Osm1 and Frd1 are soluble fumarate reductases from yeast that are critical for allowing survival under anaerobic conditions. Although they maintain redox balance during anaerobiosis, the underlying mechanism is not understood. Here, we report the crystal structure of a eukaryotic soluble fumarate reductase, which is unique among soluble fumarate reductases as it lacks a heme domain. Structural and enzymatic analyses indicate that Osm1 has a specific binding pocket for flavin molecules, including FAD, FMN, and riboflavin, catalyzing their oxidation while reducing fumarate to succinate. Moreover, ER-resident Osm1 can transfer electrons from the Ero1 FAD cofactor to fumarate either by free FAD or by a direct interaction, allowing de novo disulfide bond formation in the absence of oxygen. We conclude that soluble eukaryotic fumarate reductases can maintain an oxidizing environment under anaerobic conditions, either by oxidizing cellular flavin cofactors or by a direct interaction with flavoenzymes such as Ero1.

Aneuploidy, an unbalanced karyotype in which one or more chromosomes are present in excess or reduced copy number, causes an array of known phenotypes including proteotoxicity, genomic instability, and slowed proliferation. However, the molecular consequences of aneuploidy are poorly understood and an unbiased investigation into aneuploid cell biology is lacking. We performed high-throughput screens for genes the deletion of which has a synthetic fitness cost in aneuploid Saccharomyces cerevisiae cells containing single extra chromosomes. This analysis identified genes that, when deleted, decrease the fitness of specific disomic strains as well as those that impair the proliferation of a broad range of aneuploidies. In one case, a chromosome-specific synthetic growth defect could be explained fully by the specific duplication of a single gene on the aneuploid chromosome, highlighting the ability of individual dosage imbalances to cause chromosome-specific phenotypes in aneuploid cells. Deletion of other genes, particularly those involved in protein transport, however, confers synthetic sickness on a broad array of aneuploid strains. Indeed, aneuploid cells, regardless of karyotype, exhibit protein secretion and cell-wall integrity defects. Thus, we were able to use this screen to identify novel cellular consequences of aneuploidy, dependent on both specific chromosome imbalances and caused by many different aneuploid karyotypes. Interestingly, the vast majority of cancer cells are highly aneuploid, so this approach could be of further use in identifying both karyotype-specific and nonspecific stresses exhibited by cancer cells as potential targets for the development of novel cancer therapeutics.

Oxidative protein folding in the endoplasmic reticulum (ER) has emerged as a potentially significant source of cellular reactive oxygen species (ROS). Recent studies suggest that levels of ROS generated as a byproduct of oxidative folding rival those produced by mitochondrial respiration. Mechanisms that protect cells against oxidant accumulation within the ER have begun to be elucidated yet many questions still remain regarding how cells prevent oxidant-induced damage from ER folding events. Here we report a new role for a central well-characterized player in ER homeostasis as a direct sensor of ER redox imbalance. Specifically we show that a conserved cysteine in the lumenal chaperone BiP is susceptible to oxidation by peroxide, and we demonstrate that oxidation of this conserved cysteine disrupts BiP's ATPase cycle. We propose that alteration of BiP activity upon oxidation helps cells cope with disruption to oxidative folding within the ER during oxidative stress.

The endoplasmic reticulum (ER) provides an environment optimized for oxidative protein folding through the action of Ero1p, which generates disulfide bonds, and Pdi1p, which receives disulfide bonds from Ero1p and transfers them to substrate proteins. Feedback regulation of Ero1p through reduction and oxidation of regulatory bonds within Ero1p is essential for maintaining the proper redox balance in the ER. In this paper, we show that Pdi1p is the key regulator of Ero1p activity. Reduced Pdi1p resulted in the activation of Ero1p by direct reduction of Ero1p regulatory bonds. Conversely, upon depletion of thiol substrates and accumulation of oxidized Pdi1p, Ero1p was inactivated by both autonomous oxidation and Pdi1p-mediated oxidation of Ero1p regulatory bonds. Pdi1p responded to the availability of free thiols and the relative levels of reduced and oxidized glutathione in the ER to control Ero1p activity and ensure that cells generate the minimum number of disulfide bonds needed for efficient oxidative protein folding.

