Biogenesis of vacuolar membrane glycoproteins of yeast Saccharomyces cerevisiae.
To investigate the biogenesis of the yeast vacuole, we have sought novel marker proteins localized to the vacuolar membrane. Glycoproteins were prepared from vacuolar membrane vesicles by concanavalin A-Sepharose column chromatography and used to raise monoclonal antibodies. The antibodies obtained recognize several vacuolar proteins that have N-linked oligosaccharide chains. A set of the antibodies reacts with a vacuolar glycoprotein with a major molecular species of 72 kDa (vgp72), which appears to associate peripherally with the vacuolar membrane. The biosynthesis of vgp72 has been examined in detail by pulse-chase experiments and by analyses using various secretory mutants (sec18, sec7, and sec1) and a vacuolar protease mutant (pep4). vgp72 first appears in the endoplasmic reticulum as a 74-kDa species and is quickly modified in the Golgi apparatus to two distinct species: a 79-kDa form, and a heterogeneously glycosylated form (90-150 kDa). Subsequently, both species are proteolytically processed in the vacuole giving rise to a 72-kDa species as well as heavily glycosylated form. Thus, the biogenesis of vgp72 utilizes the early part of the secretory pathway as is the case of vacuolar soluble enzymes. A unique feature is that two species that are different in the extent of glycosylation appear to follow the same destination to the vacuolar membrane.
Nishikawa
S
S
Department of Biology, Faculty of Science, University of Tokyo, Japan.
Umemoto
N
N
Ohsumi
Y
Y
Nakano
A
A
Anraku
Y
Y
D000118:Acetylglucosaminidase; D000911:Antibodies, Monoclonal; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D005455:Fluorescent Antibody Technique; D005838:Genotype; D007425:Intracellular Membranes / Q000378:metabolism / Q000648:ultrastructure; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000302:isolation & purification; D008970:Molecular Weight; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D014617:Vacuoles / Q000378:metabolism* / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D008562:Membrane Glycoproteins; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase