A kinase cascade on the yeast lysosomal vacuole regulates its membrane dynamics: conserved kinase Env7 is phosphorylated by casein kinase Yck3.

Membrane fusion/fission is a highly dynamic and conserved process that responds to intra- and extracellular signals. Whereas the molecular machineries involved in membrane fusion/fission have been dissected, regulation of membrane dynamics remains poorly understood. The lysosomal vacuole of budding yeast (Saccharomyces cerevisiae) has served as a seminal model in studies of membrane dynamics. We have previously established that yeast ENV7 encodes an ortholog of STK16-related kinases that localizes to the vacuolar membrane and downregulates vacuolar membrane fusion. Additionally, we have previously reported that Env7 phosphorylation in vivo depends on YCK3, a gene that encodes a vacuolar membrane casein kinase I (CKI) homolog that nonredundantly functions in fusion regulation. Here, we report that Env7 physically interacts with and is directly phosphorylated by Yck3. We also establish that Env7 vacuole fusion/fission regulation and vacuolar localization are mediated through its Yck3-dependent phosphorylation. Through extensive site-directed mutagenesis, we map phosphorylation to the Env7 C terminus and confirm that Ser-331 is a primary and preferred phosphorylation site. Phospho-deficient Env7 mutants were defective in negative regulation of membrane fusion, increasing the number of prominent vacuoles, whereas a phosphomimetic substitution at Ser-331 increased the number of fragmented vacuoles. Bioinformatics approaches confirmed that Env7 Ser-331 is within a motif that is highly conserved in STK16-related kinases and that it also anchors an SXXS CKI phosphorylation motif (328SRFS331). This study represents the first report on the regulatory mechanism of an STK16-related kinase. It also points to regulation of vacuolar membrane dynamics via a novel Yck3-Env7 kinase cascade.

Yeast ENV9 encodes a conserved lipid droplet (LD) short-chain dehydrogenase involved in LD morphology.

Lipid droplets (LDs) have emerged as dynamic and interactive organelles with important roles in lipid metabolism and membrane biogenesis. Here, we report that Saccharomyces cerevisiae Env9 is a novel conserved oxidoreductase involved in LD morphology. Microscopic and biochemical studies confirm localization of tagged Env9 to LDs and implicate its C-terminal hydrophobic domain (aa241-265) in its membrane association and stability. Confocal studies reveal a role for Env9 in LD morphology. Env9 positively affects both formation of large LDs upon overexpression and LD proliferation under poor carbon source. In silico bioinformatic and modeling approaches establish that ENV9 is a widely conserved member of the short-chain dehydrogenase (SDR) superfamily. Bayesian phylogenetic studies strongly support ENV9 as an ortholog of human SDR retinol dehydrogenase 12 (RDH12). Dehydrogenase activity of Env9 was confirmed by in vitro oxidoreductase assays. RDH12 mutations have been linked to Leber Congenital Amaurosis. Similar site-directed point mutations in the predicted Env9 oxidoreductase active site (N146L) or cofactor-binding site (G23-24A) abolished its reductase activity in vitro, consistent with those reported in other retinol dehydrogenases. The same residues were essential for affecting LD size and number in vivo. Taken together, our results implicate oxidoreductase activity of Env9 in its cellular role in LD morphology.

Hygromycin B hypersensitive (hhy) mutants implicate an intact trans-Golgi and late endosome interface in efficient Tor1 vacuolar localization and TORC1 function.

Saccharomyces cerevisiae vacuoles are functionally analogous to mammalian lysosomes. Both also serve as physical platforms for Tor Complex 1 (TORC1) signal transduction, the master regulator of cellular growth and proliferation. Hygromycin B is a eukaryotic translation inhibitor. We recently reported on hygromycin B hypersensitive (hhy) mutants that fail to grow at subtranslation inhibitory concentrations of the drug and exhibit vacuolar defects (Banuelos et al. in Curr Genet 56:121-137, 2010). Here, we show that hhy phenotype is not due to increased sensitivity to translation inhibition and establish a super HHY (s-HHY) subgroup of genes comprised of ARF1, CHC1, DRS2, SAC1, VPS1, VPS34, VPS45, VPS52, and VPS54 that function exclusively or inclusively at trans-Golgi and late endosome interface. Live cell imaging of s-hhy mutants revealed that hygromycin B treatment disrupts vacuolar morphology and the localization of late endosome marker Pep12, but not that of late endosome-independent vacuolar SNARE Vam3. This, along with normal post-late endosome trafficking of the vital dye FM4-64, establishes that severe hypersensitivity to hygromycin B correlates specifically with compromised trans-Golgi and late endosome interface. We also show that Tor1p vacuolar localization and TORC1 anabolic functions, including growth promotion and phosphorylation of its direct substrate Sch9, are compromised in s-hhy mutants. Thus, an intact trans-Golgi and late endosome interface is a requisite for efficient Tor1 vacuolar localization and TORC1 function.

