larger COREnetwork that is upregulated during beer fermentation (Figure 4). The activation of a CORE networkmay impact different aspects of fermentative metabolism that are crucial for yeast viability and/orvitality and further use in serial repitching. For example, it was observed in
C. elegans thatmitochondrial proteotoxicity increases fatty acid synthesis and promotes lipid accumulation, acondition associated with mitochondrial-to-cytosolic stress response that is essential for
C. elegans survival (Kim et al., 2016). Similarly, we observed in our transcriptome analysis an increase in theexpression of genes related to lipid biosynthesis in lager yeast (Figures S11 and S12), pointing to apotentially conserved CORE network in eukaryotes. Furthermore, the roles of inter-organellarproteostasis mechanisms in the replicative and chronological life span of yeast cells have beendemonstrated previously (Perić et al., 2016; Chadwick et al., 2019), which are very likely to affectbrewing. Finally, a number of important questions remain about how the CORE network maymodulate other organelles (e.g., nucleus and vacuole) during beer fermentation (Figure 4). Asdescribed above, some components of organellar proteostasis influence transcriptional activity inthe nucleus. Recently, Andréasson et al. (2019) demonstrated an important connection betweenmitochondria and nucleus for proteostasis and cell metabolism. However, little is known aboutepigenetic modulation during proteotoxic stress induced by ethanol. In the same sense, how theCORE network connects with vacuoles is an open question (Figure 4). Noteworthy, it wasdemonstrated that in conditions of lipid imbalance, unfolded ER proteins can be removed by lipiddroplets and targeted to the vacuole for degradation by microlipophagy (Vevea et al., 2015).However, the impact of this mechanism remains to be determined in beer fermentation.To evaluate the importance of each component of the CORE network for beer fermentation,it is indispensable to get high quality RNA-seq data from different
S. pastorianus strains in20onditions of industrial yeast propagation and beer fermentation. It can be potentially achieved bytagging the major genes of
S. cerevisiae and
S. eubayanus linked to the CORE network followed byinterspecies hybridization to generate
S. pastorianus strains by different techniques, like HyPr(Alexander et al., 2016) and testing them in brewery environment. On the other hand, the use ofdifferent proteome techniques to evaluate the contribution of CORE components is also welcome aswell as the generation of
S. pastorianus mutant strains for CORE components by uding CRISPR-Cas9 technology (de Vries et al., 2017). In conclusion, a better understanding of the CORE network in the context of beerfermentation and/or ethanol stress will allow us to improve different aspects of brewing, fromethanol tolerance in VHG/HG fermentation to yeast reuse, potentially allowing us to select yeaststrains with high tolerance to ethanol or diminished aging, which will ultimately improve beer yieldand quality.
Author Contributions
DB contributed to the design, acquisition, analysis, and interpretation of data for the work;DB, BT, and MM contributed to drafting the work and prepared the final work; DB prepared thefigures and all authors approved the final manuscript.
Conflict of Interest Statement
The authors declare that this work was conducted in the absence of any commercial orfinancial relationships that could be construed as a potential conflict of interest.
References
Abe, F., and Horikoshi, K. (2005). Enhanced production of isoamyl alcohol and isoamyl acetate by ubiquitination-deficient
Saccharomyces cerevisiae mutants.
Cell. Mol. Biol. Lett.
10, 383–388.Alexander, W. G., Peris, D., Pfannenstiel, B. T., Opulente, D. A., Kuang, M., and Hittinger, C. T. (2016). Efficient engineering of marker-free synthetic allotetraploids of
Saccharomyces. ungal Genet. Biol. FG B
89, 10–17. doi:10.1016/j.fgb.2015.11.002.Alexandre, H., Ansanay-Galeote, V., Dequin, S., and Blondin, B. (2001). Global gene expression during short-term ethanol stress in
Saccharomyces cerevisiae . FEBS Lett.
Trends Biochem. Sci.
33, 141–150. doi:10.1016/j.tibs.2007.12.003.Andréasson, C., Ott, M., and Büttner, S. (2019). Mitochondria orchestrate proteostatic and metabolic stress responses.
EMBO Rep. doi:10.15252/embr.201947865.Aranda, A., Querol, A., and del Olmo, Marcel. li (2002). Correlation between acetaldehyde and ethanol resistance and expression of HSP genes in yeast strains isolated during the biologicalaging of sherry wines.
Arch. Microbiol.
Cell
85, 875–885. doi:10.1016/S0092-8674(00)81271-4.Baker, E. P., Peris, D., Moriarty, R. V., Li, X. C., Fay, J. C., and Hittinger, C. T. (2019). Mitochondrial DNA and temperature tolerance in lager yeasts.
Sci. Adv.
5, eaav1869. doi:10.1126/sciadv.aav1869.Borkovich, K. A., Farrelly, F. W., Finkelstein, D. B., Taulien, J., and Lindquist, S. (1989). Hsp82 is an essential protein that is required in higher concentrations for growth of cells at higher temperatures.
Mol. Cell. Biol.
9, 3919–3930. doi:10.1128/MCB.9.9.3919.Braakman, I., and Hebert, D. N. (2013). Protein folding in the endoplasmic reticulum.
Cold Spring Harb. Perspect. Biol.
5, a013201–a013201. doi:10.1101/cshperspect.a013201.Brejning, J., Arneborg, N., and Jespersen, L. (2005). Identification of genes and proteins induced during the lag and early exponential phase of lager brewing yeasts.
J. Appl. Microbiol.
98, 261–271. doi:10.1111/j.1365-2672.2004.02472.x.Brodsky, J. L., and Wojcikiewicz, R. J. (2009). Substrate-specific mediators of ER associated degradation (ERAD).
Curr. Opin. Cell Biol.
21, 516–521. doi:10.1016/j.ceb.2009.04.006.Buchberger, A., Bukau, B., and Sommer, T. (2010). Protein quality control in the cytosol and the endoplasmic reticulum: brothers in arms.
Mol. Cell
40, 238–252. doi:10.1016/j.molcel.2010.10.001.Cashikar, A. G., Duennwald, M., and Lindquist, S. L. (2005). A chaperone pathway in protein disaggregation: Hsp26 alters the nature of protein aggregates to facilitate reactivation by Hsp104.
J. Biol. Chem.
Curr. Genet. doi:10.1007/s00294-019-01019-0.Chapman, R. E., and Walter, P. (1997). Translational attenuation mediated by an mRNA intron.
Curr. Biol.
7, 850–859. doi:10.1016/S0960-9822(06)00373-3.Chartron, J. W., Clemons, W. M., and Suloway, C. J. (2012). The complex process of GETting tail-anchored membrane proteins to the ER.
Curr. Opin. Struct. Biol.
22, 217–224. doi:10.1016/j.sbi.2012.03.001. 22lerc, S., Hirsch, C., Oggier, D. M., Deprez, P., Jakob, C., Sommer, T., et al. (2009). Htm1 protein generates the N-glycan signal for glycoprotein degradation in the endoplasmic reticulum.
