RESUMEN
Mass spectrometry, an analytical technique that measures the mass-to-charge ratio of ionized atoms or molecules, dates back more than 100 years, and has both qualitative and quantitative uses for determining chemical and structural information. Quantitative proteomic mass spectrometry on biological samples focuses on identifying the proteins present in the samples, and establishing the relative abundances of those proteins. Such protein inventories create the opportunity to discover novel biomarkers and disease targets. We have previously introduced a normalized, label-free method for quantification of protein abundances under a shotgun proteomics platform (Griffin et al., 2010). The introduction of this method for quantifying and comparing protein levels leads naturally to the issue of modeling protein abundances in individual samples. We here report that protein abundance levels from two recent proteomics experiments conducted by the authors can be adequately represented by Sichel distributions. Mathematically, Sichel distributions are mixtures of Poisson distributions with a rather complex mixing distribution, and have been previously and successfully applied to linguistics and species abundance data. The Sichel model can provide a direct measure of the heterogeneity of protein abundances, and can reveal protein abundance differences that simpler models fail to show.
RESUMEN
Proteolytic activation of membrane-bound transcription factors has emerged as an important mechanism for the regulation of gene expression. Two membrane-bound transcription factors regulated in this manner are the Saccharomyces cerevisiae proteins Mga2p and Spt23p, which direct transcription of the Delta9-fatty acid desaturase gene OLE1. We now show that a membrane-associated complex containing the highly conserved Npl4p, Ufd1p, and Cdc48p proteins mediates the proteasome-regulated cleavage of Mga2p and Spt23p. Mutations in NPL4, UFD1, and CDC48 cause a block in Mga2p and Spt23p processing, with concomitant loss of OLE1 expression. Taken together, our data indicate that the Npl4 complex may serve to target the proteasome to the ubiquitinated endoplasmic reticulum membrane-bound proteins Mga2p and Spt23p. Given the recent finding that NPL4 is allelic to the ERAD gene HRD4, we further propose that this NPL4 function extends to all endoplasmic reticulum-membrane-associated targets of the proteasome.
Asunto(s)
Secuencia Conservada/genética , Cisteína Endopeptidasas/metabolismo , Complejos Multienzimáticos/metabolismo , Proteínas de Complejo Poro Nuclear , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Transactivadores , Factores de Transcripción/genética , Proteínas Adaptadoras del Transporte Vesicular , Adenosina Trifosfatasas , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans , Proteínas de Ciclo Celular/metabolismo , Secuencia Conservada/fisiología , Proteínas de Unión al ADN/genética , Drosophila , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Ácido Graso Desaturasas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica/genética , Humanos , Membranas Intracelulares , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Mutación/genética , Membrana Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático , Complejo de la Endopetidasa Proteasomal , Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , Saccharomyces cerevisiae , Estearoil-CoA Desaturasa , Factores de Transcripción/metabolismo , Proteína que Contiene Valosina , LevadurasRESUMEN
