Allyson F. O'Donnell

α-arrestins Aly1 and Aly2 Regulate Intracellular Trafficking in Response to Nutrient Signaling

Arrestins, known regulators of endocytosis, take on novel functions in nutrient-regulated endosomal recycling. Yeast α-arrestins, Aly1 and Aly2, redistribute the Gap1 permease from endosomes to the cell surface and interact with clathrin/AP-1. Aly2 is regulated by the Npr1 kinase and acts through mechanisms distinct from Aly1.

Specific α-Arrestins Negatively Regulate Saccharomyces cerevisiae Pheromone Response by Down-Modulating the G-Protein-Coupled Receptor Ste2

ABSTRACT G-protein-coupled receptors (GPCRs) are integral membrane proteins that initiate responses to extracellular stimuli by mediating ligand-dependent activation of cognate heterotrimeric G proteins. In yeast, occupancy of GPCR Ste2 by peptide pheromone α-factor initiates signaling by releasing a stimulatory Gβγ complex (Ste4-Ste18) from its inhibitory Gα subunit (Gpa1). Prolonged pathway stimulation is detrimental, and feedback mechanisms have evolved that act at the receptor level to limit the duration of signaling and stimulate recovery from pheromone-induced G1 arrest, including upregulation of the expression of an α-factor-degrading protease (Bar1), a regulator of G-protein signaling protein (Sst2) that stimulates Gpa1-GTP hydrolysis, and Gpa1 itself. Ste2 is also downregulated by endocytosis, both constitutive and ligand induced. Ste2 internalization requires its phosphorylation and subsequent ubiquitinylation by membrane-localized protein kinases (Yck1 and Yck2) and a ubiquitin ligase (Rsp5). Here, we demonstrate that three different members of the α-arrestin family (Ldb19/Art1, Rod1/Art4, and Rog3/Art7) contribute to Ste2 desensitization and internalization, and they do so by discrete mechanisms. We provide genetic and biochemical evidence that Ldb19 and Rod1 recruit Rsp5 to Ste2 via PPXY motifs in their C-terminal regions; in contrast, the arrestin fold domain at the N terminus of Rog3 is sufficient to promote adaptation. Finally, we show that Rod1 function requires calcineurin-dependent dephosphorylation.

A Calcineurin-dependent Switch Controls the Trafficking Function of α-Arrestin Aly1/Art6

Background: In response to nutrient signals, α-arrestins selectively regulate trafficking of membrane transporters. Results: Aly1 is a substrate of the phosphatase calcineurin, and dephosphorylation triggers Aly1-dependent internalization of the permease Dip5. Conclusion: Endocytic function of α-arrestins is stimulated by removal of inhibitory phosphorylation. Significance: These insights define a molecular mechanism controlling the function of an α-arrestin in endocytosis, which is critical for cellular adaptation. Proper regulation of plasma membrane protein endocytosis by external stimuli is required for cell growth and survival. In yeast, excess levels of certain nutrients induce endocytosis of the cognate permeases to prevent toxic accumulation of metabolites. The α-arrestins, a family of trafficking adaptors, stimulate ubiquitin-dependent and clathrin-mediated endocytosis by interacting with both a client permease and the ubiquitin ligase Rsp5. However, the molecular mechanisms that control α-arrestin function are not well understood. Here, we show that α-arrestin Aly1/Art6 is a phosphoprotein that specifically interacts with and is dephosphorylated by the Ca2+- and calmodulin-dependent phosphoprotein phosphatase calcineurin/PP2B. Dephosphorylation of Aly1 by calcineurin at a subset of phospho-sites is required for Aly1-mediated trafficking of the aspartic acid and glutamic acid transporter Dip5 to the vacuole, but it does not alter Rsp5 binding, ubiquitinylation, or stability of Aly1. In addition, dephosphorylation of Aly1 by calcineurin does not regulate the ability of Aly1 to promote the intracellular sorting of the general amino acid permease Gap1. These results suggest that phosphorylation of Aly1 inhibits its vacuolar trafficking function and, conversely, that dephosphorylation of Aly1 by calcineurin serves as a regulatory switch to promote Aly1-mediated trafficking to the vacuole.

