Two large-scale yeast two-hybrid screens were undertaken to identify protein-protein interactions between full-length open reading frames predicted from the Saccharomyces cerevisiae genome sequence. In one approach, we constructed a protein array of about 6,000 yeast transformants, with each transformant expressing one of the open reading frames as a fusion to an activation domain. This array was screened by a simple and automated procedure for 192 yeast proteins, with positive responses identified by their positions in the array. In a second approach, we pooled cells expressing one of about 6,000 activation domain fusions to generate a library. We used a high-throughput screening procedure to screen nearly all of the 6,000 predicted yeast proteins, expressed as Gal4 DNA-binding domain fusion proteins, against the library, and characterized positives by sequence analysis. These approaches resulted in the detection of 957 putative interactions involving 1,004 S. cerevisiae proteins. These data reveal interactions that place functionally unclassified proteins in a biological context, interactions between proteins involved in the same biological function, and interactions that link biological functions together into larger cellular processes. The results of these screens are shown here.

http://web.cs.mun.ca/~harold/Courses/Old/CS6754.W04/Diary/403623.pdf

A comprehensive analysis of protein–protein interactions in Saccharomyces cerevisiae

Protein-protein interactions play crucial roles in the execution of various biological functions. Accordingly, their comprehensive description would contribute considerably to the functional interpretation of fully sequenced genomes, which are flooded with novel genes of unpredictable functions. We previously developed a system to examine two-hybrid interactions in all possible combinations between the approximately 6,000 proteins of the budding yeast Saccharomyces cerevisiae. Here we have completed the comprehensive analysis using this system to identify 4,549 two-hybrid interactions among 3,278 proteins. Unexpectedly, these data do not largely overlap with those obtained by the other project [Uetz, P., et al. (2000) Nature (London) 403, 623-627] and hence have substantially expanded our knowledge on the protein interaction space or interactome of the yeast. Cumulative connection of these binary interactions generates a single huge network linking the vast majority of the proteins. Bioinformatics-aided selection of biologically relevant interactions highlights various intriguing subnetworks. They include, for instance, the one that had successfully foreseen the involvement of a novel protein in spindle pole body function as well as the one that may uncover a hitherto unidentified multiprotein complex potentially participating in the process of vesicular transport. Our data would thus significantly expand and improve the protein interaction map for the exploration of genome functions that eventually leads to thorough understanding of the cell as a molecular system.

/pdf/a-comprehensive-two-hybrid-analysis-to-explore-the-yeast-2tmnxjrhto.pdf

A comprehensive two-hybrid analysis to explore the yeast protein interactome

Our cells and tissues are challenged constantly by exposure to extreme conditions that cause acute and chronic stress. Consequently, survival has necessitated the evolution of stress response networks to detect, monitor, and respond to environmental changes (Morimoto et al. 1990, 1994a; Baeuerle 1995; Baeuerle and Baltimore 1996; Feige et al. 1996; Morimoto and Santoro 1998). Prolonged exposure to stress interferes with efficient operations of the cell, with negative consequences on the biochemical properties of proteins that, under ideal conditions, exist in thermodynamically stable states. In stressed environments, proteins can unfold, misfold, or aggregate. Therefore, the changing demands on the quality control of protein biogenesis, challenges protein homeostasis, for which the heat shock response, through the elevated synthesis of molecular chaperones and proteases, repairs protein damage and assists in the recovery of the cell. The inducible transcription of heat shock genes is the response to a plethora of stress signals (Lis and Wu 1993; Morimoto 1993; Wu 1995) (Fig. 1), including (1) environmental stresses, (2) nonstress conditions, and (3) pathophysiology and disease states. Although changes in heat shock protein (HSP) expression are associated with certain diseases (Morimoto et al. 1990), these observations leave open the question of whether this is an adaptation to the particular pathophysiological state, a reflection of the suboptimal cellular environment associated with the disease, or serves to warn other cells and tissues of imminent danger. The protective role of HSPs is a measure of their capacity to assist in the repair of protein damage. Whether in prokaryotes, plants, or animals, overexpression of one or more HSPs is often sufficient to protect cells and tissues against otherwise lethal exposures to diverse environmental stresses including hydrogen peroxide and other oxidants, toxic chemicals, extreme temperatures, and ethanol-induced toxicity (Parsell and Lindquist 1994). In vertebrate tissue culture cells and animal models, elevating HSPs level, either by modulation of the heat shock response or by constitutive overexpression of specific heat shock proteins, restricts or substantially reduces the level of pathology and cell death (Mizzen and Welch 1988; Huot et al. 1991; Jaattela et al. 1992; Parsell and Lindquist 1994; Mestril et al. 1994; Plumier et al. 1995; Marber et al. 1995; Mehlen et al. 1995; Mosser et al. 1997). This has led to the recognition that HSPs, via their chaperoning effects on proteins, protect cells from many forms of stress-induced cell damage and could influence the course of disease.