J. Cell Biol.
Curr. Biol.
7, 607–610. doi:10.1016/S0960-9822(06)00261-2.Cray, J. A., Stevenson, A., Ball, P., Bankar, S. B., Eleutherio, E. C., Ezeji, T. C., et al. (2015). Chaotropicity: a key factor in product tolerance of biofuel-producing microorganisms.
Curr. Opin. Biotechnol.
33, 228–259. doi:10.1016/j.copbio.2015.02.010.Davis, A. J., Alder, N. N., Jensen, R. E., and Johnson, A. E. (2007). The Tim9p/10p and Tim8p/13p complexes bind to specific sites on Tim23p during mitochondrial protein import.
Mol. Biol. Cell
18, 475–486. doi:10.1091/mbc.e06-06-0546.de Vries, A. R. G., de Groot, P. A., van den Broek, M., and Daran, J.-M. G. (2017). CRISPR-Cas9 mediated gene deletions in lager yeast
Saccharomyces pastorianus.
Microb. Cell Factories
16. doi:10.1186/s12934-017-0835-1.Dekker, P. J. T., Ryan, M. T., Brix, J., Müller, H., Hönlinger, A., and Pfanner, N. (1998). Preprotein translocase of the outer mitochondrial membrane: molecular dissection and assembly of the general import pore complex.
Mol. Cell. Biol.
18, 6515–6524. doi:10.1128/MCB.18.11.6515.Denic, V., Dotsch, V., and Sinning, I. (2013). Endoplasmic reticulum targeting and insertion of tail-anchored membrane proteins by the GET pathway.
Cold Spring Harb. Perspect. Biol.
5, a013334–a013334. doi:10.1101/cshperspect.a013334.English, A. R., and Voeltz, G. K. (2013). Endoplasmic reticulum structure and interconnections withother organelles.
Cold Spring Harb. Perspect. Biol.
5, a013227–a013227. doi:10.1101/cshperspect.a013227.Ernandes, J. R., Williams, J. W., Russell, I., and Stewart, G. G. (1993). Respiratory deficiency in brewing yeast strains—effects on fermentation, flocculation, and beer flavor components.
J. Am. Soc. Brew. Chem.
51, 16–20. doi:10.1094/ASBCJ-51-0016.Esteban-Pretel, G., Marín, M. P., Romero, A. M., Ponsoda, X., Ballestin, R., Canales, J. J., et al. (2011). Protein traffic is an intracellular target in alcohol toxicity.
Pharmaceuticals
4, 741–757. doi:10.3390/ph4050741.Ferris, S. P., Kodali, V. K., and Kaufman, R. J. (2014). Glycoprotein folding and quality-control mechanisms in protein-folding diseases.
Dis. Model. Mech.
7, 331–341. doi:10.1242/dmm.014589.Gardner, R. G., Nelson, Z. W., and Gottschling, D. E. (2005). Degradation-mediated protein quality control in the nucleus.
Cell
Mol. Cell
44, 811–818. doi:10.1016/j.molcel.2011.09.025.Ghosh, P., Liu, Q.-H., and Lakshman, M. R. (1995). Long-term ethanol exposure impairs glycosylation of both N- and O-glycosylated proteins in rat liver.
Metabolism
44, 890–898. doi:10.1016/0026-0495(95)90242-2. 23ibson, B. R., Lawrence, S. J., Boulton, C. A., Box, W. G., Graham, N. S., Linforth, R. S. T., et al. (2008). The oxidative stress response of a lager brewing yeast strain during industrial propagation and fermentation: oxidative stress response of lager brewing yeast.
FEMS YeastRes.
8, 574–585. doi:10.1111/j.1567-1364.2008.00371.x.Gibson, B. R., Lawrence, S. J., Leclaire, J. P. R., Powell, C. D., and Smart, K. A. (2007). Yeast responses to stresses associated with industrial brewery handling.
FEMS Microbiol. Rev.
31, 535–569. doi:10.1111/j.1574-6976.2007.00076.x.Gilbert, H. F. (1997). Protein disulfide isomerase and assisted protein folding.
J. Biol. Chem.
J. Cell Sci.
Saccharomyces cerevisiae : implications to protein foldingpathways in the cell.
Mol. Syst. Biol.
5, 275. doi:10.1038/msb.2009.26.Gorter de Vries, A. R., Voskamp, M. A., van Aalst, A. C. A., Kristensen, L. H., Jansen, L., van den Broek, M., et al. (2019). Laboratory evolution of a
Saccharomyces cerevisiae × S. eubayanus hybrid under simulated lager-brewing conditions.
Front. Genet.
10. doi:10.3389/fgene.2019.00242.Gorter de Vries, A. R., Pronk, J. T., and Daran, J.-M. G. (2019). Lager-brewing yeasts in the era of modern genetics.
FEMS Yeast Res.
19. doi:10.1093/femsyr/foz063.Hallsworth, J. E. (1998). Ethanol-induced water stress in yeast.
J. Ferment. Bioeng.
85, 125–137. doi:10.1016/S0922-338X(97)86756-6.Harding, H. P., Zhang, Y., Bertolotti, A., Zeng, H., and Ron, D. (2000). Perk is essential for translational regulation and cell survival during the unfolded protein response.
Mol. Cell
5, 897–904. doi:10.1016/S1097-2765(00)80330-5.Haslbeck, M., Braun, N., Stromer, T., Richter, B., Model, N., Weinkauf, S., et al. (2004). Hsp42 is the general small heat shock protein in the cytosol of
Saccharomyces cerevisiae. EMBO J.
23, 638–649. doi:10.1038/sj.emboj.7600080.Hetz, C., Chevet, E., and Oakes, S. A. (2015). Proteostasis control by the unfolded protein response.
Nat. Cell Biol.
17, 829–838. doi:10.1038/ncb3184.Hiraishi, H., Mochizuki, M., and Takagi, H. (2006). Enhancement of stress tolerance in
Saccharomyces cerevisiae by overexpression of ubiquitin ligase Rsp5 and ubiquitin-conjugating enzymes.
Biosci. Biotechnol. Biochem.
70, 2762–2765. doi:10.1271/bbb.60250.Hoek, J. B., Cahill, A., and Pastorino, J. G. (2002). Alcohol and mitochondria: a dysfunctional relationship.
Gastroenterology
Saccharomyces pastorianus orthologous genes using oligonucleotide microarrays.
J. Biosci. Bioeng.
Appl. Environ. Microbiol.
76, 1563–1573. 24oi:10.1128/AEM.03153-09.Iwahashi, H., Nwaka, S., Obuchi, K., and Komatsu, Y. (1998). Evidence for the interplay between trehalose metabolism and Hsp104 in yeast.
Appl. Environ. Microbiol.