The development of efficient methods to convert plant material (lignocellulose) to fermentable sugars is a promising avenue towards the development of renewable biofuels. Anaerobic fungi that reside within the digestive tract of large herbivores are among the most efficient and robust digesters of lignocellulosic material known in nature. Despite the powerful cellulose degrading capacity of gut fungi, remarkably little is known about the cellulolytic enzymes from these organisms due to the difficulties associated with their isolation and culture. We have cloned five such saccharolytic enzymes (bglA, cel48A, celpin, xylA, and xylB) from a cDNA library prepared from the anaerobic fungus Piromyces sp E2 for expression and extracellular secretion in the model eukaryote Saccharomyces cerevisiae. All enzymes were readily produced, but only low‐molecular weight proteins (xylA and xylB) were secreted efficiently into the extracellular medium. Piromyces sp E2 enzymes were posttranslationally modified in S. cerevisiae according to their sequence prediction, suggesting compatibility between the two systems, yet not all of the recombinant proteins were catalytically active. Activation of cellular stress mechanisms in the secretory pathway suggest enzyme misfolding during the production of cel48A and bglA, which likely limits activity. However, bglA, exhibited moderate reactivity against pNPG when produced in S. cerevisiae. Furthermore, since we aim to understand how the cellulolytic enzymes of anaerobic fungi are produced in their native system, we have implemented methods to isolate and culture anaerobic fungi obtained from the digestive tract of a horse. © 2011 American Institute of Chemical Engineers Environ Prog, 2012

Intracellular trafficking of the general amino acid permease, Gap1p, of Saccharomyces cerevisiae is regulated by amino acid abundance. When amino acids are scarce Gap1p is sorted to the plasma membrane, whereas when amino acids are abundant Gap1p is sorted from the trans-Golgi through the multivesicular endosome (MVE) and to the vacuole. Here we test the hypothesis that Gap1p itself is the sensor of amino acid abundance by examining the trafficking of Gap1p mutants with altered substrate specificity and transport activity. We show that trafficking of mutant Gap1p A297V , which does not transport basic amino acids, is also not regulated by these amino acids. Furthermore, we have identified a catalytically inactive mutant that does not respond to complex amino acid mixtures and constitutively sorts Gap1p to the plasma membrane. Previously we showed that amino acids govern the propensity of Gap1p to recycle from the MVE to the plasma membrane. Here we propose that in the presence of substrate the steady-state conformation of Gap1p shifts to a state that is unable to be recycled from the MVE. These results indicate a parsimonious regulatory mechanism by which Gap1p senses its transport substrates to set an appropriate level of transporter activity at the cell surface.

The sulfhydryl oxidase Ero1 oxidizes protein disulfide isomerase (PDI), which in turn catalyzes disulfide formation in proteins folding in the endoplasmic reticulum (ER). The extent to which other members of the PDI family are oxidized by Ero1 and thus contribute to net disulfide formation in the ER has been an open question. The yeast ER contains four PDI family proteins with at least one potential redox-active cysteine pair. We monitored the direct oxidation of each redox-active site in these proteins by yeast Ero1p in vitro. In this study, we found that the Pdi1p amino-terminal domain was oxidized most rapidly compared with the other oxidoreductase active sites tested, including the Pdi1p carboxyl-terminal domain. This observation is consistent with experiments conducted in yeast cells. In particular, the amino-terminal domain of Pdi1p preferentially formed mixed disulfides with Ero1p in vivo, and we observed synthetic lethality between a temperature-sensitive Ero1p variant and mutant Pdi1p lacking the amino-terminal active-site disulfide. Thus, the amino-terminal domain of yeast Pdi1p is on a preferred pathway for oxidizing the ER thiol pool. Overall, our results provide a rank order for the tendency of yeast ER oxidoreductases to acquire disulfides from Ero1p.