Membrane fusion relies on complex protein machineries, which act in sequence to catalyze the fusion of bilayers. The fusion of endoplasmic reticulum membranes requires the t-SNARE Ufe1p, and the AAA ATPase p97/Cdc48p. While the mechanisms of membrane fusion events have begun to emerge, little is known about how this fusion process is regulated. We provide first evidence that endoplasmic reticulum membrane fusion in yeast is regulated by the action of protein kinase C. Specifically, Pkc1p kinase activity is needed to protect the fusion machinery from ubiquitin-mediated degradation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas Fúngicas/metabolismo , Fusión de Membrana/fisiología , Proteína Quinasa C/metabolismo , Proteínas de Saccharomyces cerevisiae , Enzimas Ubiquitina-Conjugadoras , Adenosina Trifosfatasas , Ligasas/metabolismo , Mutación/genética , Mutación/fisiología , Orgánulos/metabolismo , Proteína Quinasa C/genética , Proteínas Qa-SNARE , Proteína que Contiene Valosina , LevadurasRESUMEN
Cdc15p is an essential protein kinase and functions with a group of late mitotic proteins that includes Lte1p, Tem1p, Cdc14p and Dbf2p/Dbf20p to inactivate Cdc28p-Clb2p at the end of mitosis in budding yeast [1] [2]. Cdc14p is activated and released from the nucleolus at late anaphase/telophase to dephosphorylate important regulators of Cdc28p-Clb2p such as Hct1p/Cdh1p, Sic1p and Swi5p in a CDC15-dependent manner [3] [4] [5] [6] [7]. How Cdc15p itself is regulated is not known. Here, we report that both the phosphorylation and localization of Cdc15p are cell cycle regulated. The extent of phosphorylation of Cdc15p gradually increases during cell-cycle progression until some point during late anaphase/telophase when it is rapidly dephosphorylated. We provide evidence suggesting that Cdc14p is the phosphatase responsible for the dephosphorylation of Cdc15p. Using a Cdc15p fusion protein coupled at its carboxyl terminus to green fluorescent protein (GFP), we found that Cdc15p, like its homologue Cdc7p [8] in fission yeast, localizes to the spindle pole bodies (SPBs) during mitosis. At the end of telophase, a portion of Cdc15p is located at the mother-bud neck, suggesting a possible role for Cdc15p in cytokinesis.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Tirosina Fosfatasas , Proteínas de Saccharomyces cerevisiae , Huso Acromático/metabolismo , Ciclo Celular , Mitosis/fisiología , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
The endoplasmic reticulum (ER) is a dynamic organelle central to many essential cellular functions. It is an important calcium store, which functions in cellular signal transduction cascades. It is also the site of entry for secreted proteins into the secretory pathway. Lumenal enzymes will fold and glycosylate these proteins, and if a protein is destined to be secreted, it will be packaged into membrane vesicles that bud off from the ER. The ER is also the site where most cellular lipids are synthesized. It is contiguous with the nuclear envelope, which serves as a diffusion barrier to control entry into and out of the nucleus. In the life cycle of a cell, the ER is in a constant flux of membrane traffic. What maintains the ER in the shape of an intact reticulum among this constant flux of material? We discuss the mechanisms that contribute to the biogenesis of the ER, the maintenance of the organelle, as well as processes that give the ER its characteristic shape and pattern of inheritance.
Asunto(s)
Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Animales , Diferenciación Celular/fisiología , División Celular/fisiología , HumanosRESUMEN
AAA ATPases play central roles in cellular activities. The ATPase p97, a prototype of this superfamily, participates in organelle membrane fusion. Cryoelectron microscopy and single-particle analysis revealed that a major conformational change of p97 during the ATPase cycle occurred upon nucleotide binding and not during hydrolysis as previously hypothesized. Furthermore, our study indicates that six p47 adaptor molecules bind to the periphery of the ring-shaped p97 hexamer. Taken together, these results provide a revised model of how this and possibly other AAA ATPases can translate nucleotide binding into conformational changes of associated binding partners.
Asunto(s)
Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas de Transporte Vesicular , Adenosina Difosfato/metabolismo , Adenosina Trifosfatasas/ultraestructura , Adenilil Imidodifosfato/metabolismo , Animales , Proteínas Portadoras/química , Proteínas Portadoras/ultraestructura , Bovinos , Microscopía por Crioelectrón , Procesamiento de Imagen Asistido por Computador , Proteínas de la Membrana/metabolismo , Ratones , Modelos Moleculares , Proteínas Sensibles a N-Etilmaleimida , Proteínas Nucleares/ultraestructura , Unión Proteica , Conformación Proteica , Proteínas SNARERESUMEN
During mating of Saccharomyces cerevisiae, two nuclei fuse to produce a single diploid nucleus. Two genes, KAR7 and KAR8, were previously identified by mutations that cause defects in nuclear membrane fusion. KAR7 is allelic to SEC71, a gene involved in protein translocation into the endoplasmic reticulum. Two other translocation mutants, sec63-1 and sec72Delta, also exhibited moderate karyogamy defects. Membranes from kar7/sec71Delta and sec72Delta, but not sec63-1, exhibited reduced membrane fusion in vitro, but only at elevated temperatures. Genetic interactions between kar7 and kar5 mutations were suggestive of protein-protein interactions. Moreover, in sec71 mutants, Kar5p was absent from the SPB and was not detected by Western blot or immunoprecipitation of pulse-labeled protein. KAR8 is allelic to JEMI, encoding an endoplasmic reticulum resident DnaJ protein required for nuclear fusion. Overexpression of KAR8/JEM1 (but not SEC63) strongly suppressed the mating defect of kar2-1, suggesting that Kar2p interacts with Kar8/Jem1p for nuclear fusion. Electron microscopy analysis of kar8 mutant zygotes revealed a nuclear fusion defect different from kar2, kar5, and kar7/sec71 mutants. Analysis of double mutants suggested that Kar5p acts before Kar8/Jem1p. We propose the existence of a nuclear envelope fusion chaperone complex in which Kar2p, Kar5p, and Kar8/Jem1p are key components and Sec71p and Sec72p play auxiliary roles.
Asunto(s)
Núcleo Celular/genética , Proteínas Fúngicas/genética , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Alelos , Transporte Biológico , Retículo Endoplásmico/metabolismo , Proteínas Fúngicas/metabolismo , Dosificación de Gen , Regulación Fúngica de la Expresión Génica , Proteínas del Choque Térmico HSP40 , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Fusión de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Microscopía Electrónica , Chaperonas Moleculares , Mutación , Membrana Nuclear/genética , Proteínas Nucleares/metabolismo , Canales de Translocación SEC , Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/ultraestructura , Supresión GenéticaRESUMEN
A new family of related ATPases has emerged, characterized by a highly conserved AAA motif. This motif forms a 230-amino-acid domain that contains Walker homology sequences and imparts ATPase activity. Homology between AAA-family members is confined mostly to the AAA domain, although additional homology outside the AAA motif is present among closely related proteins. AAA proteins act in a variety of cellular functions, including cell-cycle regulation, protein degradation, organelle biogenesis and vesicle-mediated protein transport. The AAA domain is required for protein function, but its exact role and the specific activity that it confers on AAA proteins is still unclear. This review describes current understanding of the AAA protein family.
Asunto(s)
Adenosina Trifosfatasas , Células Eucariotas/enzimología , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Ciclo Celular/fisiología , Células Eucariotas/citología , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
The fusion of endoplasmic reticulum (ER) membranes in yeast does not require Sec18p/NSF and Sec17p, two proteins needed for docking of vesicles with their target membrane. Instead, ER membranes require a NSF-related ATPase, Cdc48p. Since both vesicular and organelle fusion events use related ATPases, we investigated whether both fusion events are also SNARE mediated. We present evidence that the fusion of ER membranes requires Ufe1p, a t-SNARE that localizes to the ER, but no known v-SNAREs. We propose that the Ufe1 protein acts in the dual capacity of an organelle membrane fusion-associated SNARE by undergoing direct t-t-SNARE and Cdc48p interactions during organelle membrane fusion as well as a t-SNARE for vesicular traffic.