64, 4614–4617.Kamena, F., Diefenbacher, M., Kilchert, C., Schwarz, H., and Spang, A. (2008). Ypt1p is essential for retrograde Golgi-ER transport and for Golgi maintenance in
S. cerevisiae.
J. Cell Sci.
Saccharomyces cerevisiae. Yeast
28, 339–347. doi:10.1002/yea.1842.Kaufman, B. A., Kolesar, J. E., Perlman, P. S., and Butow, R. A. (2003). A function for the mitochondrial chaperonin Hsp60 in the structure and transmission of mitochondrial DNA nucleoids in
Saccharomyces cerevisiae . J. Cell Biol.
Biol. Chem.
Cell
Annu. Rev. Biochem.
82, 323–355. doi:10.1146/annurev-biochem-060208-092442.Kitagaki, H., Araki, Y., Funato, K., and Shimoi, H. (2007). Ethanol-induced death in yeast exhibits features of apoptosis mediated by mitochondrial fission pathway.
FEBS Lett.
J. Biosci. Bioeng.
J. Biosci. Bioeng.
Mol. Cell
64, 148–162. doi:10.1016/j.molcel.2016.08.020.Krzewska, J., Langer, T., and Liberek, K. (2001). Mitochondrial Hsp78, a member of the Clp/Hsp100 family in
Saccharomyces cerevisiae , cooperates with Hsp70 in protein refolding.
FEBS Lett.
Saccharomyces cerevisiae populations.
J. Am. Soc. Brew. Chem.
70, 268–274. doi:10.1094/ASBCJ-2012-0917-01.Lee, M. C. S., Miller, E. A., Goldberg, J., Orci, L., and Schekman, R. (2004). Bi-directional protein transport between the ER and Golgi.
Annu. Rev. Cell Dev. Biol.
20, 87–123. doi:10.1146/annurev.cellbio.20.010403.105307. 25ee, S., Carlson, T., Christian, N., Lea, K., Kedzie, J., Reilly, J. P., et al. (2000). The Yeast heat shock transcription factor changes conformation in response to superoxide and temperature.
Mol. Biol. Cell
11, 1753–1764. doi:10.1091/mbc.11.5.1753.Li, B.-Z., Cheng, J.-S., Qiao, B., and Yuan, Y.-J. (2010). Genome-wide transcriptional analysis of Saccharomyces cerevisiae during industrial bioethanol fermentation.
J. Ind. Microbiol. Biotechnol.
37, 43–55. doi:10.1007/s10295-009-0646-4.Liu, Q., Krzewska, J., Liberek, K., and Craig, E. A. (2001). Mitochondrial Hsp70 Ssc1: role in protein folding.
J. Biol. Chem.
EMBO J.
27, 1049–1059. doi:10.1038/emboj.2008.42.Lloyd, D., Moran, C. A., Suller, M. T. E., Dinsdale, M. G., and Hayes, A. J. (1996). Flow cytometricmonitoring of rhodamine 123 and a cyanine dye uptake by yeast during cider fermentation.
J. Inst. Brew.
Saccharomyces cerevisiae, ” in
Microbial Stress Tolerance for Biofuels , ed. Z. L. Liu (Berlin, Heidelberg: Springer Berlin Heidelberg), 77–115. doi:10.1007/978-3-642-21467-7_4.Mager, W. H., and Ferreira, P. M. (1993). Stress response of yeast.
Biochem. J.
Saccharomyces cerevisiae , differ in their functional properties.
J. Biol. Chem.
Saccharomyces cerevisiae . Biosci. Biotechnol. Biochem.
78, 1389–1391. doi:10.1080/09168451.2014.921561.Mizunaga, T., Katakura, Y., Miura, T., and Maruyama, Y. (1990). Purification and characterization of yeast protein disulfide isomerase.
J. Biochem. (Tokyo)
J. Biol. Chem.
Biochim. Biophys. Acta BBA - Mol. Cell Res.
J. Biol. Chem.
Nat. Chem. Biol.
3, 313–320. doi:10.1038/nchembio880.Mori, K. (2015). The unfolded protein response: the dawn of a new field.
Proc. Jpn. Acad. Ser. B
91, 469–480. doi:10.2183/pjab.91.469. 26athan, D. F., Vos, M. H., and Lindquist, S. (1997). In vivo functions of the
Saccharomyces cerevisiae
Hsp90 chaperone.
Proc. Natl. Acad. Sci.
94, 12949–12956. doi:10.1073/pnas.94.24.12949.Navarro-Tapia, E., Nana, R. K., Querol, A., and Pérez-Torrado, R. (2016). Ethanol cellular defense induce unfolded protein response in yeast.
Front. Microbiol.
7. doi:10.3389/fmicb.2016.00189.Navarro-Tapia, E., Pérez-Torrado, R., and Querol, A. (2017). Ethanol effects involve non-canonical unfolded protein response activation in yeast cells.
Front. Microbiol.
8. doi:10.3389/fmicb.2017.00383.Neupert, W., and Herrmann, J. M. (2007). Translocation of Proteins into Mitochondria.
Annu. Rev. Biochem.
76, 723–749. doi:10.1146/annurev.biochem.76.052705.163409.Nijtmans, L. G. J. (2000). Prohibitins act as a membrane-bound chaperone for the stabilization of mitochondrial proteins.
EMBO J.
19, 2444–2451. doi:10.1093/emboj/19.11.2444.Nillegoda, N. B., Theodoraki, M. A., Mandal, A. K., Mayo, K. J., Ren, H. Y., Sultana, R., et al. (2010). Ubr1 and Ubr2 function in a quality control pathway for degradation of unfolded cytosolic proteins.
Mol. Biol. Cell
21, 2102–2116. doi:10.1091/mbc.e10-02-0098.O’Connor-Cox, E. S. C., Lodolo, E. J., and Axcell, B. C. (1996). Mitochondrial relevance to yeast fermentative performance: a review.
J. Inst. Brew.
J. Ind. Microbiol.
10, 111–116. doi:10.1007/BF01583843.Panaretou, B., Siligardi, G., Meyer, P., Maloney, A., Sullivan, J. K., Singh, S., et al. (2002). Activation of the ATPase activity of Hsp90 by the stress-regulated cochaperone Aha1.
Mol. Cell
10, 1307–1318. doi:10.1016/S1097-2765(02)00785-2.Parsell, D. A., Taulien, J., Lindquist, S. L., Viitanen, P., Jaenicke, R., Horwich, A., et al. (1993). Therole of heat-shock proteins in thermotolerance.
Philos. Trans. R. Soc. Lond. B. Biol. Sci.
Biochem. Biophys. Res. Commun.
Cell Res.
28, 296–306. doi:10.1038/cr.2018.17.Perić, M., Dib, P. B., Dennerlein, S., Musa, M., Rudan, M., Lovrić, A., et al. (2016). Crosstalk between cellular compartments protects against proteotoxicity and extends lifespan.
Sci. Rep.