/pdf/regulation-of-the-heat-shock-transcriptional-response-cross-4fw7c90wzc.pdf

Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators

Autophagy is an intracellular degradation system, by which cytoplasmic contents are degraded in lysosomes. Autophagy is dynamically induced by nutrient depletion to provide necessary amino acids within cells, thus helping them adapt to starvation. Although it has been suggested that mTOR is a major negative regulator of autophagy, how it controls autophagy has not yet been determined. Here, we report a novel mammalian autophagy factor, Atg13, which forms a stable approximately 3-MDa protein complex with ULK1 and FIP200. Atg13 localizes on the autophagic isolation membrane and is essential for autophagosome formation. In contrast to yeast counterparts, formation of the ULK1-Atg13-FIP200 complex is not altered by nutrient conditions. Importantly, mTORC1 is incorporated into the ULK1-Atg13-FIP200 complex through ULK1 in a nutrient-dependent manner and mTOR phosphorylates ULK1 and Atg13. ULK1 is dephosphorylated by rapamycin treatment or starvation. These data suggest that mTORC1 suppresses autophagy through direct regulation of the approximately 3-MDa ULK1-Atg13-FIP200 complex.

Nutrient-dependent mTORC1 Association with the ULK1–Atg13–FIP200 Complex Required for Autophagy

Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding.

The two-hybrid system is a powerful technique for detecting protein-protein interactions that utilizes the well-developed molecular genetics of the yeast Saccharomyces cerevisiae. However, the full potential of this technique has not been realized due to limitations imposed by the components available for use in the system. These limitations include unwieldy plasmid vectors, incomplete or poorly designed two-hybrid libraries, and host strains that result in the selection of large numbers of false positives. We have used a novel multienzyme approach to generate a set of highly representative genomic libraries from S. cerevisiae. In addition, a unique host strain was created that contains three easily assayed reporter genes, each under the control of a different inducible promoter. This host strain is extremely sensitive to weak interactions and eliminates nearly all false positives using simple plate assays. Improved vectors were also constructed that simplify the construction of the gene fusions necessary for the two-hybrid system. Our analysis indicates that the libraries and host strain provide significant improvements in both the number of interacting clones identified and the efficiency of two-hybrid selections.

/pdf/genomic-libraries-and-a-host-strain-designed-for-highly-1u4u4zfe67.pdf

Genomic Libraries and a Host Strain Designed for Highly Efficient Two-Hybrid Selection in Yeast

I. INTRODUCTION The 70-kD heat shock proteins, or hsp70s, are highly conserved in all organisms studied so far, from bacteria to yeast to humans. Eukaryotes, including the budding yeast Saccharomyces cerevisiae, encode multiple hsp70s in their genomes. These related proteins are localized to a variety of cellular compartments, including the cytosol, mitochondria, and endoplasmic reticulum (ER). Functionally, the organellar hsp70s are better understood, having major roles in protein translocation and folding. The functions of the cytosolic hsp70s have been more difficult to define, perhaps because these proteins are involved in multiple processes, including translation, protein translocation, protein folding, and regulation of the heat shock response. This chapter reviews evolutionary analyses and genetic data concerning the roles of these proteins in the cytosol. II. EVOLUTION OF THE HSP70 MULTIGENE FAMILY To gain a better understanding of the evolutionary relationships among hsp70s across the biological spectrum, a comparison of 36 hsp70s from 25 diverse genera was conducted (Boorstein et al. 1994). The analysis, carried out by both distance-matrix and character-state methods, showed that the eukaryotic hsp70s comprise four distinct clusters (see Fig. 1). These clusters correspond to the intracellular localization of the proteins: the cytosol, the ER, mitochondria, and chloroplasts. Analysis of the comparisons revealed that eukaryotic hsp70s appear to have evolved from ancestral genes by two types of mechanisms. Mitochondrial and chloroplast hsp70s appear to be derived from the establishment of an endosymbiotic relationship between a eukaryotic host and bacterial cells. Mitochondrial hsp70s are encoded in the nuclei, but they share...

