RESUMEN
In a screen for temperature-sensitive (37 degrees C) mutants of Saccharomyces cerevisiae that are defective in the proper localization of the Golgi transmembrane protein Emp47p, we uncovered a constitutive loss-of-function mutation in CYS3/STR1, the gene coding for cystathionine-gamma-lyase. We showed by immunofluorescence, sucrose-gradient analysis and quantitative Western analysis that the mutant mislocalized Emp47p to the vacuole at high temperature, while Golgi structures were apparently normal and biosynthetic routing of the vacuolar carboxypeptidase Y (CPY) and the plasma membrane GPI-anchored protein Gas1p were unaffected. The effect of high temperature on Emp47p localization, as well as the temperature sensitivity of the mutant strain on rich medium, appear to be caused by oxidative stress and are correlated with severe reductions in the intracellular levels of low-molecular-weight thiols. In accordance with this conclusion, cys3-2 mutant cells were more sensitive to the oxidizing agent 1-chloro-2,4-dinitrobenzene, which also aggravated the mislocalization of Emp47p observed at high temperature. Furthermore, all the phenotypes of the mutant were completely complemented by exogenous supply of the main low-molecular-weight thiol, glutathione (GSH) and, importantly, the thiol beta-mercaptoethanol reversed the temperature sensitivity of the mutant. A comparison of our mutant with a mutant defective in GSH synthesis showed that gsh1Delta cells were similar to wild-type cells under the stress conditions tested, with the exception of one novel oxidative stress-related phenotype that is observed in both cys3-2 and gsh1Delta mutant cells - a defect in CDP-DAG metabolism upon shift to the non-permissive temperature. As most of the stress-related phenotypes of cys3-2 mutant cells are more severe than those seen in gsh1Delta cells, we conclude that cysteine as such is required and sufficient to confer some degree of protection from oxidative stress in yeast cells.
Asunto(s)
Citidina Difosfato Diglicéridos/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Glutamato-Cisteína Ligasa , Glutatión/metabolismo , Proteínas de Homeodominio/metabolismo , Estrés Oxidativo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , División Celular , Cistationina gamma-Liasa , Cisteína/farmacología , Proteínas de Unión al ADN/genética , Metanosulfonato de Etilo/farmacología , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas Fúngicas/genética , Genes Fúngicos , Glutatión/genética , Proteínas de Homeodominio/genética , Calor , Metabolismo de los Lípidos , Proteínas de la Membrana , Mutagénesis , Mutación , Fenotipo , Transporte de Proteínas , Recombinación Genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Fracciones Subcelulares , Factores de Transcripción/genética , Proteínas de Transporte VesicularRESUMEN
end13-1 was isolated in a screen for endocytosis mutants and has been shown to have a post-internalisation defect in endocytic transport as well as a defect in vacuolar protein sorting (Vps(-) phenotype), leading to secretion of newly synthesised vacuolar proteins. Here we demonstrate that END13 is identical to VPS4, encoding an AAA (ATPase associated with a variety of cellular activities)-family ATPase. We also report that the end13-1 mutation is a serine 335 to phenylalanine substitution in the AAA-ATPase domain of End13p/Vps4p. It has been reported that mutant cells lacking End13p/Vps4p (end13(vps4)Delta) accumulate endocytosed marker dyes, plasma membrane receptors and newly synthesised vacuolar hydrolase precursors in an endosomal compartment adjacent to the vacuole (prevacuolar compartment, or PVC). We find, however, that the end13 mutants have defects in transport of endocytosed fluorescent dyes, plasma membrane receptors and ligands from small peripherally located early endosomes to larger late endosomes, which are often located adjacent to the vacuole. Our results indicate that End13p/Vps4p may play an important role in multiple steps of membrane traffic through the endocytic pathway.
Asunto(s)
Adenosina Trifosfatasas , Endocitosis/fisiología , Endosomas/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte , Factor de Apareamiento , Mutagénesis/fisiología , Péptidos/metabolismo , Transporte de Proteínas/fisiología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Vacuolas/metabolismoRESUMEN
Eukaryotic 70 kDa heat shock proteins (Hsp70s) are localized in various cellular compartments and exhibit functions such as protein translocation across membranes, protein folding and assembly. Here we demonstrate that the constitutively expressed members of the yeast cytoplasmic Ssa subfamily, Ssa1/2p, are involved in the transport of the vacuolar hydrolase aminopeptidase 1 from the cytoplasm into the vacuole. The Ssap family members displayed overlapping functions in the transport of aminopeptidase 1. In SSAI and SSAII deletion mutants the precursor of aminopeptidase 1 accumulated in a dodecameric complex that is packaged in prevacuolar transport vesicles. Ssa1/2p was prominently localized to the vacuolar membrane, consistent with the role we propose for Ssa proteins in the fusion of transport vesicles with the vacuolar membrane.
