RESUMEN
Kex2 protease processes pro-alpha-factor in a late Golgi compartment in Saccharomyces cerevisiae. The first approximately 30 residues of the 115 amino acid CO2H-terminal cytosolic tail (C-tail) of the Kex2 protein (Kex2p) contain a Golgi retention signal that resembles coated-pit localization signals in mammalian cell surface receptors. Mutation of one (Tyr713) of two tyrosine residues in the C-tail or deletion of sequences adjacent to Tyr713 results in loss of normal Golgi localization. Surprisingly, loss of the Golgi retention signal resulted in transport of C-tail mutant Kex2p to the vacuole (yeast lysosome), as judged by kinetics of degradation and by indirect immunofluorescence. Analysis of the loss of Kex2 function in vivo after shutting off expression of wild-type or mutant forms proved that mutations that cause rapid vacuolar turnover do so by increasing the rate of exit of the enzyme from the pro-alpha-factor processing compartment. The most likely explanation for these results is that mutation of the Golgi retention signal in the C-tail results in transport of Kex2p to the vacuole by default. Wild-type Kex2p also was transported to the vacuole at an increased rate when overproduced, although apparently not due to saturation of a Golgi-retention mechanism. Instead, the wild-type and C-tail mutant forms of Kex2p may follow distinct paths to the vacuole.
Asunto(s)
Aparato de Golgi/metabolismo , Mutagénesis Sitio-Dirigida , Proproteína Convertasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Transducción de Señal , Subtilisinas , Tirosina , Vacuolas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Transporte Biológico , Técnica del Anticuerpo Fluorescente , Genes Fúngicos , Aparato de Golgi/ultraestructura , Cinética , Microscopía Electrónica , Modelos Biológicos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Biosíntesis de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Vacuolas/ultraestructuraRESUMEN
The Kex2 protease of the yeast Saccharomyces cerevisiae is a prototypical eukaryotic prohormone-processing enzyme that cleaves precursors of secreted peptides at pairs of basic residues. Here we have established the pathway of posttranslational modification of Kex2 protein using immunoprecipitation of the biosynthetically pulse-labeled protein from a variety of wild-type and mutant yeast strains as the principal methodology. Kex2 protein is initially synthesized as a prepro-enzyme that undergoes cotranslational signal peptide cleavage and addition of Asn-linked core oligosaccharide and Ser/Thr-linked mannose in the ER. The earliest detectable species, I1 (approximately 129 kD), undergoes rapid amino-terminal proteolytic removal of a approximately 9-kD pro-segment yielding species I2 (approximately 120 kD) before arrival at the Golgi complex. Transport to the Golgi complex is marked by extensive elaboration of Ser/Thr-linked chains and minor modification of Asn-linked oligosaccharide. During the latter phase of its lifetime, Kex2 protein undergoes a gradual increase in apparent molecular weight. This final modification serves as a marker for association of Kex2 protease with a late compartment of the yeast Golgi complex in which it is concentrated about 27-fold relative to other secretory proteins.
Asunto(s)
Proproteína Convertasas , Procesamiento Proteico-Postraduccional/fisiología , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimología , Serina Endopeptidasas/metabolismo , Subtilisinas , Transporte Biológico/fisiología , Gránulos Citoplasmáticos/metabolismo , Marcadores Genéticos , Glicosilación , Aparato de Golgi/metabolismo , Cinética , Oligosacáridos/metabolismo , Pruebas de Precipitina , Biosíntesis de Proteínas/fisiología , Precursores de Proteínas/metabolismo , Señales de Clasificación de Proteína/metabolismo , Saccharomyces cerevisiae/metabolismo , Serina Endopeptidasas/biosíntesis , TemperaturaRESUMEN
A protein sensitive to N-ethylmaleimide catalyses the fusion of transport vesicles with Golgi cisternae in a mammalian cell-free system. By cloning and sequencing its gene from Chinese hamster ovary cells and by use of in vitro assays, we show that this fusion protein is equivalent to the SEC18 gene product of the yeast Saccharomyces cerevisiae, known to be essential for vesicle-mediated transport from the endoplasmic reticulum to the Golgi apparatus. The mechanism of vesicular fusion is thus highly conserved, both between species and at different stages of transport.
Asunto(s)
Proteínas Portadoras/genética , Membrana Celular/metabolismo , Etilmaleimida/farmacología , Genes , Aparato de Golgi/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Transporte Biológico , Proteínas Portadoras/fisiología , Línea Celular , Citosol/metabolismo , Genes Fúngicos , Cinética , Datos de Secuencia Molecular , Proteínas Sensibles a N-Etilmaleimida , Saccharomyces cerevisiae/metabolismoRESUMEN
N-Ethylmaleimide (NEM) inhibits protein transport between successive compartments of the Golgi stack in a cell-free system. After inactivation of the Golgi membranes by NEM, transport can be rescued by adding back an appropriately prepared cytosol fraction. This complementation assay has allowed us to purify the NEM-sensitive factor, which we term NSF. The NEM-sensitive factor is a tetramer of 76-kDa subunits, and appears to act catalytically, one tetramer leading to the metabolism of numerous transport vesicles.
Asunto(s)
Proteínas Portadoras/aislamiento & purificación , Aparato de Golgi/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Anticuerpos Monoclonales , Transporte Biológico Activo/efectos de los fármacos , Proteínas Portadoras/farmacología , Línea Celular , Cricetinae , Citosol/análisisRESUMEN
The BC3Hl muscle cell line was previously reported to contain a broad array of fatty acid acylated proteins [Olson, E. N., Towler, D. A., & Glaser, L. (1985) J. Biol. Chem. 260, 3784-3790]. Palmitate was shown to be attached to membrane proteins posttranslationally through thiol ester linkages, whereas myristate was attached cotranslationally, or within seconds thereafter, to soluble and membrane-bound proteins through amide linkages [Olson, E. N., & Spizz, G. (1986) J. Biol. Chem. 261, 2458-2466]. The temporal and subcellular differences between palmitate and myristate acylation suggested that these two classes of acyl proteins might follow different intracellular pathways to distinct subcellular membrane systems or organelles. In this study, we examined the subcellular localization of the major fatty acylated proteins in BC3Hl cells. Palmitate-containing proteins were localized to the plasma membrane, but only a subset of myristate-containing proteins was localized to this membrane fraction. The majority of acyl proteins were nonglycosylated and resistant to digestion with extracellular proteases, suggesting that they were not exposed to the external surface of the plasma membrane. Many proteins were, however, digested during incubation of isolated membranes with proteases, which indicates that these proteins face the cytoplasm. Two-dimensional gel electrophoresis of proteins labeled with [3H]palmitate and [3H]myristate revealed that individual proteins were modified by only one of the two fatty acids and did not undergo both N-linked myristylation and ester-linked palmitylation. Together, these results suggest that the majority of cellular acyl proteins are routed to the cytoplasmic surface of the plasma membrane, and they raise the possibility that fatty acid acylation may play a role in intracellular sorting of nontransmembranous, nonglycosylated membrane proteins.