Mechanism of galactosylation in the Golgi apparatus. A Chinese hamster ovary cell mutant deficient in translocation of UDP-galactose across Golgi vesicle membranes.

Abstract

The biochemical defect in the mutant Chinese hamster ovary cell lines Clone 13 (Briles, E. B., Li, E., and Kornfeld, S. (1977). J. Biol. Chem. 252, 1107-1116) and Lec8 (Stanley, P. (1980) ACS Symp. Ser. 128, 214-221) was examined. These two mutants, which belong to the same genetic complementation group, were shown in previous studies to exhibit an 80-90% reduction in galactosylation and sialylation of proteins and lipids when compared to wild-type cells. The same studies, however, demonstrated that the mutants were not deficient in the corresponding sugar nucleotides, glycosyltransferases, and endogenous acceptors for these transferases. We now provide evidence strongly suggesting that the primary defect in Lec8 and Clone 13 cells is their inability to translocate UDP-galactose into the lumen of the Golgi apparatus. Golgi vesicles from Lec8 and Clone 13 CHO glycosylation mutants translocate in vitro UDP-galactose at only 3-5% the rate of vesicles from wild-type CHO cells. The deficiency is specific because vesicles from the mutant cells can translocate adenosine 3'-phosphate 5'-phosphosulfate, UDP-N-acetylglucosamine, and UDP-N-acetylgalactosamine at rates comparable to those of vesicles from wild-type cells. These studies also suggest that sugar nucleotides sharing a common uridine nucleotide utilize different translocators present in the Golgi membrane in vivo. The consequence of the above-described mutations and the resulting block in galactosylation of macromolecules in vivo on the translocation of CMP-sialic acid into the Golgi lumen was also examined. As expected, Golgi apparatus vesicles from Lec8 cells were unable to incorporate sialic acid into (endogenous) macromolecules. However, the vesicles were able to transport CMP-sialic acid into their lumen, although the rate of translocation was only 17% of that of wild-type-derived Golgi vesicles.