Asunto(s)
Proteínas Portadoras , Retículo Endoplásmico/metabolismo , Proteínas Fúngicas/fisiología , Glicoproteínas , Fusión de Membrana/fisiología , Proteínas de Saccharomyces cerevisiae , Proteínas de Transporte Vesicular , Adenosina Trifosfatasas , Adenosina Trifosfato/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Membranas Intracelulares , Glicoproteínas de Membrana/fisiología , Proteínas de la Membrana/análisis , Proteínas de la Membrana/fisiología , Microsomas/metabolismo , Mutación , Fragmentos de Péptidos , Proteínas Qa-SNARE , Proteínas Qb-SNARE , Proteínas Recombinantes de Fusión , Proteínas SNARE , Proteína que Contiene Valosina , Levaduras/citologíaRESUMEN
The fusion of endoplasmic reticulum (ER) membranes in yeast is an essential process required for normal progression of the nuclear cell cycle, karyogamy, and the maintenance of an intact organellar compartment. We showed previously that this process requires a novel fusion machinery distinct from the classic membrane docking/fusion machinery containing Sec17p (alpha-SNAP) and Sec18p (NSF). Here we show that Cdc48p, a cell-cycle protein with homology to Sec18p, is required in ER fusion. A temperature-sensitive cdc48 mutant is conditionally defective in ER fusion in vitro. Addition of purified Cdc48p restores the fusion of isolated cdc48 mutant ER membranes. We propose that Cdc48p is part of an evolutionarily conserved fusion/docking machinery involved in multiple homotypic fusion events.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Ciclo Celular/fisiología , Retículo Endoplásmico/fisiología , Proteínas Fúngicas/fisiología , Fusión de Membrana/fisiología , Adenosina Trifosfatasas , Animales , Especificidad de Anticuerpos , Proteínas de Ciclo Celular/inmunología , Citosol/fisiología , Retículo Endoplásmico/ultraestructura , Membranas Intracelulares/fisiología , Conejos , Proteína que Contiene Valosina , Levaduras/citologíaRESUMEN
We have isolated vesicles that mediate protein transport from the ER to Golgi membranes in perforated yeast. These vesicles, which form de novo during in vitro incubations, carry lumenal and membrane proteins that include core-glycosylated pro-alpha-factor, Bet1, Sec22, and Bos1, but not ER-resident Kar2 or Sec61 proteins. Thus, lumenal and membrane proteins in the ER are sorted prior to transport vesicle scission. Inhibition of Ypt1p-function, which prevents newly formed vesicles from docking to cis-Golgi membranes, was used to block transport. Vesicles that accumulate are competent for fusion with cis-Golgi membranes, but not with ER membranes, and thus are functionally committed to vectorial transport. A 900-fold enrichment was developed using differential centrifugation and a series of velocity and equilibrium density gradients. Electron microscopic analysis shows a uniform population of 60 nm vesicles that lack peripheral protein coats. Quantitative Western blot analysis indicates that protein markers of cytosol and cellular membranes are depleted throughout the purification, whereas the synaptobrevin-like Bet1, Sec22, and Bos1 proteins are highly enriched. Uncoated ER-derived transport vesicles (ERV) contain twelve major proteins that associate tightly with the membrane. The ERV proteins may represent abundant cargo and additional targeting molecules.
Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas Fúngicas/metabolismo , Aparato de Golgi/metabolismo , Transporte Biológico , Compartimento Celular , Técnicas In Vitro , Membranas Intracelulares/metabolismo , Fusión de Membrana , Glicoproteínas de Membrana/metabolismo , Saccharomyces cerevisiaeRESUMEN
Karyogamy is the process where haploid nuclei fuse to form a diploid nucleus during yeast mating. We devised a novel genetic screen that identified five new karyogamy (KAR) genes and three new cell fusion (FUS) genes. The kar mutants fell into two classes that represent distinct events in the yeast karyogamy pathway. Class I mutations blocked congression of the nuclei due to cytoplasmic microtubule defects. In Class II mutants, nuclear congression proceeded and the membranes of apposed nuclei were closely aligned but unfused. In vitro, Class II mutant membranes were defective in a homotypic ER/nuclear membrane fusion assay. We propose that Class II mutants define components of a novel membrane fusion complex which functions during vegetative growth and is recruited for karyogamy.
