RESUMEN
We report here an in vivo study of kinesin heavy chain (KHC) functions in yeast. We have identified in Schizosaccharomyces pombe a kinesin motor gene, klp3(+), which has the highest homology to the Neurospora crassa KHC. Using indirect immunofluorescence, HA epitope-tagged Klp3 protein is cytoplasmic and appears as one to a few distinct patches that are coincident with microtubules. The klp3 null allele is viable. In klp3 deleted cells, ER, Golgi and mitochondrial distribution appear normal. Mitochondrial distribution in S. pombe is known to be microtubule-associated. We show that latrunculin A does not cause mitochondria to aggregate, suggesting that mitochondrial distribution in fission yeast, unlike budding yeast, is not dependent upon actin-based processes. Neither latrunculin A nor thiabendazole affects ER or Golgi distribution. We also used the vital dye FM4-64 to visualize the internalization of the dye and its transport to vacuoles in fission yeast in the presence and absence of Klp3. We observed no significant difference between the wild-type and Klp3 null cells in either the dynamics of endocytosis or the distribution and fusion of vacuoles. The drug brefeldin A causes Golgi-to-ER recycling in wild-type fission yeast cells. Although recycling of Golgi to ER after brefeldin A treatment occurs in klp3 null cells, recycling is defective and the distribution pattern we see is different from that observed in the wild-type strain. We conclude that Klp3 plays a role in BFA-induced membrane transport. The nucleotide sequence of S. pombe klp3(+) was submitted to GenBank under Accession No. AF154055.
Asunto(s)
Proteínas de Caenorhabditis elegans , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Aparato de Golgi/metabolismo , Cinesinas/genética , Cinesinas/metabolismo , Schizosaccharomyces/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Transporte Biológico , Brefeldino A/farmacología , Clonación Molecular , ADN Complementario , Retículo Endoplásmico/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas Fúngicas/química , Galactosiltransferasas/genética , Galactosiltransferasas/metabolismo , Proteínas Fluorescentes Verdes , Membranas Intracelulares/metabolismo , Cinesinas/química , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Electrónica , Microtúbulos/metabolismo , Datos de Secuencia Molecular , Orgánulos/metabolismo , Plásmidos/genética , Schizosaccharomyces/genética , Tiabendazol/farmacologíaRESUMEN
We have cloned a gene encoding an alpha 1,2 galactosyltransferase activity from Schizosaccharomyces pombe. The open reading frame of the gene (gma12 for galactomannan, alpha 1,2), combined with the previous protein purification (Chappell and Warren, 1989), predicts an O-linked glycoprotein with type II transmembrane topology. By homologous gene disruption, we have demonstrated that the gma12 gene product (gma12p) is nonessential. The deletion strain (gma12-D10::ura4) has a significantly reduced level of galactosyltransferase activity relative to the parental strain, but both in situ lectin binding and in vitro biochemical assays demonstrate the presence of further galactosyltransferase activity in addition to gma12p. Although gma12p is not the only galactosyltransferase in S. pombe, it produces a unique carbohydrate structure on the surface of the yeast cells. We have generated a polyclonal antiserum against this carbohydrate epitope and shown that gma12p is capable of synthesizing the epitope both in vitro and in vivo. Electron microscopic localization of the gma12+ specific epitope in gma12+ cells revealed that gma12p synthesizes the carbohydrate structure in the Golgi apparatus, and subsequent intracellular transport distributes the epitope to later stages of the secretory pathway. The immunolocalization studies confirm the presence of one or more galactosyltransferase activities in the Golgi apparatus in fission yeast.
Asunto(s)
Galactosiltransferasas/metabolismo , Aparato de Golgi/enzimología , Schizosaccharomyces/enzimología , Secuencia de Aminoácidos , Antígenos Fúngicos/metabolismo , Secuencia de Bases , Metabolismo de los Hidratos de Carbono , Carbohidratos/inmunología , Clonación Molecular , ADN de Hongos/genética , Epítopos/metabolismo , Galactosiltransferasas/genética , Genes Fúngicos , Aparato de Golgi/ultraestructura , Inmunohistoquímica , Microscopía Electrónica , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Mapeo Restrictivo , Schizosaccharomyces/genética , Schizosaccharomyces/inmunologíaRESUMEN
A membrane-associated galactosyltransferase has been purified to homogeneity from the fission yeast, Schizosaccharomyces pombe. The enzyme has a molecular weight of 61,000 and is capable of transfering galactose from UDP-galactose (UDP-Gal) to a variety of mannose-based acceptors to form an alpha-1,2 galactosyl mannoside linkage. Immunofluorescence localization of the protein is consistent with the presence of the enzyme in the Golgi apparatus of S. pombe. This, together with the presence of terminal, alpha-linked galactose on the N-linked oligosaccharides of S. pombe secretory proteins, suggests that the galactosyltransferase is an enzyme involved in the processing of glycoproteins transported through the Golgi apparatus in fission yeast.