Curcumin Inhibits Growth of Saccharomyces cerevisiae through Iron Chelation

ABSTRACT Curcumin, a polyphenol derived from turmeric, is an ancient therapeutic used in India for centuries to treat a wide array of ailments. Interest in curcumin has increased recently, with ongoing clinical trials exploring curcumin as an anticancer therapy and as a protectant against neurodegenerative diseases. In vitro, curcumin chelates metal ions. However, although diverse physiological effects have been documented for this compound, curcumins mechanism of action on mammalian cells remains unclear. This study uses yeast as a model eukaryotic system to dissect the biological activity of curcumin. We found that yeast mutants lacking genes required for iron and copper homeostasis are hypersensitive to curcumin and that iron supplementation rescues this sensitivity. Curcumin penetrates yeast cells, concentrates in the endoplasmic reticulum (ER) membranes, and reduces the intracellular iron pool. Curcumin-treated, iron-starved cultures are enriched in unbudded cells, suggesting that the G1 phase of the cell cycle is lengthened. A delay in cell cycle progression could, in part, explain the antitumorigenic properties associated with curcumin. We also demonstrate that curcumin causes a growth lag in cultured human cells that is remediated by the addition of exogenous iron. These findings suggest that curcumin-induced iron starvation is conserved from yeast to humans and underlies curcumins medicinal properties.

2-Deoxyglucose Impairs Saccharomyces cerevisiae Growth by Stimulating Snf1-Regulated and α-Arrestin-Mediated Trafficking of Hexose Transporters 1 and 3

ABSTRACT The glucose analog 2-deoxyglucose (2DG) inhibits the growth of Saccharomyces cerevisiae and human tumor cells, but its modes of action have not been fully elucidated. Yeast cells lacking Snf1 (AMP-activated protein kinase) are hypersensitive to 2DG. Overexpression of either of two low-affinity, high-capacity glucose transporters, Hxt1 and Hxt3, suppresses the 2DG hypersensitivity of snf1Δ cells. The addition of 2DG or the loss of Snf1 reduces HXT1 and HXT3 expression levels and stimulates transporter endocytosis and degradation in the vacuole. 2DG-stimulated trafficking of Hxt1 and Hxt3 requires Rod1/Art4 and Rog3/Art7, two members of the α-arrestin trafficking adaptor family. Mutations in ROD1 and ROG3 that block binding to the ubiquitin ligase Rsp5 eliminate Rod1- and Rog3-mediated trafficking of Hxt1 and Hxt3. Genetic analysis suggests that Snf1 negatively regulates both Rod1 and Rog3, but via different mechanisms. Snf1 activated by 2DG phosphorylates Rod1 but fails to phosphorylate other known targets, such as the transcriptional repressor Mig1. We propose a novel mechanism for 2DG-induced toxicity whereby 2DG stimulates the modification of α-arrestins, which promote glucose transporter internalization and degradation, causing glucose starvation even when cells are in a glucose-rich environment.

The Running of the Buls: Control of Permease Trafficking by α-Arrestins Bul1 and Bul2