Distinct palmitoylation events at the amino-terminal conserved cysteines of Env7 direct its stability, localization, and vacuolar fusion regulation in S. cerevisiae.

Palmitoylation at cysteine residues is the only known reversible form of lipidation and has been implicated in protein membrane association as well as function. Many palmitoylated proteins have regulatory roles in dynamic cellular processes, including membrane fusion. Recently, we identified Env7 as a conserved and palmitoylated protein kinase involved in negative regulation of membrane fusion at the lysosomal vacuole. Env7 contains a palmitoylation consensus sequence, and substitution of its three consecutive cysteines (Cys(13)-Cys(15)) results in a non-palmitoylated and cytoplasmic Env7. In this study, we further dissect and define the role(s) of individual cysteines of the consensus sequence in various properties of Env7 in vivo. Our results indicate that more than one of the cysteines serve as palmitoylation substrates, and any pairwise combination is essential and sufficient for near wild type levels of Env7 palmitoylation, membrane localization, and phosphorylation. Furthermore, individually, each cysteine can serve as a minimum requirement for distinct aspects of Env7 behavior and function in cells. Cys(13) is sufficient for membrane association, Cys(15) is essential for the fusion regulatory function of membrane-bound Env7, and Cys(14) and Cys(15) are redundantly essential for protection of membrane-bound Env7 from proteasomal degradation. A role for Cys(14) and Cys(15) in correct sorting at the membrane is also discussed. Thus, palmitoylation at the N-terminal cysteines of Env7 directs not only its membrane association but also its stability, phosphorylation, and cellular function.

ENV7 and YCK3, which encode vacuolar membrane protein kinases, genetically interact to impact cell fitness and vacuole morphology.

Saccharomyces cerevisiae vacuoles serve as a model for membrane fusion and fission. Yck3, a vacuolar membrane kinase, has been implicated in regulation of vacuole fusion. Recently, we established Env7 as another vacuolar membrane protein kinase with similar but nonredundant function to Yck3. Here, we report that native Env7 localizes to the vacuole independent of Yck3, where as its phosphorylation is YCK3 dependent. We also show that env7Δyck3Δ double mutant exhibits severely compromised fitness, altered cell size and bud vacuoles, and F-class vacuolar morphology. Our results establish negative genetic interactions between ENV7 and YCK3 and suggest cooperative roles for the two conserved genes in regulation of membrane dynamics. Such genetic buffering supports a critical role for membrane flux in global cell fitness.

Saccharomyces cerevisiae Env7 is a novel serine/threonine kinase 16-related protein kinase and negatively regulates organelle fusion at the lysosomal vacuole.

Membrane fusion depends on conserved components and is responsible for organelle biogenesis and vesicular trafficking. Yeast vacuoles are dynamic structures analogous to mammalian lysosomes. We report here that yeast Env7 is a novel palmitoylated protein kinase ortholog that negatively regulates vacuolar membrane fusion. Microscopic and biochemical studies confirmed the localization of tagged Env7 at the vacuolar membrane and implicated membrane association via the palmitoylation of its N-terminal Cys13 to -15. In vitro kinase assays established Env7 as a protein kinase. Site-directed mutagenesis of the Env7 alanine-proline-glutamic acid (APE) motif Glu269 to alanine results in an unstable kinase-dead allele that is stabilized and redistributed to the detergent-resistant fraction by interruption of the proteasome system in vivo. Palmitoylation-deficient Env7C13-15S is also kinase dead and mislocalizes to the cytoplasm. Microscopy studies established that env7Δ is defective in maintaining fragmented vacuoles during hyperosmotic response and in buds. ENV7 function is not redundant with a similar role of vacuolar membrane kinase Yck3, as the two do not share a substrate, and ENV7 is not a suppressor of yck3Δ. Bayesian phylogenetic analyses strongly support ENV7 as an ortholog of the gene encoding human STK16, a Golgi apparatus protein kinase with undefined function. We propose that Env7 function in fusion/fission dynamics may be conserved within the endomembrane system.

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.

env1 Mutant of VPS35 gene exhibits unique protein localization and processing phenotype at Golgi and lysosomal vacuole in Saccharomyces cerevisiae.