6, 28751. doi:10.1038/srep28751.Pfeiffer, A., Stephanowitz, H., Krause, E., Volkwein, C., Hirsch, C., Jarosch, E., et al. (2016). A Complex of Htm1 and the oxidoreductase Pdi1 accelerates degradation of misfolded glycoproteins.
J. Biol. Chem.
FEMS Microbiol. Lett.
Aging Cell
1, 149–157. doi:10.1046/j.1474-9728.2002.00018.x.Piper, P. W., Talreja, K., Panaretou, B., Moradas-Ferreira, P., Byrne, K., Praekelt, U. M., et al. (1994). Induction of major heat-shock proteins of
Saccharomyces cerevisiae, including plasma membrane Hsp30, by ethanol levels above a critical threshold.
Microbiology
FEMS Yeast Res.
3, 149–157. doi:10.1016/S1567-1356(03)00002-3.Prasad, R., Xu, C., and Ng, D. T. W. (2018). Hsp40/70/110 chaperones adapt nuclear protein qualitycontrol to serve cytosolic clients.
J. Cell Biol.
J. Ind. Microbiol. Biotechnol.
38, 1133–1144. doi:10.1007/s10295-011-0999-3.Qing, Z., Hai, Z., Guohua, Z., Kaize, H., Zhirong, Y., and Yanling, J. (2012). Transcriptome analysisof
Saccharomyces cerevisiae at the late stage of very high gravity (VHG) fermentation.
Afr. J. Biotechnol.
11, 9641–9648. doi:10.5897/AJB12.268.Raimundo, N., and Kriško, A. (2018). Cross-organelle communication at the core of longevity.
Aging
10, 15–16. doi:10.18632/aging.101373.Rautio, J. J., Huuskonen, A., Vuokko, H., Vidgren, V., and Londesborough, J. (2007). Monitoring yeast physiology during very high gravity wort fermentations by frequent analysis of gene expression.
Yeast
24, 741–760. doi:10.1002/yea.1510.Reading, D. S., Hallberg, R. L., and Myers, A. M. (1989). Characterization of the yeast
HSP60 genecoding for a mitochondrial assembly factor.
Nature
J. Biol. Chem.
J. Clin. Invest.
J. Cell Biol.
Caenorhabditis elegans . J. Biol. Chem.
Saccharomyces cerevisiae transcriptomic response to alcohols and anaerobiosis.
G3 GenesGenomesGenetics
8, 3881–3890. doi:10.1534/g3.118.200677. 28chmitt, M. (1996). The molecular chaperone Hsp78 confers compartment-specific thermotoleranceto mitochondria.
J. Cell Biol.
EMBO J.
14, 3434–3444. doi:10.1002/j.1460-2075.1995.tb07349.x.Schreiner, B., Westerburg, H., Forné, I., Imhof, A., Neupert, W., and Mokranjac, D. (2012). Role of the AAA protease Yme1 in folding of proteins in the intermembrane space of mitochondria.
Mol. Biol. Cell
23, 4335–4346. doi:10.1091/mbc.e12-05-0420.Schuldiner, M., Collins, S. R., Thompson, N. J., Denic, V., Bhamidipati, A., Punna, T., et al. (2005). exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile.
Cell
Cell
61, 1349–1357. doi:10.1016/0092-8674(90)90698-E.Sethi, R., Iyer, S. S., Das, E., and Roy, I. (2018). Discrete roles of trehalose and Hsp104 in inhibition of protein aggregation in yeast cells.
FEMS Yeast Res.
18. doi:10.1093/femsyr/foy058.Shahsavarani, H., Sugiyama, M., Kaneko, Y., Chuenchit, B., and Harashima, S. (2012). Superior thermotolerance of
Saccharomyces cerevisiae for efficient bioethanol fermentation can be achieved by overexpression of RSP5 ubiquitin ligase.
Biotechnol. Adv.
30, 1289–1300. doi:10.1016/j.biotechadv.2011.09.002.Sharma, V. M. (2001). Quantitative target display: a method to screen yeast mutants conferring quantitative phenotypes by “mutant DNA fingerprints.”
Nucleic Acids Res.
29, 86e–886. doi:10.1093/nar/29.17.e86.Short, M. K., Hallett, J. P., Tar, K., Dange, T., Schmidt, M., Moir, R., et al. (2012). The yeast magmas ortholog Pam16 has an essential function in fermentative growth that involves sphingolipid metabolism.
PLoS ONE
7, e39428. doi:10.1371/journal.pone.0039428.Sidrauski, C., and Walter, P. (1997). The transmembrane kinase Ire1p is a site-specific endonucleasethat initiates mRNA splicing in the unfolded protein response.
Cell
90, 1031–1039. doi:10.1016/S0092-8674(00)80369-4.Silva, D., Brányik, T., Dragone, G., Vicente, A., Teixeira, J., and Almeida e Silva, J. (2008). High gravity batch and continuous processes for beer production: evaluation of fermentation performance and beer quality.
Chem. Pap.
62. doi:10.2478/s11696-007-0076-6.Silverstein, R. A., and Ekwall, K. (2005). Sin3: a flexible regulator of global gene expression and genome stability.
Curr. Genet.
47, 1–17. doi:10.1007/s00294-004-0541-5.Singer, M. A., and Lindquist, S. (1998). Multiple effects of trehalose on protein folding in vitro and in vivo.
Mol. Cell
1, 639–648. doi:10.1016/S1097-2765(00)80064-7.Smart, K. A. (2007). Brewing yeast genomes and genome-wide expression and proteome profiling during fermentation.
Yeast
24, 993–1013. doi:10.1002/yea.1553.Smart, K. A., Quain, D. E., Powell, C. D., and Van Zandycke, S. M. (2000). Replicative ageing and senescence in
Saccharomyces cerevisiae and the impact on brewing fermentations.
Microbiology
Saccharomyces cerevisiae . J. Appl. Microbiol. doi:10.1111/j.1365-2672.2009.04657.x.Stefanovic, S., and Hegde, R. S. (2007). Identification of a targeting factor for posttranslational membrane protein insertion into the ER.
Cell
J. Am. Soc. Brew. Chem.
68, 1–9. doi:10.1094/ASBCJ-2009-1214-01.Stolz, A., Besser, S., Hottmann, H., and Wolf, D. H. (2013). Previously unknown role for the ubiquitin ligase Ubr1 in endoplasmic reticulum-associated protein degradation.
Proc. Natl. Acad. Sci.
Mol. Cell
70, 1025-1037.e5. doi:10.1016/j.molcel.2018.04.027.Takemori, Y., Sakaguchi, A., Matsuda, S., Mizukami, Y., and Sakurai, H. (2006). Stress-induced transcription of the endoplasmic reticulum oxidoreductin gene
ERO1 in the yeast
Saccharomyces cerevisiae.
Mol. Genet. Genomics
Cold Spring Harb. Perspect. Biol.
