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 non-essential genome. Using these signatures, we generated a catalogue of 297 functional modules, and assigned function to 144 previously uncharacterised 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 cross-talk between biological processes, suggesting general design principles of genetic interactomes.

Cells of the budding yeast Saccharomyces cerevisiae are born carrying localized transmembrane landmark proteins that guide the subsequent establishment of a polarity axis and hence polarized growth to form a bud in the next cell cycle. In haploid cells, the relevant landmark proteins are concentrated at the site of the preceding cell division, to which they recruit Cdc24, the guanine nucleotide exchange factor for the conserved polarity regulator Cdc42. However, instead of polarizing at the division site, the new polarity axis is directed next to but not overlapping that site. Here, we show that the Cdc42 guanosine triphosphatase–activating protein (GAP) Rga1 establishes an exclusion zone at the division site that blocks subsequent polarization within that site. In the absence of localized Rga1 GAP activity, new buds do in fact form within the old division site. Thus, Cdc42 activators and GAPs establish concentric zones of action such that polarization is directed to occur adjacent to but not within the previous cell division site.

The septins are GTP-binding, filament-forming proteins that are involved in cytokinesis and other processes. In the yeast Saccharomyces cerevisiae, the septins are recruited to the presumptive bud site at the cell cortex, where they form a ring through which the bud emerges. We report here that in wild-type cells, the septins typically become detectable in the vicinity of the bud site several minutes before ring formation, but the ring itself is the first distinct structure that forms. Septin recruitment depends on activated Cdc42p but not on the normal pathway for bud-site selection. Recruitment occurs in the absence of F-actin, but ring formation is delayed. Mutant phenotypes and suppression data suggest that the Cdc42p effectors Gic1p and Gic2p, previously implicated in polarization of the actin cytoskeleton, also function in septin recruitment. Two-hybrid, in vitro protein binding, and coimmunoprecipitation data indicate that this role involves a direct interaction of the Gic proteins with the septin Cdc12p. INTRODUCTIONBud formation in the yeast Saccharomyces cerevisiae has served as an important model for studies of eukaryotic cell polarization (Hall, 1992;Nelson, 2003). In wild-type cells, nonrandom bud sites are selected by a system that involves cortical marker proteins and a signaling pathway based on the Ras-type GTPase Rsr1p Kang et al., 2004;Pruyne et al., 2004). GTP-bound Rsr1p then promotes the localization and/or localized activation of Cdc24p, the guanine-nucleotide-exchange factor (GEF) for the Rho-type GTPase Cdc42p, and of Cdc42p itself; the localized activation of Cdc42p then causes polarization of the cytoskeletal and secretory systems, which leads to the polarized growth of the bud Kozminski et al., 2003;Pruyne et al., 2004;Shimada et al., 2004).Among the proteins recruited early to the presumptive bud site are the septins. This widely conserved family of GTP-binding, filament-forming proteins functions in cytokinesis and other processes, many of which involve the organization of specialized regions of the cell cortex (Longtine et al., 1996;Gladfelter et al., 2001b;Longtine and Bi, 2003;Hall and Russell, 2004). S. cerevisiae has seven septins, five of which (Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Shs1p/Sep7p) are expressed in vegetative cells, where they form heterooligomeric complexes and localize interdependently to the bud site (Kim et al., 1991;Longtine et al., 1996;Frazier et al., 1998;Mortensen et al., 2002;Vrabioiu et al., 2004). About 10 min before bud emergence, the septins form a ring at the cell cortex. The bud then emerges through this ring, which concurrently reorganizes into an hourglassshaped collar that spans the mother-bud neck. This reorganization coincides with a major decrease in the exchangeability of septin subunits, presumably reflecting the formation of more stable higher-order structures at this time (Caviston et al., 2003;Dobbelaere et al., 2003;. The septin collar remains at the neck until cytokinesis, when it splits into two rings as the actomyosin ring contracts an...

