RESUMEN

Endocytosis is involved in a wide variety of cellular processes, and the internalization step of endocytosis has been extensively studied in both lower and higher eukaryotic cells. Studies in mammalian cells have described several endocytic pathways, with the main emphasis on clathrin-dependent endocytosis. Genetic studies in yeast have underlined the critical role of actin and actin-binding proteins, lipid modification, and the ubiquitin conjugation system. The combined results of studies of endocytosis in higher and lower eukaryotic cells reveal an interesting interplay in the two systems, including a crucial role for ubiquitin-associated events. The ubiquitylation of yeast cell-surface proteins clearly acts as a signal triggering their internalization. Mammalian cells display variations on the common theme of ubiquitin-linked endocytosis, according to the cell-surface protein considered. Many plasma membrane channels, transporters and receptors undergo cell-surface ubiquitylation, required for the internalization or later endocytic steps of some cell-surface proteins, whereas for others, internalization involves interaction with the ubiquitin conjugation system or with ancillary proteins, which are themselves ubiquitylated. Epsins and Eps15 (or Eps15 homologs), are commonly involved in the process of endocytosis in all eukaryotes, their critical role in this process stemming from their capacity to bind ubiquitin, and to undergo ubiquitylation.

Asunto(s)

Endocitosis/fisiología , Ubiquitina/fisiología , Levaduras/fisiología , Animales , Membrana Celular/fisiología , Clatrina/fisiología , Complejos de Clasificación Endosomal Requeridos para el Transporte , Proteínas de la Membrana/fisiología , Estructura Secundaria de Proteína , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-cbl , Proteínas de Saccharomyces cerevisiae/fisiología , Complejos de Ubiquitina-Proteína Ligasa/fisiología , Ubiquitina-Proteína Ligasas/fisiología , Levaduras/enzimología

RESUMEN

The nuclear transport of both proteins and RNAs has attracted considerable interest in recent years. However, regulation pathways of the nuclear transport machineries are still not well characterized. Previous studies indicated that ubiquitination is involved in poly(A)+ RNA nuclear export. For this reason, we systematically investigated ubiquitin-protein ligasess from the homologous to E6-AP carboxy terminus (HECT) family for potential individual roles in nuclear transport in Saccharomyces cerevisiae. Here we report that Rsp5, an essential yeast ubiquitin ligase involved in many cellular functions, when deleted or mutated in ligase activity, blocks the nuclear export of mRNAs. Affected messenger RNAs include both total poly(A)+ mRNA and heat-shock mRNAs. Mutation of Rsp5 does not affect nuclear protein import or export. Deletion of RSP5 blocks mRNA export, even under conditions where its essential role in unsaturated fatty acids biosynthesis is bypassed. Using domain mapping, we find that the ligase activity is required for proper mRNA export, indicating that ubiquitination by Rsp5 acts directly or indirectly to affect RNA export. The finding that Rsp5p ligase mutations cause a more pronounced defect at high temperatures suggests that ubiquitination of transport factors by Rsp5p may also be essential during stress conditions.

Asunto(s)

Núcleo Celular/metabolismo , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte , Ácido Graso Desaturasas/metabolismo , Calor , Estructura Terciaria de Proteína , Estearoil-CoA Desaturasa , Factores de Tiempo

RESUMEN

In yeast, membrane proteins from the biosynthetic and endocytic pathways must be ubiquitylated for sorting to inward-budding vesicles in late endosomes, which give rise to multivesicular bodies. A conserved protein complex containing the yeast Vps23p or its mammalian counterpart Tsg101 may act as the ubiquitin receptor.

Asunto(s)

Proteínas Portadoras/metabolismo , Endosomas/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Ubiquitina/metabolismo , Proteínas de Transporte Vesicular , Animales , Transporte Biológico , Membrana Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte , Humanos , Transporte de Proteínas , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo

RESUMEN

The Fur4p uracil permease, like most yeast plasma membrane proteins, undergoes ubiquitin-dependent endocytosis and is then targeted to the vacuole (equivalent to the mammalian lysosome) for degradation. The cell surface ubiquitination of Fur4p is mediated by the essential Rsp5p ubiquitin ligase. Ubiquitination of Fur4p occurs on two target lysines, which receive two ubiquitin moieties linked through ubiquitin Lys63, a type of linkage (termed UbK63) different from that involved in proteasome recognition. We report that pep4 cells deficient for vacuolar protease activities accumulate vacuolar unubiquitinated Fur4p. In contrast, pep4 cells lacking the Doa4p ubiquitin isopeptidase accumulate ubiquitin-conjugated Fur4p. These data suggest that Fur4p undergoes Doa4p-dependent deubiquitination prior to vacuolar degradation. Compared to pep4 cells, pep4 doa4 cells have huge amounts of membrane-bound ubiquitin conjugates. This indicates that Doa4p plays a general role in the deubiquitination of membrane-bound proteins, as suggested by reports describing the suppression of some doa4 phenotypes in endocytosis and vacuolar protein sorting mutants. Some of the small ubiquitin-linked peptides that are a hallmark of Doa4 deficiency are not present in rsp5 mutant cells or after overproduction of a variant ubiquitin modified at Lys 63 (UbK63R). These data suggest that the corresponding peptides are degradation products of Rsp5p substrates and probably of ubiquitin conjugates carrying UbK63 linkages. Doa4p thus appears to be involved in the deubiquitination of endocytosed plasma membrane proteins, some of them carrying UbK63 linkages.

Asunto(s)

Endocitosis/fisiología , Endopeptidasas/fisiología , Proteínas Fúngicas/fisiología , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Nucleótidos , Proteínas de Saccharomyces cerevisiae , Complejos de Ubiquitina-Proteína Ligasa , Ubiquitinas/metabolismo , Proteínas de Transporte Vesicular , Proteínas Portadoras/metabolismo , Endopeptidasas/genética , Endopeptidasas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ligasas/metabolismo , Péptidos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ubiquitina Tiolesterasa , Ubiquitina-Proteína Ligasas , Vacuolas/metabolismo

RESUMEN

Skp1p-cullin-F-box protein (SCF) complexes are ubiquitin-ligases composed of a core complex including Skp1p, Cdc53p, Hrt1p, the E2 enzyme Cdc34p, and one of multiple F-box proteins which are thought to provide substrate specificity to the complex. Here we show that the F-box protein Rcy1p is required for recycling of the v-SNARE Snc1p in Saccharomyces cerevisiae. Rcy1p localized to areas of polarized growth, and this polarized localization required its CAAX box and an intact actin cytoskeleton. Rcy1p interacted with Skp1p in vivo in an F-box-dependent manner, and both deletion of its F box and loss of Skp1p function impaired recycling. In contrast, cells deficient in Cdc53p, Hrt1p, or Cdc34p did not exhibit recycling defects. Unlike the case for F-box proteins that are known to participate in SCF complexes, degradation of Rcy1p required neither its F box nor functional 26S proteasomes or other SCF core subunits. Importantly, Skp1p was the only major partner that copurified with Rcy1p. Our results thus suggest that a complex composed of Rcy1p and Skp1p but not other SCF components may play a direct role in recycling of internalized proteins.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Proteínas F-Box , Proteínas Fúngicas/metabolismo , Proteínas de la Membrana/metabolismo , Péptido Sintasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Sitios de Unión , Proteínas de Unión al ADN/genética , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas de la Membrana/genética , Proteínas R-SNARE , Proteínas Ligasas SKP Cullina F-box , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular

RESUMEN

The Saccharomyces cerevisiae RSP5 gene encodes an essential HECT E3 ubiquitin-protein ligase. Rsp5p contains an N-terminal C2 domain, three WW domains in the central portion of the molecule, and a C-terminal catalytic HECT domain. A diverse group of substrates of Rsp5p and vertebrate C2 WW-domain-containing HECT E3s have been identified, including both nuclear and membrane-associated proteins. We determined the intracellular localization of Rsp5p and the determinants necessary for localization, in order to better understand how Rsp5p activities are coordinated. Using both green fluorescent protein fusions to Rsp5p and immunogold electron microscopy, we found that Rsp5p was distributed in a punctate pattern at the plasma membrane, corresponding to membrane invaginations that are likely sites of endosome formation, as well as at perivacuolar sites. The latter appeared to correspond to endocytic intermediates, as these structures were not seen in a sla2/end4-1 mutant, and double-immunogold labeling demonstrated colocalization of Rsp5p with the endosomal markers Pep12p and Vps32p. The C2 domain was an important determinant of localization; however, mutations that disrupted HECT domain function also caused mislocalization of Rsp5p, indicating that enzymatic activity is linked to localization. Deletion of the C2 domain partially stabilized Fur4p, a protein previously shown to undergo Rsp5p- and ubiquitin-mediated endocytosis; however, Fur4p was still ubiquitinated at the plasma membrane when the C2 domain was deleted from the protein. Together, these results indicate that Rsp5p is located at multiple sites within the endocytic pathway and suggest that Rsp5p may function at multiple steps in the ubiquitin-mediated endocytosis pathway.