4, a013193–a013193. doi:10.1101/cshperspect.a013193.Tomás, M., Marín, M. P., Martínez-Alonso, E., Esteban-Pretel, G., Díaz-Ruiz, A., Vázquez-Martínez, R., et al. (2012). Alcohol induces Golgi fragmentation in differentiated PC12 cellsby deregulating Rab1-dependent ER-to-Golgi transport.
Histochem. Cell Biol.
HAC1
RNA.
PLoS Genet.
8, e1002862. doi:10.1371/journal.pgen.1002862.van Meer, G., Voelker, D. R., and Feigenson, G. W. (2008). Membrane lipids: where they are and how they behave.
Nat. Rev. Mol. Cell Biol.
9, 112–124. doi:10.1038/nrm2330.Verghese, J., Abrams, J., Wang, Y., and Morano, K. A. (2012). Biology of the heat shock response and protein chaperones: budding yeast (
Saccharomyces cerevisiae ) as a model system.
Microbiol. Mol. Biol. Rev.
76, 115–158. doi:10.1128/MMBR.05018-11.Vevea, J. D., Garcia, E. J., Chan, R. B., Zhou, B., Schultz, M., Di Paolo, G., et al. (2015). Role for lipid droplet biogenesis and microlipophagy in adaptation to lipid imbalance in yeast.
Dev. Cell
35, 584–599. doi:10.1016/j.devcel.2015.11.010.Wang, F., Chan, C., Weir, N. R., and Denic, V. (2014). The Get1/2 transmembrane complex is an endoplasmic-reticulum membrane protein insertase.
Nature
Biol. Chem.
Biochem. Biophys. Res. Commun.
Nature
Mol. Cell. Biol.
18, 1967–1977. doi:10.1128/MCB.18.4.1967.Welti, M., and Hülsmeier, A. J. (2014). Ethanol-induced impairment in the biosynthesis of n-linked glycosylation: ethanol interference with N-linked glycosylation.
J. Cell. Biochem.
Saccharomyces cerevisiae.
J. Biol. Chem.
EMBO J.
21, 4411–4419. doi:10.1093/emboj/cdf445.Zhao, Y., MacGurn, J. A., Liu, M., and Emr, S. (2013). The ART-Rsp5 ubiquitin ligase network comprises a plasma membrane quality control system that protects yeast cells from proteotoxic stress. eLife
2, e00459. doi:10.7554/eLife.00459.Zolkiewski, M., Zhang, T., and Nagy, M. (2012). Aggregate reactivation mediated by the Hsp100 chaperones.
Arch. Biochem. Biophys.
Saccharomyces cerevisiae . Genetics igures legendsFigure 1. (A) Differentially upregulated Pan-genes associated with proteostasis observed inthe lager yeast CB11 strain during beer fermentation. The mean expression values are indicated bylog2 fold change ± standard deviation (SD) on the y-axis and in the inset. Gene names are indicatedon the x-axis. (B) Heatmap plot showing the clustered differentially upregulated genes associatedwith proteostasis observed in CB11 during beer fermentation and the associated clustered biologicalprocesses from gene ontology analysis (see Figure S1). Heatmap rows and columns were groupedusing the Euclidean distance method and complete linkage.
Figure 2. (A) Differentially upregulated Pan-genes associated with chaperones and foldingproteins observed in the lager yeast CB11 strain during beer fermentation. The mean expressionvalues are indicated by log2 fold change ± standard deviation (SD) on the y-axis and in the inset.Gene names are indicated on the x-axis. (B) Heatmap plot showing the clustered differentiallyupregulated genes associated with chaperones and folding proteins observed in CB11 during beerfermentation and the associated clustered biological processes from gene ontology analysis (seeFigure S1). Heatmap rows and columns were grouped using the Euclidean distance method andcomplete linkage.
Figure 3. (A) Number of chaperones and folding protein coding Pan-genes found to beupregulated in different organelles of the lager yeast CB11 strain during beer fermentation, incomparison to yeast propagation. (B) Number of coding Pan-genes upregulated in CB11 duringbeer fermentation, in comparison to yeast propagation, that are linked to the major chaperoneprotein families.
Figure 4.
A model for inter-organellar/cross-organellar communication/responseproteostasis (CORE network) in brewing yeast. During beer fermentation and/or yeast reuse, theendoplasmic reticulum (ER), mitochondria, and cytosol regulate proteostasis/protein quality by32onitoring their environments and communicating with one another by means of the COREnetwork. In conditions of proteotoxicity induced by ethanol during beer fermentation, the COREnetwork is activated and is composed of different proteins/pathways, such as heat shock proteins(HSPs), endoplasmic reticulum-associated protein degradation (ERAD), the stress-induced,homeostatically regulated protein degradation (SHRED) pathway, E3 ubiquitin ligases, and theprohibitin complex. Trehalose, a molecular chaperone necessary for proteotoxic response, is alsopart of the CORE network. Additionally, each organelle has its own particular mechanisms ofprotein quality control/proteostasis. The impact of the CORE network in the proteostasis responseof vacuoles of brewing yeast is not well understood, but may be associated with microlipophagy.Finally, proteotoxicity induced by ethanol regulates transcriptional activity and epigeneticmechanisms in the nucleus, which are influenced by CORE network components. Moreover, theCORE network activity and proteotoxicity are potentially linked to aging in brewing yeast cells. 33igure 1. 34igure 2. 35igure 3. 36igure 4. 378
UPPLEMENTARY MATERIAL 1Experimental procedures
DNA microarray gene expression and gene ontology analysis
DNA microarray gene expression (GSE) datasets (GSE9423, GSE10205, and GSE16376)comparing lager yeast CB11 strain (
Saccharomyces pastorianus
Table S1.
Gene expression datasets (GSEs) used in this work.
