RESUMEN
Members of the syntaxin protein family participate in the docking-fusion step of several intracellular vesicular transport events. Tlg1p has been identified as a nonessential protein required for efficient endocytosis as well as the maintenance of normal levels of trans-Golgi network proteins. In this study we independently describe Tlg1p as an essential protein required for cell viability. Depletion of Tlg1p in vivo causes a defect in the transport of the vacuolar protein carboxypeptidase Y through the early Golgi. Temperature-sensitive (ts) mutants of Tlg1p also accumulate the endoplasmic reticulum/cis-Golgi form of carboxypeptidase Y at the nonpermissive temperature (38 degrees C) and exhibit underglycosylation of secreted invertase. Overexpression of Tlg1p complements the growth defect of vti1-11 at the nonpermissive temperature, whereas incomplete complementation was observed with vti1-1, further suggesting a role for Tlg1p in the Golgi apparatus. Overexpression of Sed5p decreases the viability of tlg1 ts mutants compared with wild-type cells, suggesting that tlg1 ts mutants are more susceptible to elevated levels of Sed5p. Tlg1p is able to bind His6-tagged Sec17p (yeast alpha-SNAP) in a dose-dependent manner and enters into a SNARE complex with Vti1p, Tlg2p, and Vps45p. Morphological analyses by electron microscopy reveal that cells depleted of Tlg1p or tlg1 ts mutants incubated at the restrictive temperature accumulate 40- to 50-nm vesicles and experience fragmentation of the vacuole.
Asunto(s)
Adenosina Trifosfatasas , Proteínas Portadoras/metabolismo , Proteínas Fúngicas/metabolismo , Aparato de Golgi/metabolismo , Proteínas de Transporte de Membrana , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular , Secuencia de Bases , Transporte Biológico , Proteínas Portadoras/genética , División Celular , Clonación Molecular , Proteínas Fúngicas/genética , Glicósido Hidrolasas/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Mutación , Proteínas Qa-SNARE , Proteínas Qb-SNARE , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida , Temperatura , Vacuolas/metabolismo , Vacuolas/ultraestructura , beta-FructofuranosidasaRESUMEN
Specific transport between secretory compartments requires that vesicular carriers contain targeting proteins known as SNAREs. Ten v-SNAREs have been identified in the genome of the yeast Saccharomyces cerevisiae by sequence analysis. We report here the characterization of Gos1p, a v-SNARE localized to the Golgi compartment and likely homolog of the mammalian protein GOS-28/GS28. Gos1p is a type II membrane protein with characteristic SNARE sequence hallmarks and is functionally a SNARE protein. Gos1p was originally identified as a 28 kDa protein in an immunoprecipitate of the cis-Golgi t-SNARE Sed5p. This interaction between Sed5p and Gos1p is direct as demonstrated by in vitro binding with recombinant proteins. Deletion of GOS1 results in viable haploids with modest growth and secretory defects. Close examination of the secretory phenotype of GOS1-disrupted cells suggests that Gos1p may play a role in multiple transport steps, specifically ER-Golgi and/or intra-Golgi transport.
Asunto(s)
Proteínas Fúngicas/fisiología , Aparato de Golgi/fisiología , Proteínas de la Membrana/fisiología , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiología , Transporte Biológico/genética , Carboxipeptidasas/metabolismo , Catepsina A , División Celular/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Aparato de Golgi/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Células HeLa , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutagénesis , Proteínas Qa-SNARE , Proteínas Qb-SNARE , Proteínas SNARE , Saccharomyces cerevisiae/ultraestructuraRESUMEN
Promoters that control gene expression in Saccharomyces cerevisiae only in a sporulation-specific manner have previously been isolated from a genomic yeast DNA library fused to a promoterless Escherichia coli lacZ gene. Two novel sporulation-specific genes, SPS18 and SPS19, were isolated using this technique. These genes are divergently controlled by the same promoter but with SPS18 expressed at four times the level of SPS19. Deletion analysis has shown that the promoter elements that exert sporulation control on each of the genes overlap, having a common 25 bp sequence located within the intergenic region. SPS18 encodes a 34-KDa protein of 300 amino acids that contains a putative zinc-binding domain and a region of highly basic residues that could target the protein to the nucleus. SPS19 encodes a 31-KDa protein of 295 amino acids, which has a peroxisomal targeting signal (SKL) at its C terminus; this protein belongs to the family of non-metallo short-chain alcohol dehydrogenases. A null mutation deleting the intergenic promoter prevented expression of both genes, and when homozygous in diploids, reduced the extent of sporulation four-fold; the spores that did form were viable, but failed to become resistant to ether, and were more sensitive to lytic enzymes. This phenotype reflects a defect in spore wall maturation, indicating that the product of at least one of the genes functions during the process of spore wall formation. Therefore these genes belong to the class of late sporulation-specific genes that are sequentially activated during the process of meiosis and spore formation.
Asunto(s)
Genes Fúngicos/genética , Regiones Promotoras Genéticas/genética , Saccharomyces cerevisiae/genética , Esporas Fúngicas/genética , Transcripción Genética/genética , Secuencia de Aminoácidos , Secuencia de Bases , Datos de Secuencia Molecular , Fenotipo , Eliminación de Secuencia , TATA BoxRESUMEN
Reporter genes consisting of sporulation-specific promoters fused to lacZ were used as markers to monitor the sporulation pathway of the yeast Saccharomyces cerevisiae. Strains transformed with these lacZ gene fusions expressed beta-galactosidase (assayable on plates using the substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside, X-gal) in a sporulation-dependent manner. Mutagenesis experiments performed on transformed strains resulted in the recovery of a number of novel sporulation mutants. Three classes of mutants were obtained: those which overexpressed the reporter gene under sporulation conditions, those which did not express the gene under any conditions, and those which expressed the gene in vegetative cells not undergoing sporulation. On the basis of the blue colony-colour produced in the presence of X-gal these have been described as superblue, white, and blue vegetative mutants, respectively. These were further characterised using earlier reporter genes and other marker systems. This study established that the multicopy reporter plasmids chosen do not interfere with sporulation; they are valid tools for monitoring the pathway and they provide a way to isolate mutations not readily selected by other markers.
Asunto(s)
Clonación Molecular/métodos , Genes Fúngicos , Genes Reporteros , Saccharomyces cerevisiae/genética , Metanosulfonato de Etilo/farmacología , Operón Lac , Mutagénesis , Saccharomyces cerevisiae/fisiología , Esporas Fúngicas/genética , Transformación Genética , Rayos UltravioletaRESUMEN
A library of random yeast genomic DNA:lacZ fusions has been constructed using an episomal yeast-Escherichia coli shuttle vector (pCS1). Plasmid pCS1 requires insertion of a promoter and an in frame ATG codon upstream of its resident truncated lacZ gene to regulate expression in yeast. Yeast genomic DNA fragments of 4-6 kb were generated by partial digestion with Sau3A and ligated into the unique BamHI site of plasmid pCS1 to generate a library of 5 x 10(4) individual E. coli transformants. This library was screened to identify promoter-lacZ fusions that were expressed uniquely during sporulation. Of 342 yeast transformants that exhibited beta-galactosidase activity, two were found to express the lacZ gene in a sporulation-specific manner. This paper presents the characterization of two genomic yeast DNA fragments containing promoters that control lacZ expression during the sporulation process. Expression from the promoter present in plasmid pJC18 occurred from 11-21 hours into the sporulation process, while the promoter in plasmid pJC217 was active from 4-14 hours. Staining of nuclear DNA to correlate nuclear morphology with timing of gene expression showed when each of these promoters was active in terms of the morphological stages of sporulation.