A genome-scale genetic interaction map was constructed by examining 5.4 million gene-gene pairs for synthetic genetic interactions, generating quantitative genetic interaction profiles for approximately 75% of all genes in the budding yeast, Saccharomyces cerevisiae. A network based on genetic interaction profiles reveals a functional map of the cell in which genes of similar biological processes cluster together in coherent subsets, and highly correlated profiles delineate specific pathways to define gene function. The global network identifies functional cross-connections between all bioprocesses, mapping a cellular wiring diagram of pleiotropy. Genetic interaction degree correlated with a number of different gene attributes, which may be informative about genetic network hubs in other organisms. We also demonstrate that extensive and unbiased mapping of the genetic landscape provides a key for interpretation of chemical-genetic interactions and drug target identification.

The highly conserved Rag family GTPases have a role in reporting amino acid availability to the TOR (target of rapamycin) signaling complex, which regulates cell growth and metabolism in response to environmental cues. The yeast Rag proteins Gtr1p and Gtr2p were shown in multiple independent studies to interact with the membrane-associated proteins Gse1p (Ego3p) and Gse2p (Ego1p). However, mammalian orthologs of Gse1p and Gse2p could not be identified. We determined the crystal structure of Gse1p and found it to match the fold of two mammalian proteins, MP1 (mitogen-activated protein kinase scaffold protein 1) and p14, which form a heterodimeric complex that had been assigned a scaffolding function in mitogen-activated protein kinase pathways. The significance of this structural similarity is validated by the recent identification of a physical and functional association between mammalian Rag proteins and MP1/p14. Together, these findings reveal that key components of the TOR signaling pathway are structurally conserved between yeast and mammals, despite divergence of sequence to a degree that thwarts detection through simple homology searches.

The high capacity general amino acid permease, Gap1p, in Saccharomyces cerevisiae is distributed between the plasma membrane and internal compartments according to availability of amino acids. When internal amino acid levels are low, Gap1p is localized to the plasma membrane where it imports available amino acids from the medium. When sufficient amino acids are imported, Gap1p at the plasma membrane is endocytosed and newly synthesized Gap1p is delivered to the vacuole; both sorting steps require Gap1p ubiquitination. Although it has been suggested that identical trans-acting factors and Gap1p ubiquitin acceptor sites are involved in both processes, we define unique requirements for each of the ubiquitin-mediated sorting steps involved in delivery of Gap1p to the vacuole upon amino acid addition. Our finding that distinct ubiquitin-mediated sorting steps employ unique trans-acting factors, ubiquitination sites on Gap1p, and types of ubiquitination demonstrates a previously unrecognized level of specificity in ubiquitin-mediated protein sorting.

Oxidoreductases belonging to the protein disulfide isomerase (PDI) family promote proper disulfide bond formation in substrate proteins in the endoplasmic reticulum. In plants and metazoans, new family members continue to be identified and assigned to various functional niches. PDI-like proteins typically contain tandem thioredoxin-fold domains. The limited information available suggested that the relative orientations of these domains may be quite uniform across the family, and structural models based on this assumption are appearing. However, the X-ray crystal structure of the yeast PDI family protein Mpd1p, described here, demonstrates the radically different domain orientations and surface properties achievable with multiple copies of the thioredoxin fold. A comparison of Mpd1p with yeast Pdi1p expands our perspective on the contexts in which redox-active motifs are presented in the PDI family.

Living cells must be able to respond to physiological and environmental fluctuations that threaten cell function and viability. A cellular event prone to disruption by a wide variety of internal and external perturbations is protein folding. To ensure protein folding can proceed under a range of conditions, the cell has evolved transcriptional, translational, and posttranslational signaling pathways to maintain folding homeostasis during cell stress. This review will focus on oxidative protein folding in the endoplasmic reticulum (ER) and will discuss the features of the main facilitator of biosynthetic disulfide bond formation, Ero1. Ero1 plays an essential role in setting the redox potential in the ER and regulation of Ero1 activity is central to maintain redox homeostasis and proper ER folding activity.

Introduction of disulfide bonds into proteins entering the secretory pathway is catalyzed by Ero1p, which generates disulfide bonds de novo, and Pdi1p, which transfers disulfides to substrate proteins. A sufficiently oxidizing environment must be maintained in the endoplasmic reticulum (ER) to allow for disulfide formation, but a pool of reduced thiols is needed for isomerization of incorrectly paired disulfides. We have found that hyperoxidation of the ER is prevented by attenuation of Ero1p activity through noncatalytic cysteine pairs. Deregulated Ero1p mutants lacking certain cysteines show increased enzyme activity, a decreased lag phase in kinetic assays, and growth defects in vivo. We hypothesize that noncatalytic cysteine pairs in Ero1p sense the level of potential substrates in the ER and correspondingly modulate Ero1p activity as part of a homeostatic regulatory system governing the thiol-disulfide balance in the ER.

The general amino acid permease, Gap1p, of Saccharomyces cerevisiae transports all naturally occurring amino acids into yeast cells for use as a nitrogen source. Previous studies have shown that a nonubiquitinateable form of the permease, Gap1p(K9R,K16R), is constitutively localized to the plasma membrane. Here, we report that amino acid transport activity of Gap1p(K9R,K16R) can be rapidly and reversibly inactivated at the plasma membrane by the presence of amino acid mixtures. Surprisingly, we also find that addition of most single amino acids is lethal to Gap1p(K9R,K16R)-expressing cells, whereas mixtures of amino acids are less toxic. This toxicity appears to be the consequence of uptake of unusually large quantities of a single amino acid. Exploiting this toxicity, we isolated gap1 alleles deficient in transport of a subset of amino acids. Using these mutations, we show that Gap1p inactivation at the plasma membrane does not depend on the presence of either extracellular or intracellular amino acids, but does require active amino acid transport by Gap1p. Together, our findings uncover a new mechanism for inhibition of permease activity in response to elevated amino acid levels and provide a physiological explanation for the stringent regulation of Gap1p activity in response to amino acids.

Intracellular sorting of the general amino acid permease (Gap1p) in Saccharomyces cerevisiae depends on availability of amino acids such that at low amino acid concentrations Gap1p is sorted to the plasma membrane, whereas at high concentrations Gap1p is sorted to the vacuole. In a genome-wide screen for mutations that affect Gap1p sorting we identified deletions in a subset of components of the ESCRT (endosomal sorting complex required for transport) complex, which is required for formation of the multivesicular endosome (MVE). Gap1p-GFP is delivered to the vacuolar interior by the MVE pathway in wild-type cells, but when formation of the MVE is blocked by mutation, Gap1p-GFP efficiently cycles from this compartment to the plasma membrane, resulting in unusually high permease activity at the cell surface. Importantly, cycling of Gap1p-GFP to the plasma membrane is blocked by high amino acid concentrations, defining recycling from the endosome as a major step in Gap1p trafficking under physiological control. Mutations in LST4 and LST7 genes, previously identified for their role in Gap1p sorting, similarly block MVE to plasma membrane trafficking of Gap1p. However, mutations in other recycling complexes such as the retromer had no significant effect on the intracellular sorting of Gap1p, suggesting that Gap1p follows a genetically distinct pathway for recycling. We previously found that Gap1p sorting from the Golgi to the endosome requires ubiquitination of Gap1p by an Rsp5p ubiquitin ligase complex, but amino acid abundance does not appear to significantly alter the accumulation of polyubiquitinated Gap1p. Thus the role of ubiquitination appears to be a signal for delivery of Gap1p to the MVE, whereas amino acid abundance appears to control the cycling of Gap1p from the MVE to the plasma membrane.