The Sec1/Munc18 protein Vps45 regulates cellular levels of its SNARE binding partners Tlg2 and Snc2 in Saccharomyces cerevisiae

Intracellular membrane trafficking pathways must be tightly regulated to ensure proper functioning of all eukaryotic cells. Central to membrane trafficking is the formation of specific SNARE (soluble N-ethylmeleimide-sensitive factor attachment protein receptor) complexes between proteins on opposing lipid bilayers. The Sec1/Munc18 (SM) family of proteins play an essential role in SNARE-mediated membrane fusion, and like the SNAREs are conserved through evolution from yeast to humans. The SM protein Vps45 is required for the formation of yeast endosomal SNARE complexes and is thus essential for traffic through the endosomal system. Here we report that, in addition to its role in regulating SNARE complex assembly, Vps45 regulates cellular levels of its SNARE binding partners: the syntaxin Tlg2 and the v-SNARE Snc2: Cells lacking Vps45 have reduced cellular levels of Tlg2 and Snc2; and elevation of Vps45 levels results in concomitant increases in the levels of both Tlg2 and Snc2. As well as regulating traffic through the endosomal system, the Snc v-SNAREs are also required for exocytosis. Unlike most vps mutants, cells lacking Vps45 display multiple growth phenotypes. Here we report that these can be reversed by selectively restoring Snc2 levels in vps45 mutant cells. Our data indicate that as well as functioning as part of the machinery that controls SNARE complex assembly, Vps45 also plays a key role in determining the levels of its cognate SNARE proteins; another key factor in regulation of membrane traffic

Comparison of Internal Ribosome Entry Site (IRES) and Furin-2A (F2A) for Monoclonal Antibody Expression Level and Quality in CHO Cells

10.1371/journal.pone.0063247PLoS ONE85

Genetic interactions between a phospholipase A2 and the Rim101 pathway components in S. cerevisiae reveal a role for this pathway in response to changes in membrane composition and shape

Modulating composition and shape of biological membranes is an emerging mode of regulation of cellular processes. We investigated the global effects that such perturbations have on a model eukaryotic cell. Phospholipases A2 (PLA2s), enzymes that cleave one fatty acid molecule from membrane phospholipids, exert their biological activities through affecting both membrane composition and shape. We have conducted a genome-wide analysis of cellular effects of a PLA2 in the yeast Saccharomyces cerevisiae as a model system. We demonstrate functional genetic and biochemical interactions between PLA2 activity and the Rim101 signaling pathway in S. cerevisiae. Our results suggest that the composition and/or the shape of the endosomal membrane affect the Rim101 pathway. We describe a genetically and functionally related network, consisting of components of the Rim101 pathway and the prefoldin, retromer and SWR1 complexes, and predict its functional relation to PLA2 activity in a model eukaryotic cell. This study provides a list of the players involved in the global response to changes in membrane composition and shape in a model eukaryotic cell, and further studies are needed to understand the precise molecular mechanisms connecting them

VPS29 Is Not an Active Metallo-Phosphatase but Is a Rigid Scaffold Required for Retromer Interaction with Accessory Proteins

VPS29 is a key component of the cargo-binding core complex of retromer, a protein assembly with diverse roles in transport of receptors within the endosomal system. VPS29 has a fold related to metal-binding phosphatases and mediates interactions between retromer and other regulatory proteins. In this study we examine the functional interactions of mammalian VPS29, using X-ray crystallography and NMR spectroscopy. We find that although VPS29 can coordinate metal ions Mn2+ and Zn2+ in both the putative active site and at other locations, the affinity for metals is low, and lack of activity in phosphatase assays using a putative peptide substrate support the conclusion that VPS29 is not a functional metalloenzyme. There is evidence that structural elements of VPS29 critical for binding the retromer subunit VPS35 may undergo both metal-dependent and independent conformational changes regulating complex formation, however studies using ITC and NMR residual dipolar coupling (RDC) measurements show that this is not the case. Finally, NMR chemical shift mapping indicates that VPS29 is able to associate with SNX1 via a conserved hydrophobic surface, but with a low affinity that suggests additional interactions will be required to stabilise the complex in vivo. Our conclusion is that VPS29 is a metal ion-independent, rigid scaffolding domain, which is essential but not sufficient for incorporation of retromer into functional endosomal transport assemblies

Prion Formation and Polyglutamine Aggregation Are Controlled by Two Classes of Genes

Prions are self-perpetuating aggregated proteins that are not limited to mammalian systems but also exist in lower eukaryotes including yeast. While much work has focused around chaperones involved in prion maintenance, including Hsp104, little is known about factors involved in the appearance of prions. De novo appearance of the [PSI+] prion, which is the aggregated form of the Sup35 protein, is dramatically enhanced by transient overexpression of SUP35 in the presence of the prion form of the Rnq1 protein, [PIN+]. When fused to GFP and overexpressed in [ps−] [PIN+] cells, Sup35 forms fluorescent rings, and cells with these rings bud off [PSI+] daughters. We investigated the effects of over 400 gene deletions on this de novo induction of [PSI+]. Two classes of gene deletions were identified. Class I deletions (bug1Δ, bem1Δ, arf1Δ, and hog1Δ) reduced the efficiency of [PSI+] induction, but formed rings normally. Class II deletions (las17Δ, vps5Δ, and sac6Δ) inhibited both [PSI+] induction and ring formation. Furthermore, class II deletions reduced, while class I deletions enhanced, toxicity associated with the expanded glutamine repeats of the huntingtin protein exon 1 that causes Huntington's disease. This suggests that prion formation and polyglutamine aggregation involve a multi-phase process that can be inhibited at different steps.National Institutes of Health (U.S.) (grant GM56350)National Institutes of Health (U.S.) (NSRA F32 postdoctoral fellowship GM072340)National Institutes of Health (U.S.) (grant GM25874)Howard Hughes Medical Institut

A Genome-Wide Immunodetection Screen in S. cerevisiae Uncovers Novel Genes Involved in Lysosomal Vacuole Function and Morphology

Vacuoles of yeast Saccharomyces cerevisiae are functionally analogous to mammalian lysosomes. Both are cellular organelles responsible for macromolecular degradation, ion/pH homeostasis, and stress survival. We hypothesized that undefined gene functions remain at post-endosomal stage of vacuolar events and performed a genome-wide screen directed at such functions at the late endosome and vacuole interface – ENV genes. The immunodetection screen was designed to identify mutants that internally accumulate precursor form of the vacuolar hydrolase carboxypeptidase Y (CPY). Here, we report the uncovering and initial characterizations of twelve ENV genes. The small size of the collection and the lack of genes previously identified with vacuolar events are suggestive of the intended exclusive functional interface of the screen. Most notably, the collection includes four novel genes ENV7, ENV9, ENV10, and ENV11, and three genes previously linked to mitochondrial processes – MAM3, PCP1, PPE1. In all env mutants, vesicular trafficking stages were undisturbed in live cells as assessed by invertase and active α-factor secretion, as well as by localization of the endocytic fluorescent marker FM4-64 to the vacuole. Several mutants exhibit defects in stress survival functions associated with vacuoles. Confocal fluorescence microscopy revealed the collection to be significantly enriched in vacuolar morphologies suggestive of fusion and fission defects. These include the unique phenotype of lumenal vesicles within vacuoles in the novel env9Δ mutant and severely fragmented vacuoles upon deletion of GET4, a gene recently implicated in tail anchored membrane protein insertion. Thus, our results establish new gene functions in vacuolar function and morphology, and suggest a link between vacuolar and mitochondrial events