Characterization of a mutation and an alternative splicing of UDP-galactose transporter in MDCK-RCAr cell line.
The UDP-galactose (UDP-Gal) transporter present in the Golgi apparatus is a member of a transporter family comprising hydrophobic proteins with multiple transmembrane domains. Co-immunoprecipitation experiments showed that the full-length UDP-Gal transporter protein forms oligomeric structures in the MDCK cell. A ricin-resistant mutant of the MDCK cell line (MDCK-RCA(r)) is deficient in galactose linked to macromolecules because of a lower UDP-Gal transport rate into the Golgi apparatus. We cloned this mutated protein and found that it contains a stop codon close to the 5' terminus of its open reading frame. We also detected a shorter splicing variant of the UDP-Gal transporter which contains a 183-nt in-frame deletion in both the wild-type and the mutant mRNA. We showed that the protein, when overexpressed, is localized in the Golgi apparatus and could partially correct the phenotype of the MDCK-RCA(r) and CHO-Lec8 mutant cell lines. The level of mRNA of the UDP-Gal transporter is much lower (25-30 copies per cell) than those of the CMP-sialic acid transporter (100 copies per cell), UDP-N-acetylglucosamine transporter (80 copies per cell), and GDP-fucose transporter (65 copies per cell). The transcript level of the shorter splicing variant of the UDP-Gal transporter is extremely rare in wild-type MDCK cells (a few copies per cell), but it is significantly increased in the mutant, RCA-resistant cells.
Olczak
Mariusz
M
Laboratory of Biochemistry, Institute of Biochemistry and Molecular Biology, Wroclaw University, Tamka 2, 50-137 Wroclaw, Poland. olczak@bf.uni.wroc.pl
Guillen
Eduardo
E
D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004285:Dogs; D005091:Exons / Q000235:genetics; D006056:Golgi Apparatus / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D010641:Phenotype; D037121:Plant Lectins / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D000099237:UDP-Galactose Translocators
D009004:Monosaccharide Transport Proteins; D037121:Plant Lectins; D012333:RNA, Messenger; D000099237:UDP-Galactose Translocators; C017707:Ricinus communis agglutinin-1