Doctor of Philosophy, University of California San Francisco (2006)
Bachelor of Science, Stanford University, MATH-BS (2000)
Tobias Meyer, Postdoctoral Faculty Sponsor
To broadly explore mitochondrial structure and function as well as the communication of mitochondria with other cellular pathways, we constructed a quantitative, high-density genetic interaction map (the MITO-MAP) in Saccharomyces cerevisiae. The MITO-MAP provides a comprehensive view of mitochondrial function including insights into the activity of uncharacterized mitochondrial proteins and the functional connection between mitochondria and the ER. The MITO-MAP also reveals a large inner membrane-associated complex, which we term MitOS for mitochondrial organizing structure, comprised of Fcj1/Mitofilin, a conserved inner membrane protein, and five additional components. MitOS physically and functionally interacts with both outer and inner membrane components and localizes to extended structures that wrap around the inner membrane. We show that MitOS acts in concert with ATP synthase dimers to organize the inner membrane and promote normal mitochondrial morphology. We propose that MitOS acts as a conserved mitochondrial skeletal structure that differentiates regions of the inner membrane to establish the normal internal architecture of mitochondria.
View details for DOI 10.1083/jcb.201107053
View details for Web of Science ID 000296205300014
View details for PubMedID 21987634
Human stromal interaction molecule (STIM) proteins are parts of elaborate eukaryotic Ca(2+) signaling systems that include numerous plasma membrane (PM), endoplasmic reticulum (ER), and mitochondrial Ca(2+) transporters, channels and regulators. STIM2 and STIM1 function as Ca(2+) sensors with different sensitivities for ER Ca(2+). They translocate to ER-PM junctions and open PM Orai Ca(2+) influx channels when receptor-mediated Ca(2+) release lowers ER Ca(2+) levels. The resulting increase in cytosolic Ca(2+) leads to the activation of numerous Ca(2+) effector proteins that in turn regulate differentiation, cell contraction, secretion and other cell functions. In this review, we use an evolutionary perspective to survey molecular activation mechanisms in the Ca(2+) signaling system, with a particular focus on regulatory motifs and functions of the two STIM proteins. We discuss the presence and absence of STIM genes in different species, the order of appearance of STIM versus Orai, and the evolutionary addition of new signaling domains to STIM proteins.
View details for DOI 10.1016/j.tcb.2011.01.002
View details for Web of Science ID 000290054000002
View details for PubMedID 21288721
Defining the functional relationships between proteins is critical for understanding virtually all aspects of cell biology. Large-scale identification of protein complexes has provided one important step towards this goal; however, even knowledge of the stoichiometry, affinity and lifetime of every protein-protein interaction would not reveal the functional relationships between and within such complexes. Genetic interactions can provide functional information that is largely invisible to protein-protein interaction data sets. Here we present an epistatic miniarray profile (E-MAP) consisting of quantitative pairwise measurements of the genetic interactions between 743 Saccharomyces cerevisiae genes involved in various aspects of chromosome biology (including DNA replication/repair, chromatid segregation and transcriptional regulation). This E-MAP reveals that physical interactions fall into two well-represented classes distinguished by whether or not the individual proteins act coherently to carry out a common function. Thus, genetic interaction data make it possible to dissect functionally multi-protein complexes, including Mediator, and to organize distinct protein complexes into pathways. In one pathway defined here, we show that Rtt109 is the founding member of a novel class of histone acetyltransferases responsible for Asf1-dependent acetylation of histone H3 on lysine 56. This modification, in turn, enables a ubiquitin ligase complex containing the cullin Rtt101 to ensure genomic integrity during DNA replication.
View details for DOI 10.1038/nature05649
View details for Web of Science ID 000245626800043
View details for PubMedID 17314980
Defining protein complexes is critical to virtually all aspects of cell biology. Two recent affinity purification/mass spectrometry studies in Saccharomyces cerevisiae have vastly increased the available protein interaction data. The practical utility of such high throughput interaction sets, however, is substantially decreased by the presence of false positives. Here we created a novel probabilistic metric that takes advantage of the high density of these data, including both the presence and absence of individual associations, to provide a measure of the relative confidence of each potential protein-protein interaction. This analysis largely overcomes the noise inherent in high throughput immunoprecipitation experiments. For example, of the 12,122 binary interactions in the general repository of interaction data (BioGRID) derived from these two studies, we marked 7504 as being of substantially lower confidence. Additionally, applying our metric and a stringent cutoff we identified a set of 9074 interactions (including 4456 that were not among the 12,122 interactions) with accuracy comparable to that of conventional small scale methodologies. Finally we organized proteins into coherent multisubunit complexes using hierarchical clustering. This work thus provides a highly accurate physical interaction map of yeast in a format that is readily accessible to the biological community.