2 Cytosolic hsp70s of Saccharomyces cerevisiae : Roles in Protein Synthesis, Protein Translocation, Proteolysis, and Regulation

Strains carrying deletions in both the SSA1 and SSA2 HSP70 genes of Saccharomyces cerevisiae exhibit pleiotropic phenotypes, including the inability to grow at 37 degrees C or higher, reduced growth rate at permissive temperatures, increased HSP gene expression, and constitutive thermotolerance. A screen for extragenic suppressors of the ssa1 ssa2 slow-growth phenotype identified a spontaneous dominant suppressor mutation, EXA3-1 (R.J. Nelson, M. Heschl, and E.A. Craig, Genetics 131:277-285, 1992). Here we report that EXA3-1 is an allele of HSF1, which encodes the heat shock transcription factor (HSF). Strains containing the EXA3-1 allele in a wild-type background exhibit a 10- to 15-fold reduction in HSF activity during steady-state growth conditions as well as a delay in the accumulation of the SSA4, HSP26, and HSP104 mRNAs after a heat shock. EXA3-1-mediated suppression is the result of a single amino acid substitution of a highly conserved residue in the HSF DNA-binding domain which drastically reduces the ability of HSF to bind to heat shock elements as evaluated by band shift analysis. Together, these results indicate that the poor growth of ssa1 ssa2 strains is the result, at least in part, of the overproduction of a deleterious heat shock protein(s). This conclusion is supported by the fact that the levels of at least some heat shock proteins are reduced in ssa1 ssa2 cells containing the EXA3-1 allele. Surprisingly, strains containing the EXA3-1 allele in a wild-type HSP70 background grow early as well as the wild-type strain over a wide temperature range, displaying only a slight reduction in growth rate at 37 degrees Celsius, indicating that cells contain significantly more HSF activity than is require for growth under steady-state conditions.

/pdf/a-heat-shock-transcription-factor-with-reduced-activity-2aljs62mva.pdf

A Heat Shock Transcription Factor with Reduced Activity Suppresses a Yeast HSP70 Mutant

Genes encoding ribosomal proteins and other components of the translational apparatus are coregulated to efficiently adjust the protein synthetic capacity of the cell. Ssb, a Saccharomyces cerevisiae Hsp70 cytosolic molecular chaperone, is associated with the ribosome-nascent chain complex. To determine whether this chaperone is coregulated with ribosomal proteins, we studied the mRNA regulation of SSB under several environmental conditions. Ssb and the ribosomal protein rpL5 mRNAs were up-regulated upon carbon upshift and down-regulated upon amino acid limitation, unlike the mRNA of another cytosolic Hsp70, Ssa. Ribosomal protein and Ssb mRNAs, like many mRNAs, are down-regulated upon a rapid temperature upshift. The mRNA reduction of several ribosomal protein genes and Ssb was delayed by the presence of an allele, EXA3-1, of the gene encoding the heat shock factor (HSF). However, upon a heat shock the EXA3-1 mutation did not significantly alter the reduction in the mRNA levels of two genes encoding proteins unrelated to the translational apparatus. Analysis of gene fusions indicated that the transcribed region, but not the promoter of SSB, is sufficient for this HSF-dependent regulation. Our studies suggest that Ssb is regulated like a core component of the ribosome and that HSF is required for proper regulation of SSB and ribosomal mRNA after a temperature upshift.

/pdf/ssb-encoding-a-ribosome-associated-chaperone-is-coordinately-44baze0d2m.pdf

John Halladay

Papers

Genomic Libraries and a Host Strain Designed for Highly Efficient Two-Hybrid Selection in Yeast

2 Cytosolic hsp70s of Saccharomyces cerevisiae : Roles in Protein Synthesis, Protein Translocation, Proteolysis, and Regulation

A Heat Shock Transcription Factor with Reduced Activity Suppresses a Yeast HSP70 Mutant

SSB, Encoding a Ribosome-Associated Chaperone, Is Coordinately Regulated with Ribosomal Protein Genes

Complex Multigene Family of Functionally Distinct Hsp70s of Yeast