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Mitchell L, Lau A, Lambert J-P, Zhou H, Fong Y, Couture J-F, Figeys D, Baetz K PLoS ONE. 2011;6(10):e25336 Yeast/SGA In the budding yeast Saccharomyces cerevisiae, the lysine acetyltransferase NuA4 has been linked to a host of cellular processes through the acetylation of histone and non-histone targets. To discover proteins regulated by NuA4-dependent acetylation, we performed genome-wide synthetic dosage lethal screens to identify genes whose overexpression is toxic to non-essential NuA4 deletion mutants. The resulting genetic network identified a novel link between NuA4 and septin proteins, a group of highly conserved GTP-binding proteins that function in cytokinesis. We show that acetyltransferase-deficient NuA4 mutants have defects in septin collar formation resulting in the development of elongated buds through the Swe1-dependent morphogenesis checkpoint. We have discovered multiple sites of acetylation on four of the five yeast mitotic septins, Cdc3, Cdc10, Cdc12 and Shs1, and determined that NuA4 can acetylate three of the four in vitro. In vivo we find that acetylation levels of both Shs1 and Cdc10 are reduced in a catalytically inactive esa1 mutant. Finally, we determine that cells expressing a Shs1 protein with decreased acetylation in vivo have defects in septin localization that are similar to those observed in NuA4 mutants. These findings provide the first evidence that yeast septinproteins are acetylated and that NuA4 impacts septin dynamics.

Bleackley MR, Young BP, Loewen CJR, MacGillivray RTA Metallomics. 2011 Feb;3(2):195–205 Yeast/SGA Biological systems have developed with a strong dependence on transition metals for accomplishing a number of biochemical reactions. Iron, copper, manganese and zinc are essential for virtually all forms of life with their unique chemistries contributing to a variety of physiological processes including oxygen transport, generation of cellular energy and protein structure and function. Properties of these metals (and to a lesser extent nickel and cobalt) that make them so essential to life also make them extremely cytotoxic in many cases through the formation of damaging oxygen radicals via Fenton chemistry. While life has evolved to exploit the chemistries of transition metals to drive physiological reactions, systems have concomitantly evolved to protect against the damaging effects of these same metals. Saccharomyces cerevisiae is a valuable tool for studyingmetal homeostasis with many of the genes identified thus far having homologs in higher eukaryotes including humans. Using high density arrays, we have screened a haploid S. cerevisiae deletion set containing 4786 non-essential gene deletions for strains sensitive to each of Fe, Cu, Mn, Ni, Zn and Co and then integrated the six screens using cluster analysis to identify pathways that are unique to individual metals and others with function shared between metals. Genes with no previous implication in metal homeostasis were found to contribute to sensitivity to each metal. Significant overlap was observed between the strains that were sensitive to Mn, Ni, Zn and Co with many of these strains lacking genes for the high affinity Fe transport pathway and genes involved in vacuolar transport and acidification. The results from six genome-wide metal tolerance screens show that there is some commonality between the cellular defenses against the toxicity of Mn, Ni, Zn and Co with Fe and Cu requiring different systems. Additionally, potential new factors been identified that function in tolerance to each of the six metals.

Klosinska MM, Crutchfield CA, Bradley PH, Rabinowitz JD, Broach JR Genes Dev. 2011 Feb 15;25(4):336–49 Yeast/SGA We conducted a phenotypic, transcriptional, metabolic, and genetic analysis of quiescence in yeast induced by starvation of prototrophic cells for one of three essential nutrients (glucose, nitrogen, or phosphate) and compared those results with those obtained with cells growing slowly due to nutrient limitation. These studies address two related questions: (1) Is quiescence a state distinct from any attained during mitotic growth, and (2) does the nature of quiescence differ depending on the means by which it is induced? We found that either limitation or starvation for any of the three nutrients elicits all of the physiological properties associated with quiescence, such as enhanced cell wall integrity and resistance to heat shock and oxidative stress. Moreover, the starvations result in a common transcriptional program, which is in large part a direct extrapolation of the changes that occur during slow growth. In contrast, the metabolic changes that occur upon starvation and the genetic requirements for surviving starvation differ significantly depending on the nutrient for which the cell is starved. The genes needed by cells to survive starvation do not overlap the genes that are induced upon starvation. We conclude that cells do not access a unique and discrete G(0) state, but rather are programmed, when nutrients are scarce, to prepare for a range of possible future stressors. Moreover, these survival strategies are not unique to quiescence, but are engaged by the cell in proportion to nutrient scarcity.