View details for DOI 10.1074/mcp.M600381-MCP200
View details for Web of Science ID 000245214900006
View details for PubMedID 17200106
Abundant nonfibrillar oligomeric intermediates are a common feature of amyloid formation, and these oligomers, rather than the final fibers, have been suggested to be the toxic species in some amyloid diseases. Whether such oligomers are critical intermediates for fiber assembly or form in an alternate, potentially separable pathway, however, remains unclear. Here we study the polymerization of the amyloidogenic yeast prion protein Sup35. Rapid polymerization occurs in the absence of observable intermediates, and both targeted kinetic and direct single-molecule fluorescence measurements indicate that fibers grow by monomer addition. A three-step model (nucleation, monomer addition, and fiber fragmentation) accurately accounts for the distinctive kinetic features of amyloid formation, including weak concentration dependence, acceleration by agitation, and sigmoidal shape of the polymerization time course. Thus, amyloid growth can occur by monomer addition in a reaction distinct from and competitive with formation of potentially toxic oligomeric intermediates.
View details for DOI 10.1371/journal.pbio.0020321
View details for Web of Science ID 000224737100014
View details for PubMedID 15383837
To date, cross-species comparisons of genetic interactomes have been restricted to small or functionally related gene sets, limiting our ability to infer evolutionary trends. To facilitate a more comprehensive analysis, we constructed a genome-scale epistasis map (E-MAP) for the fission yeast Schizosaccharomyces pombe, providing phenotypic signatures for ~60% of the nonessential genome. Using these signatures, we generated a catalog of 297 functional modules, and we assigned function to 144 previously uncharacterized genes, including mRNA splicing and DNA damage checkpoint factors. Comparison with an integrated genetic interactome from the budding yeast Saccharomyces cerevisiae revealed a hierarchical model for the evolution of genetic interactions, with conservation highest within protein complexes, lower within biological processes, and lowest between distinct biological processes. Despite the large evolutionary distance and extensive rewiring of individual interactions, both networks retain conserved features and display similar levels of functional crosstalk between biological processes, suggesting general design principles of genetic interactomes.
View details for DOI 10.1016/j.molce1.2012.05.028
View details for Web of Science ID 000305095400017
View details for PubMedID 22681890
We present a genetic interaction map of pairwise measures including ∼40% of nonessential S. pombe genes. By comparing interaction maps for fission and budding yeast, we confirmed widespread conservation of genetic relationships within and between complexes and pathways. However, we identified an important subset of orthologous complexes that have undergone functional "repurposing": the evolution of divergent functions and partnerships. We validated three functional repurposing events in S. pombe and mammalian cells and discovered that (1) two lumenal sensors of misfolded ER proteins, the kinase/nuclease Ire1 and the glucosyltransferase Gpt1, act together to mount an ER stress response; (2) ESCRT factors regulate spindle-pole-body duplication; and (3) a membrane-protein phosphatase and kinase complex, the STRIPAK complex, bridges the cis-Golgi, the centrosome, and the outer nuclear membrane to direct mitotic progression. Each discovery opens new areas of inquiry and-together-have implications for model organism-based research and the evolution of genetic systems.
View details for DOI 10.1016/j.cell.2012.04.028
View details for Web of Science ID 000305119600017
View details for PubMedID 22682253
Recent work has identified several posttranslational modifications (PTMs) on chromatin-remodeling complexes. Compared with our understanding of histone PTMs, significantly less is known about the functions of PTMs on remodeling complexes, because identification of their specific roles often is hindered by the presence of redundant pathways. Remodels the Structure of Chromatin (RSC) is an essential, multifunctional ATP-dependent chromatin-remodeling enzyme of Saccharomyces cerevisiae that preferentially binds acetylated nucleosomes. RSC is itself acetylated by Gcn5 on lysine 25 (K25) of its Rsc4 subunit, adjacent to two tandem bromodomains. It has been shown that an intramolecular interaction between the acetylation mark and the proximal bromodomain inhibits binding of the second bromodomain to acetylated histone H3 tails. We report here that acetylation does not significantly alter the catalytic activity of RSC or its ability to recognize histone H3-acetylated nucleosomes preferentially in vitro. However, we find that Rsc4 acetylation is crucial for resistance to DNA damage in vivo. Epistatic miniarray profiling of the rsc4-K25R mutant reveals an interaction with mutants in the INO80 complex, a mediator of DNA damage and replication stress tolerance. In the absence of a core INO80 subunit, rsc4-K25R mutants display sensitivity to hydroxyurea and delayed S-phase progression under DNA damage. Thus, Rsc4 helps promote resistance to replication stress, and its single acetylation mark regulates this function. These studies offer an example of acetylation of a chromatin-remodeling enzyme controlling a biological output of the system rather than regulating its core enzymatic properties.
