Sean R. Collins

Global landscape of protein complexes in the yeast Saccharomyces cerevisiae

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.

Functional dissection of protein complexes involved in yeast chromosome biology using a genetic interaction map

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.

An ER-Mitochondria Tethering Complex Revealed by a Synthetic Biology Screen

Making Connections Endoplasmic reticulum (ER)–mitochondria connections have been implicated in many physiological processes, including calcium homeostasis, signaling, membrane biogenesis, and apoptosis. Kornmann et al. (p. 477, published online 25 June; see the Perspective by Wiedemann et al.) looked for a proteinaceous link between the ER and mitochondria and, using combinations of synthetic biology and classical yeast genetics, found a protein complex that tethers the two organelles. A large-scale genetic interaction map suggests that these ER-mitochondria connections are important for interorganellar phospholipid exchange. A protein complex zippers mitochondria to endoplasmic reticulum for phospholipid transfer. 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.

Exploration of the Function and Organization of the Yeast Early Secretory Pathway through an Epistatic Miniarray Profile

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.

Cotranscriptional Set2 Methylation of Histone H3 Lysine 36 Recruits a Repressive Rpd3 Complex

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.

The physical basis of how prion conformations determine strain phenotypes

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.

Comprehensive characterization of genes required for protein folding in the endoplasmic reticulum.

Protein folding in the endoplasmic reticulum is a complex process whose malfunction is implicated in disease and aging. By using the cells 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.

Mechanism of Prion Propagation: Amyloid Growth Occurs by Monomer Addition

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.

Orm family proteins mediate sphingolipid homeostasis.

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.

A comprehensive strategy enabling high-resolution functional analysis of the yeast genome

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.