MAMMALIAN GOLGI-APPARATUS UDP-N-ACETYLGLUCOSAMINE TRANSPORTER - MOLECULAR-CLONING BY PHENOTYPIC CORRECTION OF A YEAST MUTANT

Transporters in the Golgi apparatus membrane translocate nucleotide su gars from the cytosol into the Golgi lumen before these can be substra tes for the glycosylation of proteins, lipids, and proteoglycans, We h ave cloned the mammalian Golgi membrane transporter for uridine diphos phate-N-acetylglucosamine by phenotypic correction with cDNA from MDCK cells of a recently characterized Kluyveromyces lactis mutant deficie nt in Golgi transport of the above nucleotide sugar. Phenotypically co rrected transformants were separated from mutants in a fluorescent-act ivated 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 fou nd to restore the wild-type cell lectin binding phenotype, which rever ted to the mutant one upon loss of the plasmid, The DNA fragment conta ined an ORF encoding a hydrophobic, multitransmembrane spanning protei n of 326 aa that had only 22% amino acid sequence identity with the co rresponding transporter from K, lactis but showed 53% amino acid seque nce identity to the mammalian UDP-galactose transporters and 40% to th e CMP-sialic acid transporter. Golgi vesicles from the transformant re gained their ability to transport UDP-GlcNAc in an assay in vitro. The above results demonstrate that the mammalian Golgi UDP-GlcNAc transpo rter gene has all of the necessary information for the protein to be e xpressed and targeted functionally to the Golgi apparatus of yeast and that two proteins with very different amino acid sequences may transp ort the same solute within the same Golgi membrane.