How do changes in the environment trigger selective internalization of membrane proteins? This question is of broad biological importance since efficient endocytosis impacts many facets of cell physiology. In this issue of Molecular and Cellular Biology, Merhi and André (11) provide a compelling answer. They define a molecular pathway that leads from nutrient uptake through intracellular signaling to internalization of a specific nutrient permease. Their findings elegantly connect a cast of regulatory characters, including the nutrient-regulated TORC1 complex, a protein kinase (Npr1) that is a TORC1 target, and the 14-3-3 phosphoserine-binding proteins (11). Importantly, they demonstrate that two members of the -arrestin or arrestin-related trafficking adaptor (ART) family, Bul1 and Bul2, serve as linchpins connecting nutrient signaling to protein trafficking (11). The pathway that they describe bears striking similarity to the recently reported control of two other -arrestins (1, 10), suggesting that a conserved mechanism regulates arrestin-mediated trafficking. A Gap in our knowledge. In Saccharomyces cerevisiae, the general amino acid permease Gap1 is a nonspecific transporter of all L-amino acids and many amino acid analogs (5). Since studies of Gap1 began over 40 years ago in the Grenson lab (5), the trafficking of Gap1 has served as a model to identify basic features of signal-induced endocytosis. Subsequent work from many groups, notably the Kaiser and André labs, has demonstrated that Rsp5, now known to be a protein-ubiquitin ligase (the mammalian ortholog is Nedd4) (14), and Npr1, now known to be a protein kinase (as reviewed in reference 9), antagonistically control Gap1 delivery to the plasma membrane in response to changes in the available nitrogen source (6, 17). When cells are grown on a nonpreferred (or poor) nitrogen source, like proline, Gap1 localizes to the plasma membrane, where its ability to transport a broad spectrum of amino acids assists in nitrogen scavenging. Under these conditions, Gap1 is stabilized at the cell surface by active Npr1 kinase (3, 16). Npr1 is negatively regulated by TORC1, which is active when amino acids are abundant (9, 15). Conversely, growth on a preferred (or good) nitrogen source like ammonium promotes Rsp5-mediated ubiquitylation of Gap1 and stimulates its endocytosis (17). Like many membrane proteins, Gap1 lacks the sequence motifs (consensus PPXY or LPXY [14]) needed to bind Rsp5 directly. Instead, Bul1 and Bul2, which have these motifs, likely recruit Rsp5 to stimulate Gap1 ubiquitylation and internalization (6, 13, 17). However, a detailed molecular mechanism connecting nutrient supply to Gap1 trafficking remained elusive. It was not clear how nitrogen quality regulated Gap1 localization or what controlled Bul-mediated ubiquitylation of Gap1. Ammonium ion uptake leads the way. In this work, Merhi and André (11) make extensive use of the well-established nitrogen regulation of Gap1 trafficking: they grow cells on a nonpreferred nitrogen source (proline), where Gap1 is localized to the plasma membrane, and then add NH4 , a preferred nitrogen source, which induces Gap1 ubiquitylation and internalization. They demonstrate, first, that NH4 -induced Gap1 ubiquitylation and endocytosis require uptake through the ammonium ion permeases Mep1, Mep2, and Mep3 and production of glutamate, mainly by glutamate dehydrogenase Gdh1 during growth on glucose (Gdh3 does so under nonfermentative conditions). Although Gdh2 is thought to have mainly a catabolic role (converting glutamate to -ketoglutarate), Mehri and André found that when NH4 is plentiful, it can also contribute to glutamate synthesis (11). Glutamate, in turn, is the nitrogen donor for synthesis of many amino acids. Thus, the authors hypothesize that when NH4 is added to proline-grown cells, it may increase amino acid levels and activate TORC1, which promotes robust growth and proliferation under nutrient-replete conditions. How intracellular amino acids activate TORC1 is not fully understood. Recent work demonstrates that leucine bound to its leucyl-tRNA synthetase (LeuRS) interacts with the Rag GTPase in the yeast EGO complex, which in turn activates TORC1 (2). Perhaps, addition of NH4 and its conversion to glutamate increase leucine levels and/or or other amino acid levels to stimulate TORC1 via a similar mechanism, but this remains to be determined. Active TORC1 destabilizes Gap1 by stimulating endocytosis of the existing permease (as reviewed in reference 9); TORC1 inhibits Npr1 in a switch-like manner by directly phosphorylating negative regulatory sites in Npr1 and concomitantly preventing dephosphorylation of those sites by sequestering the phosphatase needed to dephosphorylate Npr1, Sit4 (as reviewed in reference 9). It was known that loss of Npr1 function causes enhanced ubiquitylation and internalization of Gap1 (3, 16). Here, the authors show that Npr1 remains dephosphorylated, even after NH4 addition, if (i) TORC1 is pharmacologically inhibited with rapamycin or (ii) NH4 uptake is prevented (in mep1 mep2 mep3 triple mutant cells) (11). Thus, the authors begin to reveal Npr1 regulation: NH4 internalization and conversion to glutamate and perhaps other amino acids activate TORC1 through an undefined mechanism (which may be similar to the tRNA-synthetase/EGO activation pathway [2]), and this in turn inhibits Npr1 and promotes Gap1 internalization (Fig. 1). It will be interesting to see in future studies if components of this same signaling pathway are important for Gap1 endocytosis when it is induced by amino acids trans-

