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.
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.
Ishida
N
N
Department of Physiological Chemistry, The Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo, 113-8613, Japan.
Yoshioka
S
S
Iida
M
M
Sudo
K
K
Miura
N
N
Aoki
K
K
Kawakita
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D002871:Chromosome Banding; D003001:Cloning, Molecular; D006224:Cricetinae; D006056:Golgi Apparatus; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D000099237:UDP-Galactose Translocators
D009004:Monosaccharide Transport Proteins; D000099237:UDP-Galactose Translocators