Asunto(s)

Endocitosis/fisiología , Ligasas/fisiología , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiología , Complejos de Ubiquitina-Proteína Ligasa , Complejos de Clasificación Endosomal Requeridos para el Transporte , Proteínas Fúngicas/fisiología , Proteínas Fluorescentes Verdes , Inmunohistoquímica , Proteínas Luminiscentes , Mutación , Transducción de Señal , Ubiquitina-Proteína Ligasas

RESUMEN

CCZ1 was previously identified by the sensitivity of ccz1(delta) mutants to high concentrations of Caffeine and the divalent ions Ca(2+ )and Zn(2+). In this paper we show that deletion of CCZ1 leads to aberrant vacuole morphology, similar to the one reported for the family of vacuolar protein sorting (vps) mutants of class B. The ccz1(&Dgr;) cells display severe vacuolar protein sorting defects for both the soluble carboxipeptidase Y and the membrane-bound alkaline phosphatase, which are delivered to the vacuole by distinct routes. Ccz1p is a membranous protein and the vast majority of Ccz1p resides in late endosomes. These results, along with a functional linkage found between the CCZ1 and YPT7 genes, indicate that the site of Ccz1p function is at the last step of fusion of multiple transport intermediates with the vacuole.

Asunto(s)

Proteínas de Transporte de Catión , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Intercambiadores de Sodio-Hidrógeno , Vacuolas/metabolismo , Proteínas de Transporte Vesicular , Proteínas de Unión al GTP rab/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Calcio/metabolismo , ATPasas Transportadoras de Calcio/genética , ATPasas Transportadoras de Calcio/metabolismo , Proteínas Portadoras/análisis , Proteínas Portadoras/metabolismo , Compartimento Celular/fisiología , Endocitosis/fisiología , Proteínas Fúngicas/análisis , Eliminación de Gen , Dosificación de Gen , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares , Mutagénesis/fisiología , Fenotipo , ATPasas Transportadoras de Calcio de la Membrana Plasmática , Plásmidos , Receptores de Superficie Celular/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Vacuolas/química , Zinc/metabolismo , Proteínas de Unión al GTP rab/genética

RESUMEN

Sequencing of the entire genome of S. cerevisiae has revealed the existence of five proteins containing EH domains. These are protein-protein interaction modules first described in mammalian Eps15, a protein that is involved in clathrin-dependent endocytosis. Two of the yeast proteins, End3p and Pan1p, are required for the internalization step of endocytosis. We report characterization of the nonessential ORF YBL047c which, like Eps15, encodes a protein with three N-terminal EH domains. Deletion of YBL047c leads to a defective fluid-phase endocytosis and to defective internalization of the pheromone (alpha)-factor and uracil permease. We therefore named YBL047c EDE1, for EH Domains and Endocytosis. Ede1p expressed as a chromosomally encoded fusion to the green fluorescent protein is localized in punctate cortical spots that only partially colocalize with actin patches. This localization is maintained when actin is depolymerized. Deletion of EDE1 impairs the diploid budding pattern, but has only a small impact on actin cytoskeleton organization, in contrast to the effects observed in pan1 cells and many end mutants impaired in proteins colocalizing with cortical actin patches. Genetic interaction was observed between EDE1 and RSP5, which encodes the ubiquitin ligase Rsp5p essential for ubiquitin-dependent endocytosis of many plasma membrane proteins, thus further emphasizing the functional link between Rsp5p and the EH domain proteins. We also observed genetic interaction between EDE1, and END3 or PAN1, suggesting that Ede1p might be part of a yeast EH network implicated in endocytosis.

