RESUMO
It has been proposed that in rat and murine tissues glucosidase II (GII) is formed by two subunits, GIIalpha and GIIbeta, respectively, responsible for the catalytic activity and the retention of the enzyme in the endoplasmic reticulum (ER). To test this proposal we disrupted genes (gls2alpha(+) and gls2beta(+)) encoding GIIalpha and GIIbeta homologs in Schizosaccharomyces pombe. Both mutant cells (gls2alpha and gls2beta) were completely devoid of GII activity in cell-free assays. Nevertheless, N-oligosaccharides formed in intact gls2alpha cells were identified as Glc(2)Man(9)GlcNAc(2) and Glc(2)Man(8)GlcNAc(2), whereas gls2beta cells formed, in addition, small amounts of Glc(1)Man(9)GlcNAc(2). It is suggested that this last compound was formed by GIIalpha transiently present in the ER. Monoglucosylated oligosaccharides facilitated glycoprotein folding in S. pombe as mutants, in which formation of monoglucosylated glycoproteins was completely (gls2alpha) or severely (gls2beta and UDP-Glc:glycoprotein:glucosyltransferase null) diminished, showed ER accumulation of misfolded glycoproteins when grown in the absence of exogenous stress as revealed by (a) induction of binding protein-encoding mRNA and (b) accumulation of glycoproteins bearing ER-specific oligosaccharides. Moreover, the same as in mammalian cell systems, formation of monoglucosylated oligosaccharides decreased the folding rate and increased the folding efficiency of glycoproteins as pulse-chase experiments revealed that carboxypeptidase Y arrived at a higher rate but in decreased amounts to the vacuoles of gls2alpha than to those of wild type cells.
Assuntos
Dobramento de Proteína , alfa-Glucosidases/química , Animais , Deleção de Genes , Regulação Enzimológica da Expressão Gênica , Camundongos , Ratos , Schizosaccharomyces , alfa-Glucosidases/genéticaRESUMO
Interaction of monoglucosylated oligosaccharides with ER lectins (calnexin and/or calreticulin) facilitates glycoprotein folding but this interaction is not essential for cell viability under normal conditions. We obtained two distinct single Schizosaccharomyces pombe mutants deficient in either one of the two pathways leading to the formation of monoglucosylated oligosaccharides. The alg6 mutant does not glucosy- late lipid-linked oligosaccharides and transfers Man9GlcNAc2 to nascent polypeptide chains and the gpt1 mutant lacks UDP-Glc:glycoprotein glucosyltransferase (GT). Both single mutants grew normally at 28 degreesC. On the other hand, gpt1/alg6 double-mutant cells grew very slowly and with a rounded morphology at 28 degreesC and did not grow at 37 degreesC. The wild-type phenotype was restored by transfection of the double mutant with a GT-encoding expression vector or by addition of 1 M sorbitol to the medium, indicating that the double mutant is affected in cell wall formation. It is suggested that facilitation of glycoprotein folding mediated by the interaction of monoglucosylated oligosaccharides with calnexin is essential for cell viability under conditions of extreme ER stress such as underglycosylation of proteins caused by the alg6 mutation and high temperature. In contrast, gls2/alg6 double-mutant cells that transfer Man9GlcNAc2 and that are unable to remove the glucose units added by GT as they lack glucosidase II (GII), grew at 37 degreesC and had, when grown at 28 degreesC, a phenotype of growth and morphology almost identical to that of wild-type cells. These results indicate that facilitation of glycoprotein folding mediated by the interaction of calnexin and monoglucosylated oligosaccharides does not necessarily require cycles of reglucosylation-deglucosylation catalyzed by GT and GII.
Assuntos
Retículo Endoplasmático/metabolismo , Glucosiltransferases/metabolismo , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/metabolismo , Sequência de Carboidratos , Glucosiltransferases/genética , Glicosilação , Dados de Sequência Molecular , Mutagênese , Oligossacarídeos/metabolismo , Fenótipo , Dobramento de Proteína , Schizosaccharomyces/genética , TemperaturaRESUMO
A key element in the quality control of glycoprotein folding is the UDP-Glc:glycoprotein glucosyltransferase (GT), which in cell-free assays exclusively glucosylates misfolded glycoproteins. In order to test if such a protein conformation is a sufficient condition for in vivo glucosylation of all N-linked oligosaccharides by GT, a Schizosaccharomyces pombe double mutant (gls2/alg6) was constructed. With this mutant, Man9GlcNAc2 is transferred to proteins and no removal of glucose units added by GT occurs as it lacks glucosidase II. The same proportion of glucosylated (Glc1Man9GlcNAc2) and unglucosylated (Man9GlcNAc2 and Man8GlcNAc2) endoplasmic reticulum (ER)-specific compounds was produced when cells were pre-incubated for 10, 20 or 30 min and further incubated with [14C]glucose for 10 min at 28 degrees C with or without 5 mM dithiothreitol (DTT), thus indicating not only that DTT did not affect protein glucosylation but also that no increased glucosylation of glycoproteins occurred in the presence of the drug. Monitoring Golgi-specific modifications of oligosaccharides after pulse-chase experiments performed in the presence or absence of 5 mM DTT showed that exit of the bulk of glycoproteins synthesized from the ER and thence their proper folding had been prevented by the drug. Cells pulse-chase labeled at 37 degrees C in the absence of DTT also yielded glucosylated and unglucosylated protein-linked oligosaccharides without Golgi-specific modifications. It was concluded that a misfolded protein conformation is not a sufficient condition for in vivo glucosylation of all N-linked oligosaccharides by GT.
Assuntos
Glucosiltransferases/metabolismo , Dobramento de Proteína , Ditiotreitol/farmacologia , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Glucose/metabolismo , Glucosiltransferases/genética , Resposta ao Choque Térmico , Mutação , Oligossacarídeos/metabolismo , Conformação Proteica/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMO
Previous work indicated that diacylglycerol (DG) molecules translocate across the cytoplasm of mammalian cells, a process relevant to the signalling role of this lipid as protein kinase C activator. Here we investigated the possible mechanism underlying DG translocation. We examined the interaction between 1,2-di-[1-14C]oleoyl-sn-glycerol and rat liver cytosol (rlc) using assays based on Lipidex-1000 and on coelution on Sepharose CL 6B. We measured high DG binding activity and found that it resides in cytosolic proteins and not in cytosolic lipids. Chromatography of rlc proteins on Sepharose CL 6B showed profiles in which the activity measured by either method coincided. Further, we showed that the DG-rlc protein interaction results in the stabilization of DG in a micellar form, eluting in the void volume of Sepharose CL 6B. Such stabilized micelles are reminiscent of insect lipophorins and may represent a new, thus far unrecognized, mode of lipid transport within living cells.
Assuntos
Diglicerídeos/metabolismo , Lipoproteínas/metabolismo , Micelas , Animais , Transporte Biológico , Cromatografia em Agarose , Citosol/metabolismo , Dextranos/metabolismo , Feminino , Fígado/ultraestrutura , Ácido Oleico/metabolismo , RatosRESUMO
Several distinct types of voltage-gated and second-messenger-operated K+, Ca2+, Na+ and Cl- channels exist in electrically non excitable cells such as those of the hematopoietic lineage. In these cells ion channels mediate cellular functions involving intracellular biochemical responses, rather than rapid electrical signaling. The presence of the channels is required for several basic functions, such as activation, secretion of lymphokines, mitogenesis, the regulation of cell volume and the mechanisms of resistance to chemotherapeutic agents. Here IN we review the patch-clamp method for studying many characteristics of these ionic channels, particularly in blood cells.