Source a GEO samples files Sample name Sample Organism Strain
GSE9423 GSM239499 Fermentation 30 hours Ferm_30h_B
Saccharomyces pastorianus
CB11GSE9423 GSM239503 Fermentation 60 hours Ferm_60h_C
Saccharomyces pastorianus
CB11GSE9423 GSM239512 Propagation 30 hours Prop_30h_C
Saccharomyces pastorianus
CB11GSE9423 GSM239504 Fermentation 8 hours Ferm_8h_A
Saccharomyces pastorianus
CB11GSE9423 GSM239514 Propagation 8 hours Prop_8h_B
Saccharomyces pastorianus
CB11GSE9423 GSM239501 Fermentation 60 hours Ferm_60h_A
Saccharomyces pastorianus
CB11GSE9423 GSM239510 Propagation 30 hours Prop_30h_A
Saccharomyces pastorianus
CB11GSE9423 GSM239515 Propagation 8 hours Prop_8h_C
Saccharomyces pastorianus
CB11GSE9423 GSM239506 Fermentation 8 hours Ferm_8h_C
Saccharomyces pastorianus
CB11GSE9423 GSM239505 Fermentation 8 hours Ferm_8h_B
Saccharomyces pastorianus
CB11GSE9423 GSM239511 Propagation 30 hours Prop_30h_B
Saccharomyces pastorianus
CB11GSE9423 GSM239502 Fermentation 60 hours Ferm_60h_B
Saccharomyces pastorianus
CB11GSE9423 GSM239508 Propagation 0 hours Prop_0h_B
Saccharomyces pastorianus
CB11GSE9423 GSM239500 Fermentation 30 hours Ferm_30h_C
Saccharomyces pastorianus
CB11GSE9423 GSM239507 Propagation 0 hours Prop_0h_A
Saccharomyces pastorianus
CB11GSE9423 GSM239509 Propagation 0 hours Prop_0h_C
Saccharomyces pastorianus
CB11 ource a GEO samples files Sample name Sample Organism Strain
GSE9423 GSM239513 Propagation 8 hours Prop_8h_A
Saccharomyces pastorianus
CB11GSE10205 GSM257787 Fermentation 102 hours Ferm_102h_A
Saccharomyces pastorianus
CB11GSE10205 GSM257776 Fermentation 8 hours Ferm_8h_A
Saccharomyces pastorianus
CB11GSE10205 GSM257778 Fermentation 8 hours Ferm_8h_C
Saccharomyces pastorianus
CB11GSE10205 GSM257789 Fermentation 102 hours Ferm_102h_C
Saccharomyces pastorianus
CB11GSE10205 GSM257780 Fermentation 30 hours Ferm_30h_B
Saccharomyces pastorianus
CB11GSE10205 GSM257782 Fermentation 60 hours Ferm_60 h_B
Saccharomyces pastorianus
CB11GSE10205 GSM257786 Fermentation 80 hours Ferm_80h_C
Saccharomyces pastorianus
CB11GSE10205 GSM257785 Fermentation 80 hours Ferm_80h_B
Saccharomyces pastorianus
CB11GSE10205 GSM257781 Fermentation 60 hours Ferm_60h_A
Saccharomyces pastorianus
CB11GSE10205 GSM257783 Fermentation 60 hours Ferm_60h_C
Saccharomyces pastorianus
CB11GSE10205 GSM257784 Fermentation 80 hours Ferm_80h_A
Saccharomyces pastorianus
CB11GSE10205 GSM257777 Fermentation 8 hours Ferm_8h_B
Saccharomyces pastorianus
CB11GSE10205 GSM257779 Fermentation 30 hours Ferm_30h_A
Saccharomyces pastorianus
CB11GSE10205 GSM257788 Fermentation 102 hours Ferm_102h_B
Saccharomyces pastorianus
CB11GSE16376 GSM410831 Propagation 0 hours Prop_0h_A
Saccharomyces pastorianus
CB11GSE16376 GSM410832 Propagation 0 hours Prop_0h_B
Saccharomyces pastorianus
CB11GSE16376 GSM410833 Propagation 0 hours Prop_0h_C
Saccharomyces pastorianus
CB11GSE16376 GSM410834 Propagation 4 hours Prop_4h_A
Saccharomyces pastorianus
CB11GSE16376 GSM410835 Propagation 4 hours Prop_4h_B
Saccharomyces pastorianus
CB11GSE16376 GSM410836 Propagation 4 hours Prop_4h_C
Saccharomyces pastorianus
CB11GSE16376 GSM410837 Propagation 8 hours Prop_8h_A
Saccharomyces pastorianus
CB11GSE16376 GSM410838 Propagation 8 hours Prop_8h_B
Saccharomyces pastorianus
CB11GSE16376 GSM410839 Propagation 8 hours Prop_8h_C
Saccharomyces pastorianus
CB11GSE16376 GSM410840 Propagation 30 hours Prop_30h_A
Saccharomyces pastorianus
CB11GSE16376 GSM410841 Propagation 30 hours Prop_30h_B
Saccharomyces pastorianus
CB11GSE16376 GSM410842 Propagation 30 hours Prop_30h_C
Saccharomyces pastorianus
CB11 a In blue color, the GSE9423 used for single DNA microarray analysis. In red, the GSE10205 and GSE16376 used forDNA microarray meta-analysis.
Saccharomyces
Genome Database (Figure S1). Further, theproteostasis- and chaperones-linked DEGs obtained from DNA microarray single- and meta-analysis were applied to select a list of commonly observed DEGs in both analysis, and were calledas proteostasis Pan-DEGs (Figure S1). The major biological processes and cellular componentassociated to proteostasis- and chaperones-linked DEGs lists from DNA microarray single- andmeta-analysis and Pan-DEGs were further determined using the R package clusterProfile and
Saccharomyces cerevisiae protein data from UniProt (Yu et al., 2012) (Figure S1). The degree offunctional enrichment for a given biological process category was quantitatively assessed ( p -value <0.01) using a hypergeometric distribution. Multiple test correction was also assessed by applyingFDR algorithm (Benjamini and Hochberg, 1995) at a significance level of p < 0.05. Semanticcomparison among biological processes and cellular component associated to DEGs were madeusing R package GOSemSim (Yu et al., 2010) using false discovery rate (FDR) < 0.01 and q-value< 0.05 (Figure S1). Networks containing the subcellular targets of proteostasis- and chaperones-associated DEGs from DNA microarray single analysis, meta-analysis and Pan-DEGs weregenerated using the R package igraph and Cytoscape 3.7.2 (Shannon et al., 2003; Csardi andNepusz, 2006, [CSL STYLE ERROR: reference with no printed form.]) (Figure S1). Heatmapscombining proteostasis- and chaperones-associated DEGs from DNA microarray single- and meta-analysis and Pan-DEGs values and GOs were designed with R package ComplexHeatmap (Gu etal., 2016), where rows and columns were grouped using Euclidean distance method and complete41inkage (Figure S1). All Figures displayed in this supplementary material as well as in themanuscript can be downloaded at https://github.com/bonattod/Proteostasis_data_analysis.git limmaFermentation 8 hFermentation 30 hFermentation 60 hFermentation 80 hFermentation 102 h Propagation 0 h Propagation 4 hPropagation 8 hPropagation 30 hFermentation x h versus Propagation y hCONTRASTSSAMPLESTOTAL = 20 CONTRASTSGSE10205GEOqueryarrayQualityMetrics GSE16376GEOqueryarrayQualityMetricsclusterProfileGoSemSimSGDAnnotated proteostasis- associated genesGene OntologiesProteostasis-associated DEGsDEGsGSE9423GEOqueryarrayQualityMetricslimmaclusterProfileGoSemSimSGDAnnotated proteostasis- associated genesProteostasis-associated DEGsSAMPLESFermentation 8 hFermentation 30 hFermentation 60 h Propagation 0 h Propagation 8 hPropagation 30 hCONTRASTSFermentation x h versus Propagation y hTOTAL = 9 CONTRASTSGene OntologiesDEGs Proteostasis Pan-DEGsclusterProfileGoSemSimGene Ontologies DNA microarray analysis DNA microarray meta-analysis
UNIPROTAnnotated subcellular protein localizationigraphCytoscapeCellular compartmentUNIPROTAnnotated subcellular protein localizationigraphCytoscapeCellular compartment UNIPROTAnnotated subcellular protein localizationigraphCytoscapeCellular compartment
Figure S1.