Asunto(s)

Endocitosis/fisiología , Proteínas Fúngicas/fisiología , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiología , Complejos de Ubiquitina-Proteína Ligasa , Actinas/metabolismo , Proteínas del Citoesqueleto , Citoesqueleto , Complejos de Clasificación Endosomal Requeridos para el Transporte , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas de Microfilamentos , Polímeros , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo

RESUMEN

In addition to its well-known role in recognition by the proteasome, ubiquitin-conjugation is also involved in downregulation of membrane receptors, transporters and channels. In most cases, ubiquitination of these plasma membrane proteins leads to their internalization followed by targeting to the lysosome/vacuole for degradation. A crucial role in ubiquitination of many plasma membrane proteins appears to be played by ubiquitin-protein ligases of the Nedd4/Rsp5p family. All family members carry an N-terminal Ca2+-dependent lipid/protein binding (C2) domain, two to four WW domains and a C-terminal catalytic Hect-domain. Nedd4 is involved in downregulation of the epithelial Na+ channel, by binding of its WW domains to specific PY motifs of the channel. Rsp5p, the unique family member in S. cerevisiae, is involved in ubiquitin-dependent endocytosis of a great number of yeast plasma membrane proteins. These proteins lack apparent PY motifs, but carry acidic sequences, and/or phosphorylated-based sequences that might be important, directly or indirectly, for their recognition by Rsp5p. In contrast to polyubiquitination leading to proteasomal recognition, a number of Rsp5p targets carry few ubiquitins per protein, and moreover with a different ubiquitin linkage. Accumulating evidence suggests that, at least in yeast, ubiquitin itself may constitute an internalization signal, recognized by a hypothetical receptor. Recent data also suggest that Nedd4/Rsp5p might play a role in the endocytic process possibly involving its C2 domain, in addition to its role in ubiquitinating endocytosed proteins.

Asunto(s)

Proteínas de Unión al Calcio/fisiología , Endocitosis/fisiología , Ligasas/fisiología , Proteínas de la Membrana/metabolismo , Ubiquitinas/metabolismo , Animales , Membrana Celular/metabolismo , Regulación hacia Abajo , Complejos de Clasificación Endosomal Requeridos para el Transporte , Humanos , Ligasas/metabolismo , Ubiquitina-Proteína Ligasas Nedd4 , Transducción de Señal , Ubiquitina-Proteína Ligasas

RESUMEN

Uracil uptake by Saccharomyces cerevisiae is mediated by the FUR4-encoded uracil permease. The modification of uracil permease by phosphorylation at the plasma membrane is a key mechanism for regulating endocytosis of this protein. This modification in turn facilitates its ubiquitination and internalization. Following endocytosis, the permease is targeted to the lysosome/vacuole for proteolysis. We have previously shown that uracil permease is phosphorylated at several serine residues within a well characterized N-terminal PEST sequence. In this report, we provide evidence that lysine residues 38 and 41, adjacent to the PEST sequence, are the target sites for ubiquitination of the permease. Conservative substitutions at both Lys(38) and Lys(41) give variant permeases that are phosphorylated but fail to internalize. The PEST sequence contains potential phosphorylation sites conforming to the consensus sequences for casein kinase 1. Casein kinase 1 (CK1) protein kinases, encoded by the redundant YCKI and YCK2 genes, are located at the plasma membrane. Either alone supports growth, but loss of function of both is lethal. Here, we show that in CK1-deficient cells, the permease is poorly phosphorylated and poorly ubiquitinated. Moreover, CK1 overproduction rescued the defective endocytosis of a mutant permease in which the serine phosphoacceptors were replaced by threonine (a less effective phosphoacceptor), which suggests that Yck activity may play a direct role in phosphorylating the permease. Permease internalization was not greatly affected in CK1-deficient cells, despite the low level of ubiquitination of the protein. This may be due to CK1 having a second counteracting role in endocytosis as shown by the higher turnover of variant permeases with unphosphorylatable versions of the PEST sequence.

Asunto(s)

Quinasa de la Caseína I , Endocitosis , Lisina , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Nucleótidos , Proteínas Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Ubiquitinas/metabolismo , Secuencia de Aminoácidos , Transporte Biológico , Caseína Quinasas , Genes Fúngicos , Variación Genética , Semivida , Lisina/genética , Proteínas de Transporte de Membrana/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae

RESUMEN

In Saccharomyces cerevisiae, endocytic material is transported through different membrane-bound compartments before it reaches the vacuole. In a screen for mutants that affect membrane trafficking along the endocytic pathway, we have identified a novel mutant disrupted for the gene YJL204c that we have renamed RCY1 (recycling 1). Deletion of RCY1 leads to an early block in the endocytic pathway before the intersection with the vacuolar protein sorting pathway. Mutation of RCY1 leads to the accumulation of an enlarged compartment that contains the t-SNARE Tlg1p and lies close to areas of cell expansion. In addition, endocytic markers such as Ste2p and the fluorescent dyes, Lucifer yellow and FM4-64, were found in a similar enlarged compartment after their internalization. To determine whether rcy1Delta is defective for recycling, we have developed an assay that measures the recycling of previously internalized FM4-64. This method enables us to follow the recycling pathway in yeast in real time. Using this assay, it could be demonstrated that recycling of membranes is rapid in S. cerevisiae and that a major fraction of internalized FM4-64 is secreted back into the medium within a few minutes. The rcy1Delta mutant is strongly defective in recycling.

Asunto(s)

Endocitosis/fisiología , Endosomas/fisiología , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/ultraestructura , Ciclo Celular/genética , Ciclo Celular/fisiología , Membrana Celular/fisiología , Endocitosis/genética , Endosomas/ultraestructura , Proteínas F-Box , Colorantes Fluorescentes , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Eliminación de Gen , Genes Fúngicos , Glicósido Hidrolasas/metabolismo , Membranas Intracelulares/fisiología , Cinética , Factor de Apareamiento , Proteínas de la Membrana/genética , Modelos Biológicos , Péptidos/metabolismo , Saccharomyces cerevisiae/genética , Vacuolas/fisiología , Proteínas de Transporte Vesicular , beta-Fructofuranosidasa

RESUMEN

Addition of ammonium ions to yeast cells growing on proline as the sole nitrogen source induces internalization of the general amino acid permease Gap1p and its subsequent degradation in the vacuole. An essential step in this down-regulation is Gap1p ubiquitination through a process requiring the Npi1p/Rsp5p ubiquitin ligase. We show in this report that NPI2, a second gene required for NH4+-induced down-regulation of Gap1p, codes for the ubiquitin hydrolase Doa4p/Ubp4p/Ssv7p and that NH4+-induced Gap1p ubiquitination is strongly reduced in npi2 cells. The npi2 mutation results in substitution of an aromatic amino acid located in a 33-residue sequence shared by some ubiquitin hydrolases of the Ubp family. In this mutant, as in doa4(delta) cells, the amount of free monomeric ubiquitin is at least four times lower than in wild-type cells. Both ubiquitination and down-regulation of the permease can be restored in npi2 cells by over-expression of ubiquitin. In proline-grown wild-type and npi2/doa4 cells overproducing ubiquitin, Gap1p appears to be mono-ubiquitinated at two lysine acceptor sites. Addition of NH4+ triggers rapid poly-ubiquitination of Gap1p, the poly-ubiquitin chains being specifically formed by linkage through the lysine 63 residue of ubiquitin. Gap1p is thus ubiquitinated differently from the proteins targeted by ubiquitination for proteolysis by the proteasome, but in the same manner as the uracil permease, also subject to ubiquitin-dependent endocytosis. When poly-ubiquitination through Lys63 is blocked, the Gap1p permease still undergoes NH4+-induced down-regulation, but to a lesser extent.

Asunto(s)

Lisina/química , Proteínas de Transporte de Membrana/efectos de los fármacos , Compuestos de Amonio Cuaternario/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Ubiquitinas/metabolismo , Secuencia de Aminoácidos , Sistemas de Transporte de Aminoácidos , Clonación Molecular , Regulación hacia Abajo , Ligasas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Datos de Secuencia Molecular , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligasas

RESUMEN

In Saccharomyces cerevisiae the FUR4-encoded uracil permease catalyzes the first step of the pyrimidine salvage pathway. The availability of uracil has a negative regulatory effect upon its own transport. Uracil causes a decrease in the level of uracil permease, partly by decreasing the FUR4 mRNA level in a promoter-independent fashion, probably by increasing its instability. Uracil entry also triggers more rapid degradation of the existing permease by promoting high efficiency of ubiquitination of the permease that signals its internalization. A direct binding of intracellular uracil to the permease is possibly involved in this feedback regulation, as the behavior of the permease is similar in mutant cells unable to convert intracellular uracil into UMP. We used cells impaired in the ubiquitination step to show that the addition of uracil produces rapid inhibition of uracil transport. This may be the first response prior to the removal of the permease from the plasma membrane. Similar down-regulation of uracil uptake, involving several processes, was observed under adverse conditions mainly corresponding to a decrease in the cellular content of ribosomes. These results suggest that uracil of exogenous or catabolic origin down-regulates the cognate permease to prevent buildup of excess intracellular uracil-derived nucleotides.

