Per O. Ljungdahl

Sensors of extracellular nutrients in Saccharomyces cerevisiae.

Abstract. It has been known for a long time that yeast are capable of making rapid metabolic adjustments in response to changing extracellular nutrient conditions. Until recently it was thought that yeast, in contrast to mammalian cells, primarily monitored nutrient availability through the activity of intracellular sensors. Recent advances in our understanding of nutrient sensing indicate that yeast cells possess several nutrient-sensing systems localized in the plasma membrane that transduce information regarding the presence of extracellular amino acids, ammonium, and glucose. Strikingly, the transmembrane components of several of these sensors, Ssy1p, Mep2p, Snf3p, and Rgt2p, are unique members of nutrient-transport protein families. Perhaps with the exception of Mep2p, the ability of these transporter homologues to transduce nutrient-(ligand)-induced signals across the plasma membrane appears to be independent of nutrient uptake; and thus these sensor components may function analogously to traditional ligand-dependent receptors. Additionally, the G protein-coupled receptor Gpr1p has been shown to exhibit properties consistent with it being a sensor. These recent advances indicate that yeast cells obtain information regarding their growth environments using sensing systems that are more similar to those present in mammalian cells than previously thought. The fact that yeast plasma membrane nutrient sensors have only recently been discovered reveals how little is understood regarding the molecular signals that enable eukaryotic cells to adapt to changing environments.

Ssy1p and Ptr3p Are Plasma Membrane Components of a Yeast System That Senses Extracellular Amino Acids

ABSTRACT Mutations in SSY1 and PTR3 were identified in a genetic selection for components required for the proper uptake and compartmentalization of histidine in Saccharomyces cerevisiae. Ssy1p is a unique member of the amino acid permease gene family, and Ptr3p is predicted to be a hydrophilic protein that lacks known functional homologs. Both Ssy1p and Ptr3p have previously been implicated in relaying signals regarding the presence of extracellular amino acids. We have found that ssy1 andptr3 mutants belong to the same epistasis group; single andssy1 ptr3 double-mutant strains exhibit indistinguishable phenotypes. Mutations in these genes cause the nitrogen-regulated general amino acid permease gene (GAP1) to be abnormally expressed and block the nonspecific induction of arginase (CAR1) and the peptide transporter (PTR2).ssy1 and ptr3 mutations manifest identical differential effects on the functional expression of multiple specific amino acid transporters. ssy1 and ptr3 mutants have increased vacuolar pools of histidine and arginine and exhibit altered cell growth morphologies accompanied by exaggerated invasive growth. Subcellular fractionation experiments reveal that both Ssy1p and Ptr3p are localized to the plasma membrane (PM). Ssy1p requires the endoplasmic reticulum protein Shr3p, the amino acid permease-specific packaging chaperonin, to reach the PM, whereas Ptr3p does not. These findings suggest that Ssy1p and Ptr3p function in the PM as components of a sensor of extracellular amino acids.

Regulation of Amino Acid, Nucleotide, and Phosphate Metabolism in Saccharomyces cerevisiae

Ever since the beginning of biochemical analysis, yeast has been a pioneering model for studying the regulation of eukaryotic metabolism. During the last three decades, the combination of powerful yeast genetics and genome-wide approaches has led to a more integrated view of metabolic regulation. Multiple layers of regulation, from suprapathway control to individual gene responses, have been discovered. Constitutive and dedicated systems that are critical in sensing of the intra- and extracellular environment have been identified, and there is a growing awareness of their involvement in the highly regulated intracellular compartmentalization of proteins and metabolites. This review focuses on recent developments in the field of amino acid, nucleotide, and phosphate metabolism and provides illustrative examples of how yeast cells combine a variety of mechanisms to achieve coordinated regulation of multiple metabolic pathways. Importantly, common schemes have emerged, which reveal mechanisms conserved among various pathways, such as those involved in metabolite sensing and transcriptional regulation by noncoding RNAs or by metabolic intermediates. Thanks to the remarkable sophistication offered by the yeast experimental system, a picture of the intimate connections between the metabolomic and the transcriptome is becoming clear.