Experimental design used in DNA microarray single and meta-analysis.Abbreviation: differential by expressed genes (DEGs);
Saccharomyces
Genome Database (SGD);Universal Protein Resource (UniProt). 42 upplementary results
DNA microarray single- and meta-analysis
Data gathered from DNA microarray single analysis (GSE9423) showed a low number ofoverexpressed and underexpressed DEGs comparing the very beginning of propagation condition (0hour) with different times of fermentation (8, 30, and 60 hours) (Figure S2A), while the number ofDEGs in fermentation compared to different propagation times (8 and 30 hours) dramaticallyincreased (Figure S2A). A similar result was also observed for DNA microarray meta-analysiscomparing different times of fermentation (GSE10205) and propagation (GSE16376) (Figure S2B),where the number of DEGs was low when yeast cells in the first hours of propagation (0 and 4hours) were compared with yeasts in different times of fermentation (from 8 to 102 hours).Additionally, the number of DEGs in both DNA microarray single- and meta-analysis sharplyincreased when cells in different times of fermentation were compared with the same yeast strainafter 8 hours of propagation (Figures S2A and B).
Figure S2.
In (A), DNA microarray single analysis of GSE9423 dataset comparing the lageryeast CB11 strain in different times of fermentation (F) and propagation (P). In (B), DNAmicroarray meta-analysis comparing the lager yeast CB11 strain in different times of fermentation(F; GSE10205) and propagation (P; GSE16376). The time of point collection (in hours) is indicatedafter the letters “F” and “P”. The black squares above the bars indicate the total number of DEGsobserved for a given contrast. The numbers inside the blue and red bars shown the total of43nderexpressed and overexpressed DEGs observed in a specific contrast. The inset in the graphic(B) is a zoom of the first two bars.
Proteostasis- and chaperones-associated DEGs in DNA microarray single- and meta-analysis
The overexpressed DEGs from DNA microarray single and meta-analysis (Figure S2A andB) were filtered for proteostasis- and chaperones-associated genes using the annotated data from
Saccharomyces
Genome Database (Figure S1). The number of overexpressed proteostasis- andchaperones-associated DEGs observed in beer fermentation using DNA microarray single analysis(Figures S3A and S4A) and DNA microarray meta-analysis (Figure S5A and S6A) was similar.These DEGs were then subjected to a gene ontology (GO) analysis and the major biologicalprocesses were evaluated (Figures S3B to S6B). Data from GO analysis showed that similarbiological processes were obtained after semantic reduction for both proteostasis- and chaperones-associated DEGs gathered from different DNA microarray analysis (Figures S3B to S6B). 44 igure S3. (A) Differentially upregulated genes from DNA microarray single analysis(GSE9423) associated with proteostasis observed in the lager yeast CB11 strain during beerfermentation, compared to the propagation step, at different times. The mean expression values areindicated by log2 fold change ± standard deviation (SD) on the y-axis and in the inset. Gene namesare indicated on the x-axis. (B) Heatmap plot showing the clustered differentially upregulated genesassociated with proteostasis observed in CB11 during beer fermentation, compared to thepropagation step, at different times and the associated clustered biological processes from gene45ntology analysis. Heatmap rows and columns were grouped using the Euclidean distance methodand complete linkage.
Figure S4. (A) Differentially upregulated genes from DNA microarray single analysis(GSE9423) associated with chaperones and folding proteins observed in the lager yeast CB11 strainduring beer fermentation, compared to the propagation step, at different times. The mean expressionvalues are indicated by log2 fold change ± standard deviation (SD) on the y-axis and in the inset.Gene names are indicated on the x-axis. (B) Heatmap plot showing the clustered differentially46pregulated genes associated with chaperones and folding proteins observed in CB11 during beerfermentation, compared to the propagation step, at different times and the associated clusteredbiological processes from gene ontology analysis. Heatmap rows and columns were grouped usingthe Euclidean distance method and complete linkage. 47 igure S5.
A) Differentially upregulated genes from DNA microarray meta-analysis(GSE10205 versus GSE16376) associated with proteostasis observed in the lager yeast CB11 strainduring beer fermentation, compared to the propagation step, at different times. The mean expressionvalues are indicated by log2 fold change ± standard deviation (SD) on the y-axis and in the inset.Gene names are indicated on the x-axis. (B) Heatmap plot showing the clustered differentiallyupregulated genes associated with proteostasis observed in CB11 during beer fermentation,compared to the propagation step, at different times and the associated clustered biologicalprocesses from gene ontology analysis. Heatmap rows and columns were grouped using theEuclidean distance method and complete linkage. 48 igure S6. (A) Differentially upregulated genes from from DNA microarray meta-analysis(GSE10205 versus GSE16376) associated with chaperones and folding proteins observed in thelager yeast CB11 strain during beer fermentation, compared to the propagation step, at differenttimes. The mean expression values are indicated by log2 fold change ± standard deviation (SD) onthe y-axis and in the inset. Gene names are indicated on the x-axis. (B) Heatmap plot showing the49lustered differentially upregulated genes associated with chaperones and folding proteins observedin CB11 during beer fermentation, compared to the propagation step, at different times and theassociated clustered biological processes from gene ontology analysis. Heatmap rows and columnswere grouped using the Euclidean distance method and complete linkage.
Subcellular localization of proteostasis- and chaperone-associated DEGs products
The subcellular localization of proteostasis- and chaperone-associated DEGs productsindicated that most of proteins can be found in cytoplasm, nucleus, ER, and mitochondria (FiguresS7A to B and Figures S8A and C). In this sense, both DNA microarray analysis point to the samesubcellular localization (Figures S8A and C) of different chaperone families whose members areupregulated in beer fermentation (Figures 8B and D).
Figure S7.