View details for DOI 10.1073/pnas.1019735108
View details for Web of Science ID 000292251000051
View details for PubMedID 21673141
Despite the essential roles of sphingolipids both as structural components of membranes and critical signalling molecules, we have a limited understanding of how cells sense and regulate their levels. Here we reveal the function in sphingolipid metabolism of the ORM genes (known as ORMDL genes in humans)-a conserved gene family that includes ORMDL3, which has recently been identified as a potential risk factor for childhood asthma. Starting from an unbiased functional genomic approach in Saccharomyces cerevisiae, we identify Orm proteins as negative regulators of sphingolipid synthesis that form a conserved complex with serine palmitoyltransferase, the first and rate-limiting enzyme in sphingolipid production. We also define a regulatory pathway in which phosphorylation of Orm proteins relieves their inhibitory activity when sphingolipid production is disrupted. Changes in ORM gene expression or mutations to their phosphorylation sites cause dysregulation of sphingolipid metabolism. Our work identifies the Orm proteins as critical mediators of sphingolipid homeostasis and raises the possibility that sphingolipid misregulation contributes to the development of childhood asthma.
View details for DOI 10.1038/nature08787
View details for Web of Science ID 000275108400029
View details for PubMedID 20182505
Genetic interactions represent the degree to which the presence of one mutation modulates the phenotype of a second mutation. In recent years, approaches for measuring genetic interactions systematically and quantitatively have proven to be effective tools for unbiased characterization of gene function and have provided valuable data for analyses of evolution. Here, we present protocols for systematic measurement of genetic interactions with respect to organismal growth rate for two yeast species.
View details for DOI 10.1016/S0076-6879(10)70009-4
View details for Web of Science ID 000275827900009
View details for PubMedID 20946812
Communication between organelles is an important feature of all eukaryotic cells. To uncover components involved in mitochondria/endoplasmic reticulum (ER) junctions, we screened for mutants that could be complemented by a synthetic protein designed to artificially tether the two organelles. We identified the Mmm1/Mdm10/Mdm12/Mdm34 complex as a molecular tether between ER and mitochondria. The tethering complex was composed of proteins resident of both ER and mitochondria. With the use of genome-wide mapping of genetic interactions, we showed that the components of the tethering complex were functionally connected to phospholipid biosynthesis and calcium-signaling genes. In mutant cells, phospholipid biosynthesis was impaired. The tethering complex localized to discrete foci, suggesting that discrete sites of close apposition between ER and mitochondria facilitate interorganelle calcium and phospholipid exchange.
View details for DOI 10.1126/science.1175088
View details for Web of Science ID 000268255100057
View details for PubMedID 19556461
Most cellular processes are performed by proteomic units that interact with each other. These units are often stoichiometrically stable complexes comprised of several proteins. To obtain a faithful view of the protein interactome we must view it in terms of these basic units (complexes and proteins) and the interactions between them. This study makes two contributions toward this goal. First, it provides a new algorithm for reconstruction of stable complexes from a variety of heterogeneous biological assays; our approach combines state-of-the-art machine learning methods with a novel hierarchical clustering algorithm that allows clusters to overlap. We demonstrate that our approach constructs over 40% more known complexes than other recent methods and that the complexes it produces are more biologically coherent even compared with the reference set. We provide experimental support for some of our novel predictions, identifying both a new complex involved in nutrient starvation and a new component of the eisosome complex. Second, we provide a high accuracy algorithm for the novel problem of predicting transient interactions involving complexes. We show that our complex level network, which we call ComplexNet, provides novel insights regarding the protein-protein interaction network. In particular, we reinterpret the finding that "hubs" in the network are enriched for being essential, showing instead that essential proteins tend to be clustered together in essential complexes and that these essential complexes tend to be large.
