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Mutants of the CMP-sialic acid transporter causing the Lec2 phenotype
M Eckhardt1, B Gotza, R Gerardy-Schahn
1Institut für Medizinische Mikrobiologie, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.
The Journal of Biological Chemistry
|August 1, 1998
Summary
Molecular defects in the CMP-sialic acid transporter were identified in Chinese hamster ovary (CHO) Lec2 mutants. A specific point mutation (G189E) directly impairs transporter activity, crucial for sialic acid transport.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Genetics
Background:
- Chinese hamster ovary (CHO) Lec2 mutants lack Golgi CMP-sialic acid transport.
- Complementation cloning identified mouse and hamster CMP-sialic acid transporter cDNAs.
Purpose of the Study:
- To determine the molecular defects causing CMP-sialic acid transporter inactivation in Lec2 mutants.
- To analyze CMP-sialic acid transporter cDNAs from five independent Lec2 clones.
Main Methods:
- Analysis of CMP-sialic acid transporter cDNAs from Lec2 mutants.
- Epitope-tagged protein expression and immunofluorescence studies.
- Site-directed mutagenesis to probe specific amino acid effects.
Main Results:
- Deletions in three clones caused reduced protein expression and cytoplasmic localization.
- A point mutation (G189E) resulted in wild-type expression and Golgi localization, but impaired transport.
- Mutagenesis of Glycine 189 showed G189A complemented transport, while G189Q and G189I abolished activity.
Conclusions:
- Molecular defects in the CMP-sialic acid transporter include deletions and point mutations.
- The G189E mutation directly affects CMP-sialic acid transporter activity.
- Glycine 189 is critical for CMP-sialic acid transporter function.