SUMMARY Mammalian lipid homeostasis requires proteolytic activation of membrane-bound sterol regulatory element binding protein (SREBP) transcription factors through sequential action of the Golgi Site-1 and Site-2 proteases. Here, we report that while SREBP function is conserved in fungi, fission yeast employs a different mechanism for SREBP cleavage. Using genetics and biochemistry, we identified four genes defective for SREBP cleavage, dsc1–4, encoding components of a transmembrane Golgi E3 ligase complex with structural homology to the Hrd1 E3 ligase complex involved in endoplasmic reticulum-associated degradation. The Dsc complex binds SREBP and cleavage requires components of the ubiquitin-proteasome pathway: the E2 conjugating enzyme Ubc4, the Dsc1 RING E3 ligase and the proteasome. dsc mutants display conserved aggravating genetic interactions with components of the multivesicular body pathway in fission yeast and budding yeast, which lacks SREBP. Together, these data suggest that the Golgi Dsc E3 ligase complex functions in a post-ER pathway for protein degradation.

Polarization of cell growth along a defined axis is essential for the generation of cell and tissue polarity. In the budding yeast Saccharomyces cerevisiae, Axl2p plays an essential role in polarity-axis determination, or more specifically, axial budding in MATa or ␣ cells. Axl2p is a type I membrane glycoprotein containing four cadherin-like motifs in its extracellular domain. However, it is not known when and how Axl2p functions together with other components of the axial landmark, such as Bud3p and Bud4p, to direct axial budding. Here, we show that the recruitment of Axl2p to the bud neck after S/G2 phase of the cell cycle depends on Bud3p and Bud4p. This recruitment is mediated via an interaction between Bud4p and the central region of the Axl2p cytoplasmic tail. This region of Axl2p, together with its N-terminal region and its transmembrane domain, is sufficient for axial budding. In addition, our work demonstrates a previously unappreciated role for Axl2p. Axl2p interacts with Cdc42p and other polarity-establishment proteins, and it regulates septin organization in late G1 independently of its role in polarity-axis determination. Together, these results suggest that Axl2p plays sequential and distinct roles in the regulation of cellular morphogenesis in yeast cell cycle.

Maintenance of protein homeostasis is essential for cellular survival. Central to this regulation are mechanisms of protein quality control in which misfolded proteins are recognized and degraded by the ubiquitin-proteasome system. One well-studied protein quality control pathway requires endoplasmic reticulum ( Control of protein homeostasis or proteostasis is key for cell function and survival (1). An important aspect of proteostasis is protein quality control in which misfolded proteins are recognized and degraded by the ubiquitin-proteasome pathway (2). Complex mechanisms regulate whether proteins are targeted for degradation, but ultimately misfolded proteins are recognized and ubiquitylated by specific E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases (3). One well-studied protein quality control pathway is ER-associated degradation (ERAD) 1 (4 -6). ER luminal and membrane proteins are targeted for cytosolic proteasomal degradation by a set of multisubunit E3 ligases integral to the ER membrane, such as Hrd1 and Doa10 in Saccharomyces cerevisiae and Hrd1 and gp78 in mammals. Key open questions in the protein quality control field are (i) what are the physiological substrates of protein quality control pathways and (ii) how do these E3 ligases recognize proteins for degradation.The sterol regulatory element-binding protein (SREBP) family of transcription factors regulates lipid homeostasis in mammals and fungi (7). These ER membrane-bound proteins are proteolytically activated in the Golgi to release the transcription factor domain from the membrane, allowing it to

In budding yeast, Rga1 negatively regulates the Rho GTPase Cdc42 by acting as a GTPase-activating protein (GAP) for Cdc42. To gain insight into the function and regulation of Rga1, we overexpressed Rga1 and an N-terminally truncated Rga1-C538 (a.a. 538-1007) segment. Overexpression of Rga1-C538 but not full-length Rga1 severely impaired growth and cell morphology in wild-type cells. We show that Rga1 is phosphorylated during the cell cycle. The lack of phenotype for full-length Rga1 upon overexpression may result from a negative regulation by G1-specific Pho85, a cyclin-dependent kinase (CDK). From a high-copy suppressor screen, we isolated RHO3, SEC9, SEC1, SSO1, SSO2, and SRO7, genes involved in exocytosis, as suppressors of the growth defect caused by Rga1-C538 overexpression. Moreover, we detected that Rga1 interacts with Rho3 in two-hybrid and bimolecular fluorescence complementation (BiFC) assays. Rga1 preferentially interacts with the GTP-bound form of Rho3 and the interaction requires the GAP domain and additional sequence upstream of the GAP domain. Our data suggest that the interaction of Rga1 with Rho3 may regulate Rho3’s function in polarized bud growth.

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