Hph1 and Hph2 Are Novel Components of the Sec63/Sec62 Posttranslational Translocation Complex That Aid in Vacuolar Proton ATPase Biogenesis

ABSTRACT Hph1 and Hph2 are homologous integral endoplasmic reticulum (ER) membrane proteins required for Saccharomyces cerevisiae survival under environmental stress conditions. To investigate the molecular functions of Hph1 and Hph2, we carried out a split-ubiquitin-membrane-based yeast two-hybrid screen and identified their interactions with Sec71, a subunit of the Sec63/Sec62 complex, which mediates posttranslational translocation of proteins into the ER. Hph1 and Hph2 likely function in posttranslational translocation, as they interact with other Sec63/Sec62 complex subunits, i.e., Sec72, Sec62, and Sec63. hph1Δ hph2Δ cells display reduced vacuole acidification; increased instability of Vph1, a subunit of vacuolar proton ATPase (V-ATPase); and growth defects similar to those of mutants lacking V-ATPase activity. sec71Δ cells exhibit similar phenotypes, indicating that Hph1/Hph2 and the Sec63/Sec62 complex function during V-ATPase biogenesis. Hph1/Hph2 and the Sec63/Sec62 complex may act together in this process, as vacuolar acidification and Vph1 stability are compromised to the same extent in hph1Δ hph2Δ and hph1Δ hph2Δ sec71Δ cells. In contrast, loss of Pkr1, an ER protein that promotes posttranslocation assembly of Vph1 with V-ATPase subunits, further exacerbates hph1Δ hph2Δ phenotypes, suggesting that Hph1 and Hph2 function independently of Pkr1-mediated V-ATPase assembly. We propose that Hph1 and Hph2 aid Sec63/Sec62-mediated translocation of specific proteins, including factors that promote efficient biogenesis of V-ATPase, to support yeast cell survival during environmental stress.

FACT, the Bur Kinase Pathway, and the Histone Co-Repressor HirC Have Overlapping Nucleosome-Related Roles in Yeast Transcription Elongation

Gene transcription is constrained by the nucleosomal nature of chromosomal DNA. This nucleosomal barrier is modulated by FACT, a conserved histone-binding heterodimer. FACT mediates transcription-linked nucleosome disassembly and also nucleosome reassembly in the wake of the RNA polymerase II transcription complex, and in this way maintains the repression of ‘cryptic’ promoters found within some genes. Here we focus on a novel mutant version of the yeast FACT subunit Spt16 that supplies essential Spt16 activities but impairs transcription-linked nucleosome reassembly in dominant fashion. This Spt16 mutant protein also has genetic effects that are recessive, which we used to show that certain Spt16 activities collaborate with histone acetylation and the activities of a Bur-kinase/Spt4–Spt5/Paf1C pathway that facilitate transcription elongation. These collaborating activities were opposed by the actions of Rpd3S, a histone deacetylase that restores a repressive chromatin environment in a transcription-linked manner. Spt16 activity paralleling that of HirC, a co-repressor of histone gene expression, was also found to be opposed by Rpd3S. Our findings suggest that Spt16, the Bur/Spt4–Spt5/Paf1C pathway, and normal histone abundance and/or stoichiometry, in mutually cooperative fashion, facilitate nucleosome disassembly during transcription elongation. The recessive nature of these effects of the mutant Spt16 protein on transcription-linked nucleosome disassembly, contrasted to its dominant negative effect on transcription-linked nucleosome reassembly, indicate that mutant FACT harbouring the mutant Spt16 protein competes poorly with normal FACT at the stage of transcription-linked nucleosome disassembly, but effectively with normal FACT for transcription-linked nucleosome reassembly. This functional difference is consistent with the idea that FACT association with the transcription elongation complex depends on nucleosome disassembly, and that the same FACT molecule that associates with an elongation complex through nucleosome disassembly is retained for reassembly of the same nucleosome.

