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dc.contributor.authorAbeijon, Claudia
dc.contributor.authorRobbins, Phillips W.
dc.contributor.authorHirschberg, Carlos B.
dc.date2022-08-11T08:09:36.000
dc.date.accessioned2022-08-23T16:37:16Z
dc.date.available2022-08-23T16:37:16Z
dc.date.issued1996-06-11
dc.date.submitted2009-04-02
dc.identifier.citation<p>Proc Natl Acad Sci U S A. 1996 Jun 11;93(12):5963-8.</p>
dc.identifier.issn0027-8424 (Print)
dc.identifier.pmid8650202
dc.identifier.urihttp://hdl.handle.net/20.500.14038/38975
dc.description.abstractThe mannan chains of Kluyveromyces lactis mannoproteins are similar to those of Saccharomyces cerevisiae except that they lack mannose phosphate and have terminal alpha1-->2-linked N-acetylglucosamine. The biosynthesis of these chains probably occurs in the lumen of the Golgi apparatus, by analogy to S. cerevisiae. The sugar donors, GDP-mannose and UDP-GlcNAc, must first be transported from the cytosol, their site of synthesis, via specific Golgi membrane transporters into the lumen where they are substrates in the biosynthesis of these mannoproteins. A mutant of K. lactis, mnn2-2, that lacks terminal N-acetylglucosamine in its mannan chains in vivo, has recently been characterized and shown to have a specific defect in transport of UDP-GlcNAc into the lumen of Golgi vesicles in vitro. We have now cloned the gene encoding the K. lactis Golgi membrane UDP-GlcNAc transporter by complementation of the mnn2-2 mutation. The mnn2-2 mutant was transformed with a genomic library from wild-type K. lactis in a pKD1-derived vector; transformants were isolated and phenotypic correction was monitored following cell surface labeling with fluorescein isothiocyanate conjugated to Griffonia simplicifolia II lectin, which binds terminal N-acetylglucosamine, and a fluorescent activated cell sorter. A 2.4-kb DNA fragment was found to restore the wild-type lectin binding phenotype. Upon loss of the plasmid containing this fragment, reversion to the mutant phenotype occurred. The above fragment contained an open reading frame for a multitransmembrane spanning protein of 328 amino acids. The protein contains a leucine zipper motif and has high homology to predicted proteins from S. cerevisiae and C. elegans. In an assay in vitro, Golgi vesicles isolated from the transformant had regained their ability to transport UDP-GlcNAc. Taken together, the above results strongly suggest that the cloned gene encodes the Golgi UDP-GlcNAc transporter of K. lactis.
dc.language.isoen_US
dc.relation<p><a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&list_uids=8650202&dopt=Abstract">Link to Article in PubMed</a></p>
dc.relation.urlhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC39171/
dc.subjectAmino Acid Sequence
dc.subjectBase Sequence
dc.subjectCell Separation
dc.subjectCloning, Molecular
dc.subjectDNA, Recombinant
dc.subjectEscherichia coli
dc.subjectFlow Cytometry
dc.subjectGolgi Apparatus
dc.subjectKluyveromyces
dc.subjectMolecular Sequence Data
dc.subjectPhosphoenolpyruvate Sugar Phosphotransferase System
dc.subjectUridine Diphosphate
dc.subjectLife Sciences
dc.subjectMedicine and Health Sciences
dc.titleMolecular cloning of the Golgi apparatus uridine diphosphate-N-acetylglucosamine transporter from Kluyveromyces lactis
dc.typeArticle
dc.source.journaltitleProceedings of the National Academy of Sciences of the United States of America
dc.source.volume93
dc.source.issue12
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/oapubs/1803
dc.identifier.contextkey808569
html.description.abstract<p>The mannan chains of Kluyveromyces lactis mannoproteins are similar to those of Saccharomyces cerevisiae except that they lack mannose phosphate and have terminal alpha1-->2-linked N-acetylglucosamine. The biosynthesis of these chains probably occurs in the lumen of the Golgi apparatus, by analogy to S. cerevisiae. The sugar donors, GDP-mannose and UDP-GlcNAc, must first be transported from the cytosol, their site of synthesis, via specific Golgi membrane transporters into the lumen where they are substrates in the biosynthesis of these mannoproteins. A mutant of K. lactis, mnn2-2, that lacks terminal N-acetylglucosamine in its mannan chains in vivo, has recently been characterized and shown to have a specific defect in transport of UDP-GlcNAc into the lumen of Golgi vesicles in vitro. We have now cloned the gene encoding the K. lactis Golgi membrane UDP-GlcNAc transporter by complementation of the mnn2-2 mutation. The mnn2-2 mutant was transformed with a genomic library from wild-type K. lactis in a pKD1-derived vector; transformants were isolated and phenotypic correction was monitored following cell surface labeling with fluorescein isothiocyanate conjugated to Griffonia simplicifolia II lectin, which binds terminal N-acetylglucosamine, and a fluorescent activated cell sorter. A 2.4-kb DNA fragment was found to restore the wild-type lectin binding phenotype. Upon loss of the plasmid containing this fragment, reversion to the mutant phenotype occurred. The above fragment contained an open reading frame for a multitransmembrane spanning protein of 328 amino acids. The protein contains a leucine zipper motif and has high homology to predicted proteins from S. cerevisiae and C. elegans. In an assay in vitro, Golgi vesicles isolated from the transformant had regained their ability to transport UDP-GlcNAc. Taken together, the above results strongly suggest that the cloned gene encodes the Golgi UDP-GlcNAc transporter of K. lactis.</p>
dc.identifier.submissionpathoapubs/1803
dc.contributor.departmentDepartment of Biochemistry and Molecular Biology
dc.source.pages5963-8


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