Asunto(s)
Núcleo Celular/fisiología , Genes Fúngicos , Fusión de Membrana , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Núcleo Celular/ultraestructura , Cruzamientos Genéticos , Diploidia , Técnica del Anticuerpo Fluorescente , Prueba de Complementación Genética , Genotipo , Mutagénesis , Mutagénesis Insercional , Membrana Nuclear/fisiología , Membrana Nuclear/ultraestructura , Saccharomyces cerevisiae/ultraestructuraRESUMEN
We have developed assays using cells and isolated membranes to identify factors mediating fusion of the ER-nuclear membrane network in yeast. When cells containing distinctly tagged ER-nuclear envelope membranes are observed during mating, the markers of both parental membranes become colocalized in a process sharing a genetic requirement with karyogamy. Using isolated membranes, we find that fusion between ER compartments requires ATP, but not cytosol, Sec17p (alpha-SNAP), or Sec18p (NSF), the latter two being required at the fusion step in vesicular transport. Proteins tightly associated with the ER membrane are essential for fusion, as is Kar2p (BiP), an ER lumenal hsp70 homolog. BiP may activate an ER-localized fusogen, allowing nuclear fusion and karyogamy in yeast.
Asunto(s)
Adenosina Trifosfatasas , Retículo Endoplásmico/metabolismo , Proteínas Fúngicas/metabolismo , Genes Fúngicos/fisiología , Proteínas HSP70 de Choque Térmico , Proteínas de Choque Térmico/metabolismo , Fusión de Membrana/fisiología , Membrana Nuclear/metabolismo , Proteína Disulfuro Isomerasas , Proteínas de Saccharomyces cerevisiae , Proteínas de Transporte Vesicular , Adenosina Trifosfato/metabolismo , Bioensayo/métodos , Biomarcadores , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cruzamientos Genéticos , Citosol/metabolismo , Proteínas Fúngicas/genética , Eliminación de Gen , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Precursores de Proteínas/metabolismo , Canales de Translocación SEC , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida , Levaduras/citología , Levaduras/genética , Levaduras/metabolismo , Cigoto/fisiologíaRESUMEN
Osmoadaptation in S. cerevisiae occurs through intracellular accumulation of glycerol in response to an increase in osmolarity of the surrounding environment. Analysis of ssv1-2, a strain carrying a mutation in a gene required for vacuole biogenesis, protein-sorting and osmohomeostasis, shows that the strain is terminally inactivated by 1.5 M NaCl within 10 seconds while the isogenic wild type maintains slow growth and accumulates glycerol within 18 hours. This study provides the first evidence that the vacuole participates in an immediate osmoregulatory process permitting survival until the osmoadaptive glycerol accumulation allows growth under osmotically unfavorable conditions.
Asunto(s)
Saccharomyces cerevisiae/fisiología , Espectroscopía de Resonancia Magnética , Saccharomyces cerevisiae/ultraestructura , Cloruro de Sodio/toxicidad , Vacuolas/fisiología , Equilibrio HidroelectrolíticoRESUMEN
The yeast vacuole plays an important role in nitrogen metabolism, storage and intracellular macromolecular degradation. Evidence suggests that it is also involved in osmohomeostasis of the cell. We have taken a mutational approach for the analysis of vacuolar function and biogenesis by the isolation of 97 mutants unable to grow if high concentrations of salt are present in the medium. Phenotypic analysis was able to demonstrate that apart from osmosensitivity the mutations also conferred other properties such as altered vacuolar morphology and secretion of the vacuolar enzymes carboxypeptidase Y, proteinase A, proteinase B and alpha-mannosidase. The mutants fall into at least 17 complementation groups, termed ssv for salt-sensitive vacuolar mutants, of which two are identical to complementation groups isolated by others. We conclude that in Saccharomyces cerevisiae correct vacuolar biogenesis and protein targeting is required for osmotolerance as well as other important cellular processes.