Asunto(s)

Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Nucleótidos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Uracilo/metabolismo , Secuencia de Bases , Cartilla de ADN/genética , Regulación hacia Abajo/efectos de los fármacos , Genes Fúngicos , Moduladores del Transporte de Membrana , Proteínas de Transporte de Membrana/antagonistas & inhibidores , Proteínas de Transporte de Membrana/genética , Plásmidos/genética , Pirimidinas/farmacología , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Regulación hacia Arriba , Uracilo/farmacología

RESUMEN

Six open reading frames (ORFs) of unknown function from the left arm of Saccharomyces cerevisiae chromosome XV were deleted in two genetic backgrounds by disruption cassettes with long flanking homology (LFH) (Wach, 1996), within the frame of the research project EUROFAN. The LFH disruption cassettes, obtained by PCR, were made by introducing the kanMX4 marker module between two fragments homologous to the promoter and terminator regions of a given ORF. Transformants resistant to geneticin (G418) were selected. The LFH disruption cassettes were cloned in a bacterial vector. Each cognate gene was also cloned in a centromeric plasmid. Correct deletion of each gene was verified by four different PCR reactions. Sporulation and tetrad analysis of heterozygous deletants revealed that ORF YOL102c is essential. The non-growing haploid spores gave rise to microcolonies. Basic phenotypic analyses were performed on haploid deletants of both mating types of the five non-essential ORFs, YOL018c, YOL098c, YOL101c, YOL104c and YOL105c. Plate growth tests on different media at 15 degrees C, 30 degrees C or 37 degrees C did not reveal any significant differences between parental and mutant cells. Mating and sporulation efficiencies were not affected in any of the viable disruptants as compared to wild-type cells.

Asunto(s)

Cromosomas Fúngicos , Eliminación de Gen , Genes Fúngicos , Saccharomyces cerevisiae/genética , Marcadores Genéticos , Sistemas de Lectura Abierta/genética , Fenotipo , Reacción en Cadena de la Polimerasa/métodos , Saccharomyces cerevisiae/crecimiento & desarrollo , Transformación Genética

RESUMEN

The ORF YOL018c (TLG2) of Saccharomyces cerevisiae encodes a protein that belongs to the syntaxin protein family. The proteins of this family, t-SNAREs, are present on target organelles and are thought to participate in the specific interaction between vesicles and acceptor membranes in intracellular membrane trafficking. TLG2 is not an essential gene, and its deletion does not cause defects in the secretory pathway. However, its deletion in cells lacking the vacuolar ATPase subunit Vma2p leads to loss of viability, suggesting that Tlg2p is involved in endocytosis. In tlg2Delta cells, internalization was normal for two endocytic markers, the pheromone alpha-factor and the plasma membrane uracil permease. In contrast, degradation of alpha-factor and uracil permease was delayed in tlg2Delta cells. Internalization of positively charged Nanogold shows that the endocytic pathway is perturbed in the mutant, which accumulates Nanogold in primary endocytic vesicles and shows a greatly reduced complement of early endosomes. These results strongly suggest that Tlg2p is a t-SNARE involved in early endosome biogenesis.

Asunto(s)

Endocitosis/fisiología , Factores de Intercambio de Guanina Nucleótido , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Nucleótidos , Orgánulos/fisiología , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiología , ATPasas de Translocación de Protón Vacuolares , Secuencia de Aminoácidos , Cromosomas Fúngicos , Clonación Molecular , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Eliminación de Gen , Cinética , Factor de Apareamiento , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Orgánulos/ultraestructura , Péptidos/genética , Péptidos/metabolismo , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón/metabolismo , Proteínas Qa-SNARE , Proteínas Recombinantes de Fusión/biosíntesis , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido

RESUMEN

The yeast plasma membrane, uracil permease, undergoes ubiquitin-dependent endocytosis and subsequent degradation in the vacuole via a process that does not involve the proteasome. Cell-surface ubiquitination of this protein is mediated by the ubiquitin-protein ligase Npi1p/Rsp5p and involves Lys63-linked ubiquitin chains. This report describes the intracellular fate of a mutant form of uracil permease carrying a three amino acid insertion in a cytoplasmic loop. Most of this protein is not deployed beyond the ER, and is degraded by the 26S proteasome. Mutant permease degradation is almost unaffected in cells with impaired Npi1p/Rsp5p, but is dependent on the Ubc6p and Ubc7p ubiquitin-conjugating enzymes, suggesting that proteolysis of the protein requires its prior ubiquitination. Overproduction of a derivative of ubiquitin with a modified Lys48 strongly impairs mutant permease degradation. This suggests that, like other proteasome substrates, mutant permease might be polyubiquitinated with Lys48-linked ubiquitin chains. These findings provide an example of a yeast plasma membrane protein that is routed to the 'ER degradation' pathway, and highlight the versatility of the ubiquitin system.

Asunto(s)

Cisteína Endopeptidasas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Complejos Multienzimáticos/metabolismo , Mutación , Proteínas de Transporte de Nucleótidos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Ubiquitinas/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico , Proteínas Fúngicas/metabolismo , Aparato de Golgi , Ligasas/metabolismo , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana/genética , Complejo de la Endopetidasa Proteasomal , Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligasas

RESUMEN

Uptake of uracil by the yeast Saccharomyces cerevisiae is mediated by a specific permease encoded by the FUR4 gene. Uracil permease located at the cell surface is subject to two covalent modifications: phosphorylation and ubiquitination. The ubiquitination step is necessary prior to permease endocytosis and subsequent vacuolar degradation. Here, we demonstrate that a PEST-like sequence located within the cytoplasmic N terminus of the protein is essential for uracil permease turnover. Internalization of the transporter was reduced when some of the serines within the region were converted to alanines and severely impaired when all five serines within the region were mutated or when this region was absent. The phosphorylation and degree of ubiquitination of variant permeases were inversely correlated with the number of serines replaced by alanines. A serine-free version of this sequence was very poorly phosphorylated, and elimination of this sequence prevented ubiquitination. Thus, it appears that the serine residues in the PEST-like sequence are required for phosphorylation and ubiquitination of uracil permease. A PEST-like sequence in which the serines were replaced by glutamic acids allowed efficient permease turnover, suggesting that the PEST serines are phosphoacceptors.

Asunto(s)

Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Nucleótidos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimología , Sitios de Unión , Activación Enzimática/genética , Genes Fúngicos , Mutación , Saccharomyces cerevisiae/genética , Análisis de Secuencia

RESUMEN

We have recently reported that the yeast plasma membrane uracil permease undergoes cell-surface ubiquitination, which is dependent on the Npi1/Rsp5 ubiquitin-protein ligase. Ubiquitination of this permease, like that of some other transporters and receptors, signals endocytosis of the protein, leading to its subsequent vacuolar degradation. This process does not involve the proteasome, which binds and degrades ubiquitin-protein conjugates carrying Lys48-linked ubiquitin chains. The data presented here show that ubiquitination and endocytosis of uracil permease are impaired in yeast cells lacking the Doa4p ubiquitin-isopeptidase. Both processes were rescued by overexpression of wild-type ubiquitin. Mutant ubiquitins carrying Lys-->Arg mutations at Lys29 and Lys48 restored normal permease ubiquitination. In contrast, a ubiquitin mutated at Lys63 did not restore permease polyubiquitination. Ubiquitin-permease conjugates are therefore extended through the Lys63 of ubiquitin. When polyubiquitination through Lys63 is blocked, the permease still undergoes endocytosis, but at a reduced rate. We have thus identified a natural target of Lys63-linked ubiquitin chains. We have also shown that monoubiquitination is sufficient to induce permease endocytosis, but that Lys63-linked ubiquitin chains appear to stimulate this process.

