Mutants of the CMP-sialic acid transporter causing the Lec2 phenotype.
Chinese hamster ovary (CHO) mutants belonging to the Lec2 complementation group are unable to translocate CMP-sialic acid to the lumen of the Golgi apparatus. Complementation cloning in these cells has recently been used to isolate cDNAs encoding the CMP-sialic acid transporter from mouse and hamster. The present study was carried out to determine the molecular defects leading to the inactivation of CMP-sialic acid transport. To this end, CMP-sialic acid transporter cDNAs derived from five independent clones of the Lec2 complementation group, were analyzed. Deletions in the coding region were observed for three clones, and single mutants were found to contain an insertion and a point mutation. Epitope-tagged variants of the wild-type transporter protein and of the mutants were used to investigate the effect of the structural changes on the expression and subcellular targeting of the transporter proteins. Mutants derived from deletions showed reduced protein expression and in immunofluorescence showed a diffuse staining throughout the cytoplasm in transiently transfected cells, while the translation product derived from the point-mutated cDNA (G189E) was expressed at the level of the wild-type transporter and co-localized with the Golgi marker alpha-mannosidase II. This mutation therefore seems to directly affect the transport activity. Site-directed mutagenesis was used to change glycine 189 into alanine, glutamine, and isoleucine, respectively. While the G189A mutant was able to complement CMP-sialic acid transport-deficient Chinese hamster ovary mutants, the exchange of glycine 189 into glutamine or isoleucine dramatically affected the transport activity of the CMP-sialic acid transporter.
Institut für Medizinische Mikrobiologie, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.
M
M
Eckhardt
B
B
Gotza
R
R
Gerardy-Schahn
D000818:Animals; D001692:Biological Transport / Q000235:genetics; D016466:CHO Cells; D002352:Carrier Proteins / Q000737:chemistry*; D003001:Cloning, Molecular; D006224:Cricetinae; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism; D004252:DNA Mutational Analysis; D000939:Epitopes / Q000276:immunology; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000502:physiology; D008565:Membrane Proteins / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009842:Oligopeptides; D010455:Peptides / Q000276:immunology; D012333:RNA, Messenger / Q000378:metabolism; D014162:Transfection / Q000235:genetics
D002352:Carrier Proteins; D000939:Epitopes; D008565:Membrane Proteins; D009842:Oligopeptides; D010455:Peptides; D012333:RNA, Messenger; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; C085706:FLAG peptide