文章基本信息

标题：Mammalian Golgi apparatus UDP-N-acetylglucosamine transporter: Molecular cloning by phenotypic correction of a yeast mutant
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作者：Eduardo Guillen ; Claudia Abeijon ; Carlos B. Hirschberg 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1998
卷号：95
期号：14
页码：7888-7892
DOI：10.1073/pnas.95.14.7888
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Transporters in the Golgi apparatus membrane translocate nucleotide sugars from the cytosol into the Golgi lumen before these can be substrates for the glycosylation of proteins, lipids, and proteoglycans. We have cloned the mammalian Golgi membrane transporter for uridine diphosphate-N-acetylglucosamine by phenotypic correction with cDNA from MDCK cells of a recently characterized Kluyveromyces lactis mutant deficient in Golgi transport of the above nucleotide sugar. Phenotypically corrected transformants were separated from mutants in a fluorescent-activated cell sorter after labeling of K. lactis cells with fluorescein isothiocyanate (FITC) conjugated to Griffonia simplicifolia II lectin, which binds terminal N-acetylglucosamine. A 2-kb DNA fragment was found to restore the wild-type cell lectin binding phenotype, which reverted to the mutant one upon loss of the plasmid. The DNA fragment contained an ORF encoding a hydrophobic, multitransmembrane spanning protein of 326 aa that had only 22% amino acid sequence identity with the corresponding transporter from K. lactis but showed 53% amino acid sequence identity to the mammalian UDP-galactose transporters and 40% to the CMP-sialic acid transporter. Golgi vesicles from the transformant regained their ability to transport UDP-GlcNAc in an assay in vitro. The above results demonstrate that the mammalian Golgi UDP-GlcNAc transporter gene has all of the necessary information for the protein to be expressed and targeted functionally to the Golgi apparatus of yeast and that two proteins with very different amino acid sequences may transport the same solute within the same Golgi membrane.
关键词：membranes/glycoproteins/proteoglycans/glycosylation