Genetic evidence for the heterodimeric structure of glucosidase II. The effect of disrupting the subunit-encoding genes on glycoprotein folding.
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.
D'Alessio
C
C
Instituto de Investigaciones Bioquímicas Fundación Campomar, Antonio Machado 151, 1405 Buenos Aires, Argentina.
Fernández
F
F
Trombetta
E S
ES
Parodi
A J
AJ
D000818:Animals; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic; D051379:Mice; D017510:Protein Folding; D051381:Rats; D012568:Schizosaccharomyces; D000520:alpha-Glucosidases / Q000737:chemistry* / Q000235:genetics
C034251:4-nitrophenyl-alpha-glucosidase; D000520:alpha-Glucosidases