The endosomal trafficking factors CORVET and ESCRT suppress plasma membrane residence of the renal outer medullary potassium channel (ROMK)

Protein trafficking can act as the primary regulatory mechanism for ion channels with high open probabilities, such as the renal outer medullary (ROMK) channel. ROMK, also known as Kir1.1 (KCNJ1), is the major route for potassium secretion into the pro-urine and plays an indispensable role in regulating serum potassium and urinary concentrations. However, the cellular machinery that regulates ROMK trafficking has not been fully defined. To identify regulators of the cell-surface population of ROMK, we expressed a pH-insensitive version of the channel in the budding yeast Saccharomyces cerevisiae. We determined that ROMK primarily resides in the endoplasmic reticulum (ER), as it does in mammalian cells, and is subject to ER-associated degradation (ERAD). However, sufficient ROMK levels on the plasma membrane rescued growth on low-potassium medium of yeast cells lacking endogenous potassium channels. Next, we aimed to identify the biological pathways most important for ROMK regulation. Therefore, we used a synthetic genetic array to identify non-essential genes that reduce the plasma membrane pool of ROMK in potassium-sensitive yeast cells. Genes identified in this screen included several members of the endosomal complexes required for transport (ESCRT) and the class-C core vacuole/endosome tethering (CORVET) complexes. Mass spectroscopy analysis confirmed that yeast cells lacking an ESCRT component accumulate higher potassium concentrations. Moreover, silencing of ESCRT and CORVET components increased ROMK levels at the plasma membrane in HEK293 cells. Our results indicate that components of the post-endocytic pathway influence the cell-surface density of ROMK and establish that components in this pathway modulate channel activity.

The β subunit of yeast AMP-activated protein kinase directs substrate specificity in response to alkaline stress

Saccharomyces cerevisiae express three isoforms of Snf1 kinase that differ by which β subunit is present, Gal83, Sip1 or Sip2. Here we investigate the abundance, activation, localization and signaling specificity of the three Snf1 isoforms. The relative abundance of these isoforms was assessed by quantitative immunoblotting using two different protein extraction methods and by fluorescence microscopy. The Gal83 containing isoform is the most abundant in all assays while the abundance of the Sip1 and Sip2 isoforms is typically underestimated especially in glass-bead extractions. Earlier studies to assess Snf1 isoform function utilized gene deletions as a means to inactivate specific isoforms. Here we use point mutations in Gal83 and Sip2 and a 17 amino acid C-terminal truncation of Sip1 to inactivate specific isoforms without affecting their abundance or association with the other subunits. The effect of low glucose and alkaline stresses was examined for two Snf1 phosphorylation substrates, the Mig1 and Mig2 proteins. Any of the three isoforms was capable of phosphorylating Mig1 in response to glucose stress. In contrast, the Gal83 isoform of Snf1 was both necessary and sufficient for the phosphorylation of the Mig2 protein in response to alkaline stress. Alkaline stress led to the activation of all three isoforms yet only the Gal83 isoform translocates to the nucleus and phosphorylates Mig2. Deletion of the SAK1 gene blocked nuclear translocation of Gal83 and signaling to Mig2. These data strongly support the idea that Snf1 signaling specificity is mediated by localization of the different Snf1 isoforms.