View details for DOI 10.1074/mcp.M800490-MCP200
View details for Web of Science ID 000266904900016
View details for PubMedID 19176519
Protein folding in the endoplasmic reticulum is a complex process whose malfunction is implicated in disease and aging. By using the cell's endogenous sensor (the unfolded protein response), we identified several hundred yeast genes with roles in endoplasmic reticulum folding and systematically characterized their functional interdependencies by measuring unfolded protein response levels in double mutants. This strategy revealed multiple conserved factors critical for endoplasmic reticulum folding, including an intimate dependence on the later secretory pathway, a previously uncharacterized six-protein transmembrane complex, and a co-chaperone complex that delivers tail-anchored proteins to their membrane insertion machinery. The use of a quantitative reporter in a comprehensive screen followed by systematic analysis of genetic dependencies should be broadly applicable to functional dissection of complex cellular processes from yeast to human.
View details for DOI 10.1126/science.1167983
View details for Web of Science ID 000264559800030
View details for PubMedID 19325107
Reversible protein phosphorylation is a signaling mechanism involved in all cellular processes. To create a systems view of the signaling apparatus in budding yeast, we generated an epistatic miniarray profile (E-MAP) comprised of 100,000 pairwise, quantitative genetic interactions, including virtually all protein and small-molecule kinases and phosphatases as well as key cellular regulators. Quantitative genetic interaction mapping reveals factors working in compensatory pathways (negative genetic interactions) or those operating in linear pathways (positive genetic interactions). We found an enrichment of positive genetic interactions between kinases, phosphatases, and their substrates. In addition, we assembled a higher-order map from sets of three genes that display strong interactions with one another: triplets enriched for functional connectivity. The resulting network view provides insights into signaling pathway regulation and reveals a link between the cell-cycle kinase, Cak1, the Fus3 MAP kinase, and a pathway that regulates chromatin integrity during transcription by RNA polymerase II.
View details for DOI 10.1016/j.cell.2008.12.039
View details for Web of Science ID 000263930900017
View details for PubMedID 19269370
Recent studies identified local calcium signals at the leading edge of migrating cells, suggesting a new role for calcium in cell polarization and chemotaxis.
View details for DOI 10.1016/j.devcel.2009.01.018
View details for Web of Science ID 000263589700003
View details for PubMedID 19217416
We used a quantitative, high-density genetic interaction map, or E-MAP (Epistatic MiniArray Profile), to interrogate the relationships within and between RNA-processing pathways. Due to their complexity and the essential roles of many of the components, these pathways have been difficult to functionally dissect. Here, we report the results for 107,155 individual interactions involving 552 mutations, 166 of which are hypomorphic alleles of essential genes. Our data enabled the discovery of links between components of the mRNA export and splicing machineries and Sem1/Dss1, a component of the 19S proteasome. In particular, we demonstrate that Sem1 has a proteasome-independent role in mRNA export as a functional component of the Sac3-Thp1 complex. Sem1 also interacts with Csn12, a component of the COP9 signalosome. Finally, we show that Csn12 plays a role in pre-mRNA splicing, which is independent of other signalosome components. Thus, Sem1 is involved in three separate and functionally distinct complexes.
View details for DOI 10.1016/j.molcel.2008.11.012
View details for Web of Science ID 000261539700015
View details for PubMedID 19061648
An epistasis map (E-MAP) was constructed in the fission yeast, Schizosaccharomyces pombe, by systematically measuring the phenotypes associated with pairs of mutations. This high-density, quantitative genetic interaction map focused on various aspects of chromosome function, including transcription regulation and DNA repair/replication. The E-MAP uncovered a previously unidentified component of the RNA interference (RNAi) machinery (rsh1) and linked the RNAi pathway to several other biological processes. Comparison of the S. pombe E-MAP to an analogous genetic map from the budding yeast revealed that, whereas negative interactions were conserved between genes involved in similar biological processes, positive interactions and overall genetic profiles between pairs of genes coding for physically associated proteins were even more conserved. Hence, conservation occurs at the level of the functional module (protein complex), but the genetic cross talk between modules can differ substantially.
