Indispensability of Transmembrane Domains of Golgi UDP-Galactose Transporter as Revealed by Analysis of Genetic Defects in UDP-Galactose Transporter-Deficient Murine Had-1 Mutant Cell Lines and Construction of Deletion Mutants

Abstract

UDP-galactose transporter is a membrane protein localized in the Golgi apparatus. It translocates UDP-galactose from the cytosol into the Golgi lumen, thus providing galactosyltransferases with their substrate. We characterized murine UDP-galactose transporter through molecular cloning for the following purposes: (i) to elucidate the molecular bases underlying the genetic defects of murine Had-1 mutants, which are deficient in UDP-galactose transporting activity, and (ii) to obtain information that would help us in planning rational approaches to identify functionally essential regions, based on comparison of primary structures between human and murine UDP-galactose transporters. We identified five nonsense mutations, one missense Gly178Asp mutation, and two aberrant splicing mutations. Although glycine178 is highly conserved among nucleotide-sugar transporters, a Gly178Ala variant was functional. The species-differences between human and murine UDP-galactose transporters were largely confined to the N- and C-terminal regions of the transporters. Substantial deletions in the N- and C-terminal regions did not lead to loss of UDP-galactose transporting activity, indicating that these cytosolic regions are dispensable for the transporting activity. The transporter was fused with green-fluorescent protein at the C-terminal cytosolic tail without impairing the functions of either protein. Our results demonstrate the importance of the transmembrane core region of the UDP-galactose transporter protein.

Point Mutations Identified in Lec8 Chinese Hamster Ovary Glycosylation Mutants That Inactivate Both the UDP-galactose and CMP-sialic Acid Transporters

“…As expected from previous truncation experiments (11), the cytoplasmic tails were interchangeable. However, exchanges of amino acid stretches that represent potential transmembrane domains inactivated the UGT with a single exception.…”

“…In contrast, the cytosolically located N-and C-terminal tails of murine UGT were found to be dispensable. Deletion of both ends affected neither the activity nor the subcellular destination of the UGT (11).…”

“…1B, the amino acid sequence deduced from the newly cloned gene is aligned to UGT sequences from human (9, 12), mouse (11), and yeast (13). The high conservation between the mammalian genes (overall identity Ͼ93%; see Fig.…”

“…Such an analysis in CHO Lec2 cells (defective in Golgi transport of CMP-sialic acid) and in murine Had-1 cells identified two highly conserved amino acids (Gly 189 in the hamster CST and Gly 178 in the murine UGT) that are likely to participate in the folding of active transporters (11,21).…”

Transport of UDP-galactose in Plants

“…Complementation of mutants allowed the cloning of putative UDP-galactose transporter genes from human and Schizosaccharomyces pombe (6,10). Through PCR-based approaches and screening of libraries, murine, hamster, and Drosophila cDNAs were also isolated (6,7,11). Expression of these genes in Saccharomyces cerevisiae followed by in vitro transport assays into Golgi vesicles confirmed that the gene products actually transport UDP-galactose (12).…”