Asunto(s)
Galactosiltransferasas/metabolismo , Aparato de Golgi/enzimología , Microsomas/enzimología , Saccharomycetales/enzimología , Schizosaccharomyces/enzimología , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Citosol/enzimología , Ácido Edético/farmacología , Técnica del Anticuerpo Fluorescente , Galactosiltransferasas/aislamiento & purificación , Glicoproteínas/genética , Aparato de Golgi/ultraestructura , Histocitoquímica , Cinética , Manganeso/farmacología , Peso Molecular , Procesamiento Proteico-Postraduccional , Schizosaccharomyces/ultraestructura , Especificidad por SustratoRESUMEN
Two members of the hsp70 family, termed hsc70 and BiP, have been implicated in promoting protein folding and assembly processes in the cytoplasm and the lumen of the endoplasmic reticulum, respectively. Short hydrophilic (8 to 25 residues) synthetic peptides have now been tested as possible mimics of polypeptide chain substrates to help define an enzymatic basis for these activities. Both BiP and hsc70 have specific peptide binding sites. Peptide binding elicits hydrolysis of adenosine triphosphate, with the subsequent release of bound peptide.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares , Péptidos/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Retículo Endoplásmico/metabolismo , Chaperón BiP del Retículo Endoplásmico , Hidrólisis , Microsomas Hepáticos/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Conformación ProteicaRESUMEN
Chymotryptic digestion of bovine brain uncoating ATPase produced a 60-kDa fragment that was subsequently proteolyzed to 44 kDa. Loss of clathrin cage uncoating activity paralleled the conversion of the intact 70-kDa enzyme to the 60-kDa fragment, while clathrin binding activity was lost as the 60-kDa fragment was degraded to 44 kDa. This 44-kDa fragment has been purified to homogeneity and characterized as a clathrin-independent ATPase. The 44-kDa ATPase domain has been localized within the intact enzyme by the use of amino-terminal specific antibodies. This localization relates to the conserved nature of the 70-kDa heat shock protein family, of which bovine brain uncoating ATPase is a constitutively expressed member.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Encéfalo/enzimología , Proteínas Portadoras/metabolismo , Proteínas HSP70 de Choque Térmico , Adenosina Trifosfatasas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas , Proteínas Portadoras/aislamiento & purificación , Bovinos , Quimotripsina/metabolismo , Clatrina/aislamiento & purificación , Clatrina/metabolismo , Proteínas del Choque Térmico HSC70 , RatasRESUMEN
The synthetic peptide, VGIDLGTTYSC, derived from the heat shock-induced genes human hsp70, Drosophila hsp70, S. cerevisiae YG100, and E. coli dnaK, elicited antibodies that recognized two constitutive proteins in bovine extracts. One of these proteins, 71 kd, has previously been identified as uncoating ATPase, an enzyme that releases clathrin from coated vesicles. This immunological data complemented the result that uncoating ATPase was indistinguishable from the constitutive mammalian 71 kd stress protein by either partial proteolytic mapping or two-dimensional gel analysis. In addition, affinity-purified uncoating ATPase antibodies recognize proteins in yeast identified as the gene products of the heat shock or heat shock cognate genes YG100 and YG102. The results show that uncoating ATPase is a member of the 70 kd heat shock protein family.
Asunto(s)
Adenosina Trifosfatasas/genética , Proteínas Portadoras/genética , Proteínas HSP70 de Choque Térmico , Proteínas de Choque Térmico/genética , Adenosina Difosfato Ribosa/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Proteínas Portadoras/metabolismo , Bovinos , Clatrina/metabolismo , Cricetinae , Citosol/metabolismo , Proteínas del Choque Térmico HSC70 , Humanos , Punto Isoeléctrico , Proteínas de la Membrana/genética , Microsomas/metabolismo , Peso Molecular , Fragmentos de Péptidos/análisis , Saccharomyces cerevisiae/genéticaRESUMEN
We have synthesized microgram quantities of a functional eucaryotic mRNA by in vitro transcription. For this purpose, we constructed a plasmid in which the Escherichia coli lactose promoter was 5' to the vesicular stomatitis virus (VSV) G protein gene (Rose, J. K., and C. J. Gallione, 1981, J. Virol., 39:519-528). This DNA served as the template in an in vitro transcription reaction utilizing E. coli RNA polymerase. The RNA product was capped using the vaccinia guanylyltransferase. A typical preparation of the synthetic G mRNA was equivalent to the amount of G mRNA that can be isolated from approximately 10(8) VSV-infected cells. This synthetic mRNA was translated by a wheat germ extract in the presence of microsomes, producing a polypeptide that was indistinguishable from G protein in its size, antigenicity, degree of glycosylation, and its membrane insertion. This technique should aid in identifying features needed by proteins for insertion into membranes.