View details for DOI 10.1126/science.1162609
View details for Web of Science ID 000260094500037
View details for PubMedID 18818364
Large-scale genetic interaction studies provide the basis for defining gene function and pathway architecture. Recent advances in the ability to generate double mutants en masse in Saccharomyces cerevisiae have dramatically accelerated the acquisition of genetic interaction information and the biological inferences that follow. Here we describe a method based on F factor-driven conjugation, which allows for high-throughput generation of double mutants in Escherichia coli. This method, termed genetic interaction analysis technology for E. coli (GIANT-coli), permits us to systematically generate and array double-mutant cells on solid media in high-density arrays. We show that colony size provides a robust and quantitative output of cellular fitness and that GIANT-coli can recapitulate known synthetic interactions and identify previously unidentified negative (synthetic sickness or lethality) and positive (suppressive or epistatic) relationships. Finally, we describe a complementary strategy for genome-wide suppressor-mutant identification. Together, these methods permit rapid, large-scale genetic interaction studies in E. coli.
View details for DOI 10.1038/NMETH.1240
View details for Web of Science ID 000258912700011
View details for PubMedID 19160513
Functional genomic studies in Saccharomyces cerevisiae have contributed enormously to our understanding of cellular processes. Their full potential, however, has been hampered by the limited availability of reagents to systematically study essential genes and the inability to quantify the small effects of most gene deletions on growth. Here we describe the construction of a library of hypomorphic alleles of essential genes and a high-throughput growth competition assay to measure fitness with unprecedented sensitivity. These tools dramatically increase the breadth and precision with which quantitative genetic analysis can be performed in yeast. We illustrate the value of these approaches by using genetic interactions to reveal new relationships between chromatin-modifying factors and to create a functional map of the proteasome. Finally, by measuring the fitness of strains in the yeast deletion library, we addressed an enigma regarding the apparent prevalence of gene dispensability and found that most genes do contribute to growth.
View details for DOI 10.1038/nmeth.1234
View details for Web of Science ID 000258077400015
View details for PubMedID 18622397
Many amyloid inhibitors resemble molecules that form chemical aggregates, which are known to inhibit many proteins. Eight known chemical aggregators inhibited amyloid formation of the yeast and mouse prion proteins Sup35 and recMoPrP in a manner characteristic of colloidal inhibition. Similarly, three known anti-amyloid molecules inhibited beta-lactamase in a detergent-dependent manner, which suggests that they too form colloidal aggregates. The colloids localized to preformed fibers and prevented new fiber formation in electron micrographs. They also blocked infection of yeast cells with Sup35 prions, which suggests that colloidal inhibition may be relevant in more biological milieus.
View details for Web of Science ID 000253417400015
View details for PubMedID 18223646
The yeast Mec1/Tel1 kinases, ATM/ATR in mammals, coordinate the DNA damage response by phosphorylating proteins involved in DNA repair and checkpoint pathways. Recently, ATP-dependent chromatin remodeling complexes, such as the INO80 complex, have also been implicated in DNA damage responses, although regulatory mechanisms that direct their function remain unknown. Here, we show that the Ies4 subunit of the INO80 complex is phosphorylated by the Mec1/Tel1 kinases during exposure to DNA-damaging agents. Mutation of Ies4's phosphorylation sites does not significantly affect DNA repair processes, but does influence DNA damage checkpoint responses. Additionally, ies4 phosphorylation mutants are linked to the function of checkpoint regulators, such as the replication checkpoint factors Tof1 and Rad53. These findings establish a chromatin remodeling complex as a functional component in the Mec1/Tel1 DNA damage signaling pathway that modulates checkpoint responses and suggest that posttranslational modification of chromatin remodeling complexes regulates their involvement in distinct processes.
View details for DOI 10.1016/j.cell.2007.06.010
View details for Web of Science ID 000249038100017
View details for PubMedID 17693258
The proteasome regulates histone lysine methylation and gene transcription, but how it does so is poorly understood. To better understand this process, we used the epistatic miniarray profile (E-MAP) approach to identify factors that genetically interact with proteasomal subunits. In addition to members of the Set1 complex that mediate histone H3 lysine 4 methylation (H3K4me), we found that deleting members of the CCR4/NOT mRNA processing complex exhibit synthetic phenotypes when combined with proteasome mutants. Further biochemical analyses revealed physical associations between CCR4/NOT and the proteasome in vivo. Consistent with the genetic and biochemical interactions linking CCR4/NOT with proteasome and Set1-mediated methylation, we find that loss of Not4 decreases global and gene-specific H3K4 trimethylation (H3K4me3) and decreases 19S proteasome recruitment to the PMA1 gene. Similar to proteasome regulation of histone methylation, loss of CCR4/NOT members does not affect ubiquitinated H2B. Mapping of Not4 identified the RING finger domain as essential for H3K4me3, suggesting a role for ubiquitin in this process. Consistent with this idea, loss of the Not4-interacting protein Ubc4, a known ubiquitin-conjugating enzyme, decreases H3K4me3. These studies implicate CCR4/NOT in the regulation of H3K4me3 through a ubiquitin-dependent pathway that likely involves the proteasome.