Asunto(s)
Aminopeptidasas/metabolismo , Citoplasma/metabolismo , Proteínas Fúngicas/fisiología , Proteínas HSP70 de Choque Térmico/fisiología , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismo , Adenosina Trifosfatasas , Aminopeptidasas/química , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/metabolismo , Transporte Biológico , Precursores Enzimáticos/química , Precursores Enzimáticos/metabolismo , Técnica del Anticuerpo Fluorescente , Proteínas Fúngicas/genética , Eliminación de Gen , Genes Fúngicos/genética , Genes Fúngicos/fisiología , Proteínas HSP70 de Choque Térmico/genética , Respuesta al Choque Térmico , Membranas Intracelulares/metabolismo , Peso Molecular , Fagocitosis , Unión Proteica , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimologíaRESUMEN
Vesicle specific SNAP receptors (v-SNAREs) Bos1p and Bet1p are involved in targeting of anterograde vesicles between the endoplasmic reticulum (ER) and early Golgi of Saccharomyces cerevisiae. To analyze factors that influence the targeting of these proteins, alpha-factor tagged versions of Bos1p and Bet1p were employed. The alpha-factor can be cleaved off by the Kex2p protease as soon as the hybrid proteins reach the late Golgi compartment. The data obtained by monitoring of Kex2p cleavage, by immunofluorescence microscopy and cell fractionation showed that Bos1-alpha and Bet1-alpha have different cellular localization and dynamics. Bos1-alpha is an ER protein, which recycles between the Golgi and the ER in COPI-dependent manner. Bet1-alpha is an early Golgi protein and it does not change its localization under conditions when other recycling Golgi proteins can be trapped in the ER.
Asunto(s)
Proteínas Portadoras/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Proproteína Convertasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular , Transporte Biológico , Proteínas Portadoras/genética , Proteína Coat de Complejo I/genética , Proteína Coat de Complejo I/metabolismo , Gránulos Citoplasmáticos/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas de la Membrana/genética , Mutación , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Proteínas R-SNARE , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas SNARE , Saccharomyces cerevisiae/genética , Fracciones Subcelulares/química , Subtilisinas/metabolismoRESUMEN
Emp47p is a yeast Golgi transmembrane protein with a retrograde, Golgi to ER transport di-lysine signal in its cytoplasmic tail. Emp47p has previously been shown to recycle between the Golgi complex and the ER and to require its di-lysine signal for Golgi localization. In contrast to other proteins with di-lysine signals, the Golgi-localization of Emp47p has been shown to be preserved in ret1-1 cells expressing a mutant alpha-COP subunit of coatomer. Here we demonstrate by sucrose gradient fractionation and immunofluorescence analysis that recycling of Emp47p was unimpaired in ret1-1. Furthermore we have characterized three new alleles of ret1 and showed that Golgi localization of Emp47p was intact in cells with those mutant alleles. We could correlate the ongoing recycling of Emp47p in ret1-1 with preserved in vitro binding of coatomer from ret1-1 cells to immobilized GST-Emp47p-tail fusion protein. As previously reported, the di-lysine signal of Wbp1p was not recognized by ret1-1 mutant coatomer, suggesting a possible role for alpha-COP in the differential binding to distinct di-lysine signals. In contrast to results with alpha-COP mutants, we found that Emp47p was mislocalised to the vacuole in mutants affecting beta'-, gamma-, delta-, and zeta-COP subunits of coatomer and that the mutant coatomer bound neither to the Emp47p nor to the Wbp1p di-lysine signal in vitro. Therefore, the retrograde transport of Emp47p displayed a differential requirement for individual coatomer subunits and a special role of alpha-COP for a particular transport step in vivo.
Asunto(s)
Dipéptidos/metabolismo , Hexosiltransferasas , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae , Alelos , Secuencia de Aminoácidos , Transporte Biológico Activo , Proteína Coatómero , Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Aparato de Golgi/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutación , Unión Proteica , Conformación Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Transferasas/genética , Transferasas/metabolismo , Proteínas de Transporte VesicularRESUMEN
Sec20p and Tip20p were previously identified as two interacting proteins involved in early steps of the secretory pathway in Saccharomyces cerevisiae. Here we describe a novel temperature-sensitive allele of TIP20 and analyze its phenotype. While sec20 and tip20 mutants exhibited a defect in forward ER-to-Golgi transport at the non-permissive temperature, both were also defective for retrieval of various dilysine-tagged proteins from the Golgi back to the endoplasmic reticulum (ER) at lower temperature. Dilysine-dependent Golgi localization of Emp47p was also defective in both mutants. These results suggest a role for the Sec20/Tip20p complex in retrieval of dilysine-tagged proteins back to the ER.