The yeast vacuole is functionally and structurally equivalent to the mammalian lysosome. Delivery of resident and cargo proteins to the lysosome is vital for proper cellular operations, and failure to correctly target proteins to the organelle is correlated with the development of neurodegenerative and lysosomal storage diseases. We previously reported a novel mutant screen for vacuolar trafficking defects in yeast Saccharomyces cerevisiae that resulted in the isolation of env1, an allelic mutant of VPS35. As a member of the retromer complex, Vps35p binds directly to cargos and facilitates their retrograde transport to trans Golgi from endosomes. Our previous studies established that env1 exhibits unique pleiotropic phenotype in comparison to other tested VPS35 alleles including severe growth sensitivity to hygromycin B and internal accumulation of the precursor form of the vacuolar enzyme carboxypeptidase Y. Here, through a combination of sub-cellular fractionation and indirect immunofluorescence microscopy, we confirm and extend the unique phenotype of env1 to processing and localization of additional proteins within the vacuolar trafficking pathway. In comparative studies with a null and an allelic mutant of VPS35, env1 exhibited unique processing defects of retromer-independent vacuolar membrane enzyme alkaline phosphatase at the vacuole and significant Golgi localization of retromer cargos Vps10p and Kex2p despite compromised trafficking at the Golgi and late endosome interface.

Genomic analysis of severe hypersensitivity to hygromycin B reveals linkage to vacuolar defects and new vacuolar gene functions in Saccharomyces cerevisiae.

The vacuole of Saccharomyces cerevisiae has been a seminal model for studies of lysosomal trafficking, biogenesis, and function. Several yeast mutants defective in such vacuolar events have been unable to grow at low levels of hygromycin B, an aminoglycoside antibiotic. We hypothesized that such severe hypersensitivity to hygromycin B (hhy) is linked to vacuolar defects and performed a genomic screen for the phenotype using a haploid deletion strain library of non-essential genes. Fourteen HHY genes were initially identified and were subjected to bioinformatics analyses. The uncovered hhy mutants were experimentally characterized with respect to vesicular trafficking, vacuole morphology, and growth under various stress and drug conditions. The combination of bioinformatics analyses and phenotypic characterizations implicate defects in vesicular trafficking, vacuole fusion/fission, or vacuole function in all hhy mutants. The collection was enriched for sensitivity to monensin, indicative of vacuolar trafficking defects. Additionally, all hhy mutants showed severe sensitivities to rapamycin and caffeine, suggestive of TOR kinase pathway defects. Our experimental results also establish a new role in vacuolar and vesicular functions for two genes: PAF1, encoding a RNAP II-associated protein required for expression of cell cycle-regulated genes, and TPD3, encoding the regulatory subunit of protein phosphatase 2A. Thus, our results support linkage between severe hypersensitivity to hygromycin B and vacuolar defects.

A novel immunodetection screen for vacuolar defects identifies a unique allele of VPS35 in S. cerevisiae.

The late endosome and vacuole of yeast Saccharomyces cerevisiae are functionally equivalent to the mammalian late endosome and lysosome. The late endosome is the convergence point of the biosynthetic and endocytic trafficking to the vacuole. Here, we describe a novel immunodetection screen to isolate mutants defective in trafficking the soluble hydrolase carboxypeptidase Y (CPY) at the late endosome to vacuole interface (env mutants). Mutants exhibit vacuolar morphology and endocytosis defects as assayed by electron, fluorescent, and nomarski microscopy. In biochemical assays, they internally accumulate p2CPY in a dense membrane compartment lacking vacuolar properties yet display normal secretion phenotypes. The results suggest vacuolar morphology and function defects that are exclusively at the late endosome/vacuole interface. env mutants define five complementation groups. The first gene of the collection to be cloned, ENV1 is allelic to VPS35 whose established function is in retrograde trafficking from late endosome to trans-Golgi network (TGN). Microscopic, biochemical, and growth analyses establish that env1 is distinct from other alleles of VPS35 in vacuolar morphology, growth characteristics, and internal accumulation of p2CPY. Our results indicate that ENV genes may define new gene functions at the late endosome to vacuole interface.

Cys254 and Cys49/Cys87of simian virus 40 Vp1 are essential in formation of infectious virions.