View details for DOI 10.1073/pnas.0607996104
View details for Web of Science ID 000245657600026
View details for PubMedID 17389396
Many genes can be deleted with little phenotypic consequences. By what mechanism and to what extent the presence of duplicate genes in the genome contributes to this robustness against deletions has been the subject of considerable interest. Here, we exploit the availability of high-density genetic interaction maps to provide direct support for the role of backup compensation, where functionally overlapping duplicates cover for the loss of their paralog. However, we find that the overall contribution of duplicates to robustness against null mutations is low ( approximately 25%). The ability to directly identify buffering paralogs allowed us to further study their properties, and how they differ from non-buffering duplicates. Using environmental sensitivity profiles as well as quantitative genetic interaction spectra as high-resolution phenotypes, we establish that even duplicate pairs with compensation capacity exhibit rich and typically non-overlapping deletion phenotypes, and are thus unable to comprehensively cover against loss of their paralog. Our findings reconcile the fact that duplicates can compensate for each other's loss under a limited number of conditions with the evolutionary instability of genes whose loss is not associated with a phenotypic penalty.
View details for DOI 10.1038/msb4100127
View details for Web of Science ID 000245381300009
View details for PubMedID 17389874
The use of the budding yeast Saccharomyces cerevisiae as a simple eukaryotic model system for the study of chromatin assembly and regulation has allowed rapid discovery of genes that influence this complex process. The functions of many of the proteins encoded by these genes have not yet been fully characterized. Here, we describe a high-throughput methodology that can be used to illuminate gene function and discuss its application to a set of genes involved in the creation, maintenance and remodeling of chromatin structure. Our technique, termed E-MAPs, involves the generation of quantitative genetic interaction maps that reveal the function and organization of cellular proteins and networks.
View details for DOI 10.1016/j.ymeth.2006.07.034
View details for Web of Science ID 000242902600009
View details for PubMedID 17101447
A principle that has emerged from studies of protein aggregation is that proteins typically can misfold into a range of different aggregated forms. Moreover, the phenotypic and pathological consequences of protein aggregation depend critically on the specific misfolded form. A striking example of this is the prion strain phenomenon, in which prion particles composed of the same protein cause distinct heritable states. Accumulating evidence from yeast prions such as [PSI+] and mammalian prions argues that differences in the prion conformation underlie prion strain variants. Nonetheless, it remains poorly understood why changes in the conformation of misfolded proteins alter their physiological effects. Here we present and experimentally validate an analytical model describing how [PSI+] strain phenotypes arise from the dynamic interaction among the effects of prion dilution, competition for a limited pool of soluble protein, and conformation-dependent differences in prion growth and division rates. Analysis of three distinct prion conformations of yeast Sup35 (the [PSI+] protein determinant) and their in vivo phenotypes reveals that the Sup35 amyloid causing the strongest phenotype surprisingly shows the slowest growth. This slow growth, however, is more than compensated for by an increased brittleness that promotes prion division. The propensity of aggregates to undergo breakage, thereby generating new seeds, probably represents a key determinant of their physiological impact for both infectious (prion) and non-infectious amyloids.
View details for DOI 10.1038/nature04922
View details for Web of Science ID 000239455900048
View details for PubMedID 16810177
Identification of protein-protein interactions often provides insight into protein function, and many cellular processes are performed by stable protein complexes. We used tandem affinity purification to process 4,562 different tagged proteins of the yeast Saccharomyces cerevisiae. Each preparation was analysed by both matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography tandem mass spectrometry to increase coverage and accuracy. Machine learning was used to integrate the mass spectrometry scores and assign probabilities to the protein-protein interactions. Among 4,087 different proteins identified with high confidence by mass spectrometry from 2,357 successful purifications, our core data set (median precision of 0.69) comprises 7,123 protein-protein interactions involving 2,708 proteins. A Markov clustering algorithm organized these interactions into 547 protein complexes averaging 4.9 subunits per complex, about half of them absent from the MIPS database, as well as 429 additional interactions between pairs of complexes. The data (all of which are available online) will help future studies on individual proteins as well as functional genomics and systems biology.
