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J Biol Chem, Vol. 273, Issue 32, 20189-20195, August 7, 1998
From the Institut für Medizinische Mikrobiologie,
Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany
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
Mutants of the CMP-sialic Acid Transporter Causing the Lec2
Phenotype
-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.
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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