The SV40 capsid is composed of pentameric capsomeres of Vp1. We have previously shown that disulfide linkages at Vp1 Cys9, Cys104, and Cys207 are essential in formation of infectious virions. Here, the role of the remaining four cysteines was explored. Single, double, and quadruple cys --> ser mutant genomes at Vp1 Cys49, Cys87, Cys254, and Cys267 codons were generated and transfected into CV-1 cells. The quadruple mutant Vp1 continued to localize to the nucleus and to bind DNA, but resulted in no plaques. SV40Vp1.Cys254 was the only single mutant with complete defect in plaque formation. The double mutant at Vp1.Cys49.Cys87 showed complete defect in plaque formation, while single mutants at the two residues resulted in plaques, suggesting a cumulative effect. All mutants defective in plaque formation continued to localize viral proteins in the nucleus. Taken together, our results suggest that Cys254 and the Cys49/Cys87 combination are essential in late stages of infectious virion formation.

The simian virus 40 minor structural protein Vp3, but not Vp2, is essential for infectious virion formation.

The SV40 capsid is composed of pentameric capsomeres of the major structural protein Vp1. The two minor structural proteins, Vp2 and Vp3, interact with the capsid. Here, the roles of Vp2 and Vp3 were explored during the course of SV40 infection. Start codons of Vp2, Vp3, or both Vp2 and Vp3, were destroyed by site-directed mutagenesis, and mutant genomes were transfected into CV-1 cells. SV40DeltaVp2 produced plaques and infectious virion particles with titres indistinguishable from wild-type. SV40DeltaVp3 and SV40 DeltaVp2/Vp3 were defective in plaque formation and rendered no infectious particles. All three mutants showed normal nuclear localization of T-Ag and Vp1; they also showed packaging of SV40 DNA by nuclease digestion assays. Thus, Vp3 is essential for formation of infectious SV40 particles, whereas Vp2 is not. One critical role of full-length Vp3 appears to be in virus-cell interactions at post-packaging steps of a permissive infection.

Cys(9), Cys(104) and Cys(207) of simian virus 40 Vp1 are essential for infectious virion formation in CV-1 cells.

Structural studies have implicated Cys(9), Cys(104) and Cys(207) of simian virus 40 (SV40) Vp1 in disulfide bond formation. Recently, we have shown the three cysteines to be essential for disulfide linkage of Vp1 complexes in vitro. Here, the role of the three cysteines was explored during the course of SV40 infection. Single-, double- and triple-mutant Vp1 at Cys(9), Cys(104) and Cys(207) continued to localize to the nuclei of transfected CV-1 cells and to bind DNA, but showed a range of abilities to form plaques. Only mutants containing the Cys(9)-->Ser change showed defects in plaque formation. Single mutants at Cys(9) formed small plaques; mutants at Cys(9). Cys(104), Cys(9). Cys(207) and Cys(9). Cys(104). Cys(207) formed no plaques. All three isolated revertants contained back-mutations at the Vp1 Cys(9) codon. These results further confirm the involvement of the three Vp1 cysteines in protein-protein interactions during virus assembly. Cys(9) is critical for production of wild-type infectious virions, whereas Cys(104) and Cys(207) play secondary roles.

Cys9, Cys104 and Cys207 of simian virus 40 Vp1 are essential for inter-pentamer disulfide-linkage and stabilization in cell-free lysates.

Previous studies have implicated disulfide bonds between Vp1 molecules in the stabilization of the simian virus 40 (SV40) capsid. To identify the cysteine residues involved in intermolecular disulfide interactions, systematic oligo-directed mutagenesis of cysteine codons to serine codons was initiated. Wild-type and mutant Vp1 proteins were produced in rabbit reticulocyte lysates and were allowed to interact post-translationally. Disulfide-linked Vp1 complexes were assessed via non-reducing SDS-PAGE and via sucrose-gradient sedimentation. Wild-type Vp1 forms 7S pentamers followed by 12S disulfide-linked multi-pentameric complexes in cell-free lysates. Mutagenesis of all seven cysteine codons abolished Vp1 12S complexes, but did not affect pentamer formation. A quadruple Vp1 mutant at Cys49, Cys87, Cys254 and Cys267 continued to form 12S complexes, whereas the major products of the Cys9, Cys104 and Cys207 triple mutant Vp1 were 7S pentamers. Single and double mutant Vp1 proteins at the three cysteines affected continued to form 12S complexes, but to a lesser extent. Thus, inter-pentamer disulfide bonds at Cys9, Cys104 and Cys207 are essential and sufficient for stabilization of Vp1 complexes in cell-free lysates. These results are in agreement with previous structural studies of SV40 that implicated the same three residues in disulfide linkage in the capsid. Possible parameters for the involvement of the three cysteines are discussed.