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, mannosylinositolphosphorylceramide levels were drastically reduced.

Normal, unimmunized mouse serum from several strains (BALB/c, C57/b, DBA/2, NZB, SJL, CD/1) contains an endogenous IgG antibody that localizes to the Golgi complex of rat pancreatic acinar cells. Treatment of pancreatic acini with 5 microM monensin resulted in the swelling and vacuolization of the Golgi cisternae, and in a corresponding annular staining by the mouse serum as observed by immunofluorescence, suggesting that the antigen recognized is on the Golgi complex cisternal membrane. The antiserum did not react with pancreatic secretory proteins, and its binding to smooth microsomal membranes was retained following sodium carbonate washing, supporting a Golgi membrane localization. Advantage was taken of the existence of the endogenous murine antibody for the isolation of monoclonal antibodies directed to the Golgi complex of the rat pancreas. Two antibodies, antiGolgi 1 and antiGolgi 2, are described. Both antibodies are IgMs that recognize integral membrane proteins of the trans-Golgi cisternae, with lighter and patchy staining of the pancreatic lumen membrane, as observed both by light and electron microscopy. AntiGolgi 1 recognizes predominately a protein of molecular weight 103,000-108,000, whereas antiGolgi 2 shows a strong reaction to a 180-kd band as well as the 103-108-kd protein.