Asunto(s)

Endocitosis , Endopeptidasas , Lisina/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Nucleótidos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Ubiquitinas/metabolismo , Arginina/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Cicloheximida/farmacología , Endocitosis/efectos de los fármacos , Complejos de Clasificación Endosomal Requeridos para el Transporte , Estabilidad de Enzimas , Proteínas Fúngicas/metabolismo , Lisina/química , Mutagénesis Sitio-Dirigida , Fenotipo , Inhibidores de la Síntesis de la Proteína/farmacología , Saccharomyces cerevisiae/enzimología , Ubiquitina Tiolesterasa , Ubiquitinas/química

RESUMEN

The Saccharomyces cerevisiae actin-related protein Arp2p is an essential component of the actin cytoskeleton. We have tested its potential role in the endocytic and exocytic pathways by using a temperature-sensitive allele, arp2-1. The fate of the plasma membrane transporter uracil permease was followed to determine whether Arp2p plays a role in the endocytic pathway. Inhibition of normal endocytosis as revealed by maintenance of active uracil permease at the plasma membrane and strong protection against subsequent vacuolar degradation of the protein were observed in the mutant at the restrictive temperature. Furthermore, arp2-1 cells accumulated ubiquitin-permease conjugates, formed prior to internalization. These effects were also visible at permissive temperature, whereas the actin cytoskeleton appeared to be normally polarized. The soluble hydrolase carboxypeptidase Y and the lipophilic dye FM 4-64 were targeted normally to the vacuole in arp2-1 cells. Thus, Arp2p is required for internalization but does not play a major role in later steps of endocytosis. Synthetic lethality was demonstrated between arp2-1 and the endocytic mutant end3-1, suggesting participation of Arp2p and End3p in the same process. Finally, no evidence for a major defect in secretion was apparent; invertase secretion and delivery of uracil permease to the plasma membrane were unaffected in arp2-1 cells.

Asunto(s)

Actinas/fisiología , Proteínas del Citoesqueleto , Endocitosis , Proteínas Fúngicas/fisiología , Proteínas de Transporte de Nucleótidos , Proteínas de Saccharomyces cerevisiae , Proteína 2 Relacionada con la Actina , Actinas/genética , Transporte Biológico/genética , Membrana Celular/metabolismo , Endocitosis/genética , Endosomas/genética , Endosomas/metabolismo , Genes Letales , Glicósido Hidrolasas/metabolismo , Hidrolasas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Mutación , Procesamiento Proteico-Postraduccional , Compuestos de Piridinio/química , Compuestos de Piridinio/metabolismo , Compuestos de Amonio Cuaternario/química , Compuestos de Amonio Cuaternario/metabolismo , Saccharomyces cerevisiae , Vacuolas/enzimología , Vacuolas/genética , Vacuolas/metabolismo , beta-Fructofuranosidasa

RESUMEN

The uracil permease of Saccharomyces cerevisiae is a 633 residue polytopic plasma membrane protein. Hydropathy profile analysis indicates that this protein has long hydrophilic N- and C-termini and 10-12 potential transmembrane segments. Previous results based on analysis of hybrid proteins allowed identification of the first transmembrane segment of uracil permease, and provided a preliminary indication of the cytoplasmic orientation of its N-terminus. In this work, other experimental approaches were used to confirm this orientation, and to determine that of the C-terminus. Epitopes in the N- and the C-termini of the protein were protected against trypsin degradation on intact protoplasts, but readily digested on permeabilized protoplasts. Immunofluorescent analysis showed that antibodies to the last 10 amino acids of uracil permease bind to detergent-treated protoplasts, but not to intact ones. Carboxypeptidase digested the C-terminus of uracil permease inserted into the sealed dog-pancreas microsomes. These results establish that both N- and C-termini are cytoplasmic, the permease polypeptide spanning the membrane an even number of times. The orientation of several hydrophilic loops with respect to the membrane was investigated by introducing potential glycosylation sites into these regions. We checked whether the resulting mutant proteins were glycosylated when expressed in the presence of dog-pancreas microsomes. Our data show that two loops of the protein are lumenal. Together with previous results, this work indicates that uracil permease is a 10 membrane-spanning protein, with rather small external loops and three main cytoplasmic regions (the N- and C-termini and a central 60-residue loop).

Asunto(s)

Membrana Celular/química , Proteínas Fúngicas/química , Proteínas de la Membrana/química , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Nucleótidos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Animales , Membrana Celular/enzimología , Permeabilidad de la Membrana Celular , Polaridad Celular , Perros , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas Fúngicas/efectos de los fármacos , Glicosilación , Hexosaminidasas/farmacología , Proteínas de la Membrana/efectos de los fármacos , Proteínas de Transporte de Membrana/efectos de los fármacos , Microsomas/metabolismo , Datos de Secuencia Molecular , Conformación Proteica , Pliegue de Proteína , Tripsina/farmacología