Genetic and Biochemical Analysis of the Yeast Plasma Membrane Ssy1p-Ptr3p-Ssy5p Sensor of Extracellular Amino Acids

ABSTRACT Ssy1p and Ptr3p are known components of a yeast plasma membrane system that functions to sense the presence of amino acids in the extracellular environment. In response to amino acids, this sensing system initiates metabolic signals that ultimately regulate the functional expression of several amino acid-metabolizing enzymes and transport proteins, including multiple, genetically distinct amino acid permeases. We have found that SSY5 encodes a third component of this amino acid sensing system. Mutations inSSY5 manifest phenotypes that are indistinguishable from those resulting from either single ssy1 andptr3 mutations or ssy5 ssy1 and ssy5 ptr3 double mutations. Although Ssy5p is predicted to be a soluble protein, it exhibits properties indicating that it is a peripherally associated plasma membrane protein. Each of the three sensor components, Ssy1p, Ptr3p, and Ssy5p, adopts conformations and modifications that are dependent upon the availability of amino acids and on the presence of the other two components. These results suggest that these components function as part of a sensor complex localized to the plasma membrane. Consistent with a sensor complex, the overexpression of SSY1 or the unique N-terminal extension of this amino acid permease homologue inactivates the amino acid sensor in a dominant-negative manner. Each of the components of the Ssy1p-Ptr3p-Ssy5p (SPS) signaling system undergoes rapid physical changes, reflected in altered electrophoretic mobility, when leucine is added to cells grown in media lacking amino acids. Furthermore, the levels of each SPS sensor component present in whole-cell extracts diminish upon leucine addition. The rapid physical alterations and reduced levels of sensor components are consistent with their being downregulated in response to amino acid availability. These results reveal the dynamic nature of the amino acid-initiated signals transduced by the SPS sensor.

The Coxsackievirus and Adenovirus Receptor (CAR) Forms a Complex with the PDZ Domain-containing Protein Ligand-of-Numb Protein-X (LNX)

The Coxsackievirus and adenovirus receptor (CAR) functions as a virus receptor, but its primary biological function is unknown. A yeast two-hybrid screen was used to identify Ligand-of-Numb protein-X (LNX) as a binding partner to the intracellular tail of CAR. LNX harbors several protein-protein interacting domains, including four PDZ domains, and was previously shown to bind to and regulate the expression level of the cell-fate determinant Numb. CAR was able to bind LNX both in vivo and in vitro. Efficient binding to LNX required not only the consensus PDZ domain binding motif in the C terminus of CAR but also upstream sequences. The CAR binding region in LNX was mapped to the second PDZ domain. CAR and LNX were also shown to colocalize in vivo in mammalian cells. We speculate that CAR and LNX are part of a larger protein complex that might have important functions at discrete subcellular localizations in the cell.

Characterization of potassium transport in wild‐type and isogenic yeast strains carrying all combinations of trk1, trk2 and tok1 null mutations

Saccharomyces cerevisiae cells express three defined potassium‐specific transport systems en‐coded by TRK1 , TRK2 and TOK1 . To gain a more complete understanding of the physiological function of these transport proteins, we have constructed a set of isogenic yeast strains carrying all combinations of trk1 Δ, trk2 Δ and tok1 Δ null mutations. The in vivo K + transport characteristics of each strain have been documented using growth‐based assays, and the in vitro biochemical and electrophysiological properties associated with K + transport have been determined. As has been reported previously, Trk1p and Trk2p facilitate high‐affinity potassium uptake and appear to be functionally redundant under a wide range of environmental conditions. In the absence of TRK1 and TRK2 , strains lack the ability specifically to take up K + , and trk1 Δ trk2 Δ double mutant cells depend upon poorly understood non‐specific cation uptake mechanisms for growth. Under conditions that impair the activity of the non‐specific uptake system, termed NSC1, we have found that the presence of functional Tok1p renders cells sensitive to Cs + . Based on this finding, we have established a growth‐based assay that monitors the in vivo activity of Tok1p.

Specialized membrane-localized chaperones prevent aggregation of polytopic proteins in the ER

The integral endoplasmic reticulum (ER) membrane protein Shr3p is required for proper plasma membrane localization of amino acid permeases (AAPs) in yeast. In the absence of Shr3p AAPs are uniquely retained in the ER with each of their twelve membrane-spanning segments correctly inserted in the membrane. Here, we show that the membrane domain of Shr3p specifically prevents AAPs from aggregating, and thus, plays a critical role in assisting AAPs to fold and correctly attain tertiary structures required for ER exit. Also, we show that the integral ER proteins, Gsf2p, Pho86p, and Chs7p, function similarly to Shr3p. In cells individually lacking one of these components only their cognate substrates, hexose transporters, phosphate transporters, and chitin synthase-III, respectively, aggregate and consequently fail to exit the ER membrane. These findings indicate that polytopic membrane proteins depend on specialized membrane-localized chaperones to prevent inappropriate interactions between membrane-spanning segments as they insert and fold in the lipid bilayer of the ER membrane.