Armakola M, Hart MP, Gitler AD Methods. 2011 Mar;53(3):238–45 Yeast/SGA The budding yeast Saccharomyces cerevisiae is an emerging tool for investigating the molecular pathways that underpin several human neurodegenerative disorders associated with protein misfolding. Amyotrophic lateral sclerosis (ALS) is a devastating adult onset neurodegenerative disease primarily affecting motor neurons. The protein TDP-43 has recently been demonstrated to play an important role in the disease, however, the mechanisms by which TDP-43 contributes to pathogenesis are unclear. To explore the mechanistic details that result in aberrant accumulation of TDP-43 and to discover potential strategies for therapeutic intervention, we employed a yeast TDP-43 proteinopathy model system. These studies allowed us to determine the regions of TDP-43 required for aggregation and toxicity and to define the effects of ALS-linked mutant forms of TDP-43. We have also been able to harness the power of yeast genetics to identify potent modifiers of TDP-43 toxicity using high-throughput yeast genetic screens. Here, we describe the methods and approaches that we have used in order to gain insight into TDP-43 biology and its role in disease. These approaches are readily adaptable to other neurodegenerative disease proteins.

Sun Z, Diaz Z, Fang X, Hart MP, Chesi A, Shorter J, Gitler AD PLoS Biol. 2011 Apr;9(4):e1000614 Yeast/SGA TDP-43 and FUS are RNA-binding proteins that form cytoplasmic inclusions in some forms of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Moreover, mutations in TDP-43 and FUS are linked to ALS and FTLD. However, it is unknown whether TDP-43 and FUSaggregate and cause toxicity by similar mechanisms. Here, we exploit a yeast model and purified FUS to elucidate mechanisms of FUS aggregationand toxicity. Like TDP-43, FUS must aggregate in the cytoplasm and bind RNA to confer toxicity in yeast. These cytoplasmic FUS aggregates partition to stress granule compartments just as they do in ALS patients. Importantly, in isolation, FUS spontaneously forms pore-like oligomers and filamentous structures reminiscent of FUS inclusions in ALS patients. FUS aggregation and toxicity requires a prion-like domain, but unlike TDP-43, additional determinants within a RGG domain are critical for FUS aggregation and toxicity. In further distinction to TDP-43, ALS-linked FUSmutations do not promote aggregation. Finally, genome-wide screens uncovered stress granule assembly and RNA metabolism genes that modifyFUS toxicity but not TDP-43 toxicity. Our findings suggest that TDP-43 and FUS, though similar RNA-binding proteins, aggregate and confer diseasephenotypes via distinct mechanisms. These differences will likely have important therapeutic implications.

