Title

Molecular cloning of the Golgi apparatus uridine diphosphate-N-acetylglucosamine transporter from Kluyveromyces lactis

UMMS Affiliation

Department of Biochemistry and Molecular Biology

Date

6-11-1996

Document Type

Article

Subjects

Amino Acid Sequence; Base Sequence; Cell Separation; Cloning, Molecular; DNA, Recombinant; Escherichia coli; Flow Cytometry; Golgi Apparatus; Kluyveromyces; Molecular Sequence Data; Phosphoenolpyruvate Sugar Phosphotransferase System; Uridine Diphosphate

Disciplines

Life Sciences | Medicine and Health Sciences

Abstract

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.

Rights and Permissions

Citation: Proc Natl Acad Sci U S A. 1996 Jun 11;93(12):5963-8.

Related Resources

Link to Article in PubMed

Journal Title

Proceedings of the National Academy of Sciences of the United States of America

PubMed ID

8650202