The role of the yeast plasma membrane SPS nutrient sensor in the metabolic response to extracellular amino acids

In response to discrete environmental cues, Saccharomyces cerevisiae cells adjust patterns of gene expression and protein activity to optimize metabolism. Nutrient‐sensing systems situated in the plasma membrane (PM) of yeast have only recently been discovered. Ssy1p is one of three identified components of the Ssy1p–Ptr3p–Ssy5 (SPS) sensor of extracellular amino acids. SPS sensor‐initiated signals are known to modulate the expression of a number of amino acid and peptide transporter genes (i.e. AGP1, BAP2, BAP3, DIP5, GAP1, GNP1, TAT1, TAT2 and PTR2) and arginase (CAR1). To obtain a better understanding of how cells adjust metabolism in response to extracellular amino acids in the environment and to assess the consequences of loss of amino acid sensor function, we investigated the effects of leucine addition to wild‐type and ssy1 null mutant cells using genome‐wide transcription profile analysis. Our results indicate that the previously identified genes represent only a subset of the full spectrum of Ssy1p‐dependent genes. The expression of several genes encoding enzymes in amino acid biosynthetic pathways, including the branched‐chain, lysine and arginine, and the sulphur amino acid biosynthetic pathways, are modulated by Ssy1p. Additionally, the proper transcription of several nitrogen‐regulated genes, including NIL1 and DAL80, encoding well‐studied GATA transcription factors, is dependent upon Ssy1p. Finally, several genes were identified that require Ssy1p for wild‐type expression independently of amino acid addition. These findings demonstrate that yeast cells require the SPS amino acid sensor component, Ssy1p, to adjust diverse cellular metabolic processes properly.

Membrane chaperone Shr3 assists in folding amino acid permeases preventing precocious ERAD

The yeast endoplasmic reticulum (ER) membrane-localized chaperone Shr3 plays a critical role in enabling amino acid permeases (AAPs) to fold and attain proper structures required for functional expression at the plasma membrane. In the absence of Shr3, AAPs specifically accumulate in the ER, where despite the correct insertion of their 12 transmembrane segments (TMSs), they aggregate forming large molecular weight complexes. We show that Shr3 prevents aggregation and facilitates the functional assembly of independently coexpressed N- and C-terminal fragments of the general AAP Gap1. Shr3 interacts with and maintains the first five TMSs in a conformation that can posttranslationally assemble with the remaining seven TMSs. We also show that Doa10- and Hrd1-dependent ER-associated degradation (ERAD) pathways redundantly degrade AAP aggregates. In combination, doa10Δ hrd1Δ mutations stabilize AAP aggregates and partially suppress amino acid uptake defects of shr3 mutants. Consequently, in cells with impaired ERAD, AAPs are able to attain functional conformations independent of Shr3. These findings illustrate that folding and degradation are tightly coupled processes during membrane protein biogenesis.

Divergence of Stp1 and Stp2 Transcription Factors in Candida albicans Places Virulence Factors Required for Proper Nutrient Acquisition under Amino Acid Control

ABSTRACT Candida albicans possesses a plasma membrane-localized sensor of extracellular amino acids. Here, we show that in response to amino acids, this sensor induces the proteolytic processing of two latent transcription factors, Stp1 and Stp2. Processing removes negative regulatory motifs present in the N-terminal domains of these factors. Strikingly, Stp1 and Stp2 exhibit a clear dichotomy in the genes they transactivate. The shorter active form of Stp2 activates genes required for amino acid uptake. The processed form of Stp1 activates genes required for degradation of extracellular protein and uptake of peptides, and cells lacking Stp1 do not express the secreted aspartyl protease SAP2 or the oligopeptide transporter OPT1. Consequently, stp1 null mutants are unable to grow on media with protein as the sole nitrogen source. Cells expressing the STP1* allele that encodes a protein lacking the inhibitory N-terminal domain constitutively express SAP2 and OPT1 even in the absence of extracellular proteins or peptides. Also, we show that Stp1 levels, but not Stp2 levels, are downregulated in the presence of millimolar concentrations of extracellular amino acids. These results define the hierarchy of regulatory mechanisms that differentially control two discrete pathways for the assimilation of nitrogen.