GUANOSINE DIPHOSPHATASE IS REQUIRED FOR PROTEIN AND SPHINGOLIPID GLYCOSYLATION IN THE GOLGI LUMEN OF SACCHAROMYCES-CEREVISIAE

Current models for nucleotide sugar use in the Golgi apparatus predict a critical role for the lumenal nucleoside diphosphatase. After trans fer of sugars to endogenous macromolecular acceptors, the enzyme conve rts nucleoside diphosphates to nucleoside monophosphates which in tum exit the Golgi lumen in a coupled antiporter reaction, allowing entry of additional nucleotide sugar from the cytosol. To test this model, w e cloned the gene for the S. cerevisiae guanosine diphosphatase and co nstructed a null mutation. This mutation should reduce the concentrati ons of GDP-mannose and GMP and increase the concentration of GDP in th e Golgi lumen. The alterations should in tum 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 carb oxypeptidase Y and underglycosylation of invertase. In addition, manno sylinositolphosphorylceramide levels were drastically reduced.