Ju S, Tardiff DF, Han H, Divya K, Zhong Q, Maquat LE, Bosco DA, Hayward LJ, Brown RH Jr, Lindquist S, Ringe D, Petsko GA PLoS Biol. 2011 Apr;9(4):e1001052 Yeast/SGA FUS/TLS is a nucleic acid binding protein that, when mutated, can cause a subset of familial amyotrophic lateral sclerosis (fALS). AlthoughFUS/TLS is normally located predominantly in the nucleus, the pathogenic mutant forms of FUS/TLS traffic to, and form inclusions in, the cytoplasm of affected spinal motor neurons or glia. Here we report a yeast model of human FUS/TLS expression that recapitulates multiple salient features of the pathology of the disease-causing mutant proteins, including nuclear to cytoplasmic translocation, inclusion formation, and cytotoxicity. Protein domain analysis indicates that the carboxyl-terminus of FUS/TLS, where most of the ALS-associated mutations are clustered, is required but not sufficient for the toxicity of the protein. A genome-wide genetic screen using a yeast over-expression library identified five yeast DNA/RNA binding proteins, encoded by the yeast genes ECM32, NAM8, SBP1, SKO1, and VHR1, that rescue the toxicity of human FUS/TLS without changing its expression level, cytoplasmic translocation, or inclusion formation. Furthermore, hUPF1, a human homologue of ECM32, also rescues the toxicity ofFUS/TLS in this model, validating the yeast model and implicating a possible insufficiency in RNA processing or the RNA quality control machinery in the mechanism of FUS/TLS mediated toxicity. Examination of the effect of FUS/TLS expression on the decay of selected mRNAs in yeastindicates that the nonsense-mediated decay pathway is probably not the major determinant of either toxicity or suppression.

Konopka CA, Locke MN, Gallagher PS, Pham N, Hart MP, Walker CJ, Gitler AD, Gardner RG Mol Biol Cell. 2011 Jun 15;22(12):1971–84 Yeast/SGA Nine human disorders result from the toxic accumulation and aggregation of proteins with expansions in their endogenous polyalanine (polyA) tracts. Given the prevalence of polyA tracts in eukaryotic proteomes, we wanted to understand the generality of polyA-expansion cytotoxicity by usingyeast as a model organism. In our initial case, we expanded the polyA tract within the native yeast poly(Adenine)-binding protein Pab1 from 8A to 13A, 15A, 17A, and 20A. These expansions resulted in increasing formation of Pab1 inclusions, insolubility, and cytotoxicity that correlated with the length of the polyA expansion. Pab1 binds mRNA as part of its normal function, and disrupting RNA binding or altering cytoplasmic mRNA levels suppressed the cytotoxicity of 17A-expanded Pab1, indicating a requisite role for mRNA in Pab1 polyA-expansion toxicity. Surprisingly, neither manipulation suppressed the cytotoxicity of 20A-expanded Pab1. Thus longer expansions may have a different mechanism for toxicity. We think that this difference underscores the potential need to examine the cytotoxic mechanisms of both long and short expansions in models of expansion disorders.

Short MK, Hallett JP, Tar K, Dange T, Schmidt M, Moir R, Willis IM, Jubinsky PT PLoS ONE. 2012;7(7):e39428 Yeast/SGA Magmas is a growth factor responsive gene encoding an essential mitochondrial protein in mammalian cells. Pam16, the Magmas ortholog in Saccharomyces cerevisiae, is a component of the presequence translocase-associated motor. A temperature-sensitive allele (pam16-I61N) was used to query an array of non-essential gene-deletion strains for synthetic genetic interactions. The pam16-I61N mutation at ambient temperature caused synthetic lethal or sick phenotypes with genes involved in lipid metabolism, perixosome synthesis, histone deacetylation and mitochondrial protein import. The gene deletion array was also screened for suppressors of the pam16-I61N growth defect to identify compensatory pathways. Five suppressor genes were identified (SUR4, ISC1, IPT1, SKN1, and FEN1) and all are involved in sphingolipid metabolism. pam16-I61N cells cultured in glucose at non-permissive temperatures resulted in rapid growth inhibition and G1 cell cycle arrest, but cell viability was maintained. Altered mitochondria morphology, reduced peroxisome induction in glycerol/ethanol and oleate, and changes in the levels of several sphingolipids including C18 alpha-hydroxy-phytoceramide, were also observed in the temperature sensitive strain. Deletion of SUR4, the strongest suppressor, reversed the temperature sensitive fermentative growth defect, the morphological changes and the elevated levels of C18 alpha-hydroxy phytoceramide in pam16-I61N. Deletion of the other four suppressor genes had similar effects on C18 alpha-hydroxy-phytoceramide levels and restored proliferation to thepam16-I61N strain. In addition, pam16-I61N inhibited respiratory growth, likely by reducing cardiolipin, which is essential for mitochondrial function. Our results suggest that the pleiotropic effects caused by impaired Pam16/Magmas function are mediated in part by changes in lipid metabolism.

