Guanosine diphosphatase is required for protein and sphingolipid glycosylation in the golgi lumen of Saccharomyces cerevisiae

• Published in: The Journal of cell biology 1993-07, Vol.122 (2), p.307-323
• Main Authors: Abeijon, C, Yanagisawa, K, Mandon, E.C, Hausler, A, Moremen, K, Hirschberg, C.B, Robbins, P.W
• Format: Article
• Language: English
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Amino Acid Sequence; Amino acids; APARATO GOLGI; APPAREIL DE GOLGI; Base Sequence; beta-Fructofuranosidase; Biological and medical sciences; Carboxypeptidases - biosynthesis; Cathepsin A; Cell structures and functions; Cell Wall - chemistry; Cell Wall - ultrastructure; Cellular biology; Ceramides - biosynthesis; CHITINASE; Chitinases - metabolism; Cloning, Molecular; Enzymes; FRUCTOFURANOSIDASA; FRUCTOFURANOSIDASE; Fundamental and applied biological sciences. Psychology; Gels; GENE; GENES; Genes, Fungal; Genetics; GLICOLIPIDOS; GLYCOLIPIDE; Glycoside Hydrolases - metabolism; Glycosylation; Golgi apparatus; Golgi Apparatus - metabolism; HIDROLASAS; HYDROLASE; LIPIDE; LIPIDOS; Lipids; Mannose - metabolism; Molecular and cellular biology; Molecular Sequence Data; Mutation; PEPTIDASAS; PEPTIDASE; Plasmids; Polymerase chain reaction; Proteins - metabolism; Pyrophosphatases - genetics; Pyrophosphatases - metabolism; QUITINASA; REACCIONES QUIMICAS; REACTION CHIMIQUE; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - growth & development; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae - ultrastructure; Saccharomyces cerevisiae Proteins; SECUENCIA NUCLEICA; SEQUENCE NUCLEIQUE; Sugars; Yeast; Yeasts
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.122.2.307

Current models for nucleotide sugar use in the Golgi apparatus predict a critical role for the lumenal nucleoside diphosphatase. After transfer of sugars to endogenous macromolecular acceptors, the enzyme converts nucleoside diphosphates to nucleoside monophosphates which in turn exit the Golgi lumen in a coupled antiporter reaction, allowing entry of additional nucleotide sugar from the cytosol. To test this model, we cloned the gene for the S. cerevisiae guanosine diphosphatase and constructed a null mutation. This mutation should reduce the concentrations of GDP-mannose and GMP and increase the concentration of GDP in the Golgi lumen. The alterations should in turn decrease mannosylation of proteins and lipids in this compartment. In fact, we found a partial block in O- and N-glycosylation of proteins such as chitinase and carboxypeptidase Y and underglycosylation of invertase. In addition, mannosylinositolphos-phorylceramide levels were drastically reduced