Networks describing the subcellular localization of proteostasis-coding DEGsobtained from DNA microarray single (GSE9423; A) and meta-analysis (GSE10205 versusGSE16376; B). The width of edges (thin to thick) is proportional to the mean logFC for each DEGevaluated in each analysis. The diameter of nodes representing the subcellular targets do not haveany biological and/or statistical significance. 50
LNP1 GET1PHB1YOS9KAR2 PMT1 PHO86ORM2ULI1RTN1
CellMembrane
CPR6 SSB2HSP82 HSP42SYT1YAP1 HSP104
Cytoplasm
SHQ1OTU1HCH1 SBA1 PTC2
Nucleus
GET3 EMP65CUR1HSP26 ERJ5MDJ1HSP78SNF1 ZIM17PHB2
Mitochondria
IRC25 GET2PDI1SGT2 ER YPT1 MPD1 CPR5
Golgi
SSA4SSA3EDE1AHA1GET4 TSA1 (A)
SSA3 YPT1SSA4BCK1 GET4SYT1
Cytoplasm
SGT2 OTU1AHA1 SBA1 HAC1HSP82 YAR1PTC2
Nucleus
ZIM17MDJ1XDJ1PHB2PHB1HSP78
Mitochondria
PMT1GET2YOP1KAR2 CPR5 ORM2PDI1 ER HSP42CPR6SSB2EDE1 TSA1 SLT2IRC25GET3 HSP104HSP26GET1HCH1 YAP1HSC82
Golgi
RTN1 MID2PMT2 RTN2 PHO86
CellMembrane (B) igure S8. (A) and (C) Number of chaperones and folding protein coding genes found to beupregulated in different organelles of the lager yeast CB11 strain during beer fermentation, incomparison to yeast propagation as observed from DNA microarray single (GSE9423) and meta-analysis (GSE10205 versus GSE16376), respectively. (B) and (D) Number of coding genesupregulated in CB11 during beer fermentation, in comparison to yeast propagation, that are linked51 (A) (B)(C) (D) o the major chaperone protein families as observed from DNA microarray single (GSE9423) andmeta-analysis (GSE10205 versus GSE16376), respectively.
Evaluation of proteostasis- and chaperone-associated Pan-DEGs
In order to identify common upregulated proteostasis- and chaperone-associated DEGs(Pan-DEGs) in yeast lager CB11 strain during beer fermentation, DNA microarray data from singleand meta-analysis were used (Figure S1). A high degree of overlap between DNA microarrayanalysis was observed, with 36 proteostasis-associated Pan-DEGs and 54 chaperone-associatedPan-DEGs identified (Figures S9A and B). This proteostasis- and chaperone-associated Pan-DEGswere further applied for biological processes and subcellular localization analyses (Figures 1 to 3 inthe main text of the manuscript). 52 roteostasis-associated Pan-DEGs
GSE9423 GSE10205 versus GSE16376 (A) Chaperone-associated Pan-DEGs
GSE9423 GSE10205 versus GSE16376 (B)
Figure S9.
Evaluation of overlap degree of upregulated DEGs (Pan-DEGs) in yeast lagerCB11 during beer fermentation between DNA microarray single- (GSE9423) and meta-analysis(GSE10205 versus GSE16376). (A) and (B), proteostasis- and chaperone-associated Pan-DEGs,respectively.
Additional data rp6p-Rpd3p-Pbp1p interaction network A protein-protein interaction network of Crp6p with Rpd3p and Pbp1p (Figure S10) wasobtained from STRING 11.0 (https://string-db.org). The following parameters were used fornetwork prospection:
Saccharomyces cerevisiae as selected organism; active prediction methods:databases and experiments; no more than five interactions in the first network shell; mediumconfidence score (0.400); evidence as meaning of network edges.
Figure S10.
Protein-protein network interaction of yeast
Saccharomyces cerevisiae
Cpr6pwith Rpd3p and Pbp1p. Edges color and number indicate supporting evidences from curateddatabases (light blue) and experiments (dark blue).
Evaluation of fatty acid-associated DEGs in Saccharomyces pastorianus CB11 strain during beerfermentation
Data from DNA single- (GSE9423) and meta-analysis (GSE10205 versus GSE16376)indicated that genes linked to fatty acid biosynthesis are upregulated in yeast lager CB11 strainduring beer fernentation. In this sense, 124 upregulated fatty acid biosynthesis-associated DEGswere observed in GSE9423 dataset (Figure S11), while 113 DEGs were overexpressed in theGSE10205 versus GSE16376 datasets (Figure S12). 54
RCO1 CTI6SIN3 SNT1 SAP30RPD3CPR6PBP1 igure S11. (A) Differentially upregulated genes from DNA microarray single analysis(GSE9423) associated with fatty acids biosynthesis observed in the lager yeast CB11 strain duringbeer fermentation, compared to the propagation step, at different times. The mean expression valuesare indicated by log2 fold change ± standard deviation (SD) on the y-axis and in the inset. Genenames are indicated on the x-axis.
Figure S12.
Differentially upregulated genes from DNA microarray meta-analysis(GSE10205 versus GSE16376) associated with fatty acids biosynthesis observed in the lager yeastCB11 strain during beer fermentation, compared to the propagation step, at different times. Themean expression values are indicated by log2 fold change ± standard deviation (SD) on the y-axisand in the inset. Gene names are indicated on the x-axis. 55 eferences
Benjamini, Y., and Hochberg, Y. (1995). Controlling the false discovery rate: a practical and powerful approach to multiple testing.
J. R. Stat. Soc. Ser. B Methodol.
57, 289–300.Csardi, G., and Nepusz, T. (2006). The igraph software package for complex network research.
InterJournal
Complex Systems, 1695.Davis, S., and Meltzer, P. S. (2007). GEOquery: a bridge between the Gene Expression Omnibus (GEO) and BioConductor.
Bioinformatics
23, 1846–1847. doi:10.1093/bioinformatics/btm254.Gibson, B. R., Lawrence, S. J., Boulton, C. A., Box, W. G., Graham, N. S., Linforth, R. S. T., et al. (2008). The oxidative stress response of a lager brewing yeast strain during industrial propagation and fermentation: Oxidative stress response of lager brewing yeast.
FEMS Yeast Res.
8, 574–585. doi:10.1111/j.1567-1364.2008.00371.x.Gu, Z., Eils, R., and Schlesner, M. (2016). Complex heatmaps reveal patterns and correlations in multidimensional genomic data.
Bioinformatics
32, 2847–2849. doi:10.1093/bioinformatics/btw313.Kauffmann, A., Gentleman, R., and Huber, W. (2009). arrayQualityMetrics—a bioconductor package for quality assessment of microarray data.
Bioinformatics
25, 415–416. doi:10.1093/bioinformatics/btn647.Ritchie, M. E., Phipson, B., Wu, D., Hu, Y., Law, C. W., Shi, W., et al. (2015). limma powers differential expression analyses for RNA-sequencing and microarray studies.
Nucleic Acids Res.
43, e47–e47. doi:10.1093/nar/gkv007.Shannon, P., Markiel, A., Ozier, O., Baliga, N. S., Wang, J. T., Ramage, D., et al. (2003). Cytoscape:a software environment for integrated models of biomolecular interaction networks.
Genome Res.
13, 2498–2504. doi:10.1101/gr.1239303.Yu, G., Li, F., Qin, Y., Bo, X., Wu, Y., and Wang, S. (2010). GOSemSim: an R package for measuring semantic similarity among GO terms and gene products.
Bioinformatics
26, 976–978. doi:10.1093/bioinformatics/btq064.Yu, G., Wang, L.-G., Han, Y., and He, Q.-Y. (2012). clusterProfiler: an R package for comparing biological themes among gene clusters.