View details for DOI 10.1038/nature04670
View details for Web of Science ID 000236350400036
View details for PubMedID 16554755
Recently, approaches have been developed for high-throughput identification of synthetic sick/lethal gene pairs. However, these are only a specific example of the broader phenomenon of epistasis, wherein the presence of one mutation modulates the phenotype of another. We present analysis techniques for generating high-confidence quantitative epistasis scores from measurements made using synthetic genetic array and epistatic miniarray profile (E-MAP) technology, as well as several tools for higher-level analysis of the resulting data that are greatly enhanced by the quantitative score and detection of alleviating interactions.
View details for DOI 10.1186/gb-2006-7-7-r63
View details for Web of Science ID 000241322700020
View details for PubMedID 16859555
The yeast histone deacetylase Rpd3 can be recruited to promoters to repress transcription initiation. Biochemical, genetic, and gene-expression analyses show that Rpd3 exists in two distinct complexes. The smaller complex, Rpd3C(S), shares Sin3 and Ume1 with Rpd3C(L) but contains the unique subunits Rco1 and Eaf3. Rpd3C(S) mutants exhibit phenotypes remarkably similar to those of Set2, a histone methyltransferase associated with elongating RNA polymerase II. Chromatin immunoprecipitation and biochemical experiments indicate that the chromodomain of Eaf3 recruits Rpd3C(S) to nucleosomes methylated by Set2 on histone H3 lysine 36, leading to deacetylation of transcribed regions. This pathway apparently acts to negatively regulate transcription because deleting the genes for Set2 or Rpd3C(S) bypasses the requirement for the positive elongation factor Bur1/Bur2.
View details for DOI 10.1016/j.cell.2005.10.025
View details for Web of Science ID 000233497400009
View details for PubMedID 16286008
We present a strategy for generating and analyzing comprehensive genetic-interaction maps, termed E-MAPs (epistatic miniarray profiles), comprising quantitative measures of aggravating or alleviating interactions between gene pairs. Crucial to the interpretation of E-MAPs is their high-density nature made possible by focusing on logically connected gene subsets and including essential genes. Described here is the analysis of an E-MAP of genes acting in the yeast early secretory pathway. Hierarchical clustering, together with novel analytical strategies and experimental verification, revealed or clarified the role of many proteins involved in extensively studied processes such as sphingolipid metabolism and retention of HDEL proteins. At a broader level, analysis of the E-MAP delineated pathway organization and components of physical complexes and illustrated the interconnection between the various secretory processes. Extension of this strategy to other logically connected gene subsets in yeast and higher eukaryotes should provide critical insights into the functional/organizational principles of biological systems.
View details for DOI 10.1016/j.cell.2005.08.031
View details for Web of Science ID 000233264300017
View details for PubMedID 16269340
Self-propagating beta-sheet-rich protein aggregates are implicated in a wide range of protein-misfolding phenomena, including amyloid diseases and prion-based inheritance. Two properties have emerged as common features of amyloids. Amyloid formation is ubiquitous: many unrelated proteins form such aggregates and even a single polypeptide can misfold into multiple forms--a process that is thought to underlie prion strain variation. Despite this promiscuity, amyloid propagation can be highly sequence specific: amyloid fibres often fail to catalyse the aggregation of other amyloidogenic proteins. In prions, this specificity leads to barriers that limit transmission between species. Using the yeast prion [PSI+], we show in vitro that point mutations in Sup35p, the protein determinant of [PSI+], alter the range of 'infectious' conformations, which in turn changes amyloid seeding specificity. We generate a new transmission barrier in vivo by using these mutations to specifically disfavour subsets of prion strains. The ability of mutations to alter the conformations of amyloid states without preventing amyloid formation altogether provides a general mechanism for the generation of prion transmission barriers and may help to explain how mutations alter toxicity in conformational diseases.
View details for DOI 10.1038/nature01894
View details for Web of Science ID 000184843600043
View details for PubMedID 12931190