Herzig Y, Sharpe HJ, Elbaz Y, Munro S, Schuldiner M PLoS Biol. 2012;10(5):e1001329 Yeast/SGA The endoplasmic reticulum (ER) is the site of synthesis of secreted and membrane proteins. To exit the ER, proteins are packaged into COPII vesicles through direct interaction with the COPII coat or aided by specific cargo receptors. Despite the fundamental role of such cargo receptors in protein traffic, only a few have been identified; their cargo spectrum is unknown and the signals they recognize remain poorly understood. We present here an approach we term "PAIRS" (pairing analysis of cargo receptors), which combines systematic genetic manipulations of yeast with automated microscopy screening, to map the spectrum of cargo for a known receptor or to uncover a novel receptor for a particular cargo. Using PAIRS we followed the fate of ∼150 cargos on the background of mutations in nine putative cargo receptors and identified novel cargo for most of these receptors. Deletion of the Erv14 cargo receptor affected the widest range of cargo. Erv14 substrates have a wide array of functions and structures; however, they are all membrane-spanning proteins of the late secretory pathway or plasma membrane. Proteins residing in these organelles have longer transmembrane domains (TMDs). Detailed examination of one cargo supported the hypothesis that Erv14 dependency reflects the length rather than the sequence of the TMD. The PAIRS approach allowed us to uncover new cargo for known cargo receptors and to obtain an unbiased look at specificity in cargo selection. Obtaining the spectrum of cargo for a cargo receptor allows a novel perspective on its mode of action. The rules that appear to guide Erv14 substrate recognition suggest that sorting of membrane proteins at multiple points in the secretorypathway could depend on the physical properties of TMDs. Such a mechanism would allow diverse proteins to utilize a few receptors without the constraints of evolving location-specific sorting motifs.

Finnigan GC, Cronan GE, Park HJ, Srinivasan S, Quiocho FA, Stevens TH J Biol Chem. 2012 Jun 1;287(23):19487–500 Yeast/SGA Subunit a of the yeast vacuolar-type, proton-translocating ATPase enzyme complex (V-ATPase) is responsible for both proton translocation and subcellular localization of this highly conserved molecular machine. Inclusion of the Vph1p isoform causes the V-ATPase complex to traffic to the vacuolar membrane, whereas incorporation of Stv1p causes continued cycling between the trans-Golgi and endosome. We previously demonstrated that this targeting information is contained within the cytosolic, N-terminal portion of V-ATPase subunit a (Stv1p). To identify residues responsible forsorting of the Golgi isoform of the V-ATPase, a random mutagenesis was performed on the N terminus of Stv1p. Subsequent characterization of mutant alleles led to the identification of a short peptide sequence, W(83)KY, that is necessary for proper Stv1p localization. Based on three-dimensional homology modeling to the Meiothermus ruber subunit I, we propose a structural model of the intact Stv1p-containing V-ATPasedemonstrating the accessibility of the W(83)KY sequence to retrograde sorting machinery. Finally, we characterized the sorting signal within the context of a reconstructed Stv1p ancestor (Anc.Stv1). This evolutionary intermediate includes an endogenous W(83)KY sorting motif and issufficient to compete with sorting of the native yeast Stv1p V-ATPase isoform. These data define a novel sorting signal that is both necessary andsufficient for trafficking of the V-ATPase within the Golgi/endosomal network.