The genes for the Golgi apparatus N-acetylglucosaminyltransferase and the UDP-N-acetylglucosamine transporter are contiguous in Kluyveromyces lactis.
The mannan chains of Kluyveromyces lactis mannoproteins are similar to those of Saccharomyces cerevisiae except that they lack mannose phosphate and have terminal alpha(1-->2)-linked N-acetylglucosamine. Previously, Smith et al. (Smith, W. L. Nakajima, T., and Ballou, C. E. (1975) J. Biol. Chem. 250, 3426-3435) characterized two mutants, mnn2-1 and mnn2-2, which lacked terminal N-acetylglucosamine in their mannoproteins. The former mutant lacks the Golgi N-acetylglucosaminyltransferase activity, whereas the latter one was recently found to be deficient in the Golgi UDP-GlcNAc transporter activity. Analysis of extensive crossings between the two mutants led Ballou and co-workers (reference cited above) to conclude that these genes were allelic or tightly linked. We have now cloned the gene encoding the K. lactis Golgi membrane N-acetylglucosaminyltransferase by complementation of the mnn2-1 mutation and named it GNT1. The mnn2-1 mutant was transformed with a 9.5-kilobase (kb) genomic fragment previously shown to contain the gene encoding the UDP-GlcNAc transporter; transformants were isolated, and phenotypic correction was monitored after cell surface labeling with fluorescein isothiocyanate-conjugated Griffonia simplicifolia II lectin, which binds terminal N-acetylglucosamine, and a fluorescence-activated cell sorter. The above 9.5-kb DNA fragment restored the wild-type lectin binding phenotype of the transferase mutant; further subcloning of this fragment yielded a smaller one containing an opening reading frame of 1,383 bases encoding a protein of 460 amino acids with an estimated molecular mass of 53 kDa, which also restored the wild-type phenotype. Transformants had also regained the ability to transfer N-acetylglucosamine to 3-0-alpha-D-mannopyranosyl-D-mannopyranoside. The gene encoding the above transferase was found to be approximately 1 kb upstream from the previously characterized MNN2 gene encoding the UDP-GlcNAc Golgi transporter. Each gene can be transcribed independently by their own promoter. To our knowledge this is the first demonstration of two Golgi apparatus functionally related genes being contiguous in a genome.
Guillen
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E
Department of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, Boston, Massachusetts 02118-2394, USA.
Abeijon
C
C
Hirschberg
C B
CB
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D016681:Genome, Fungal; D006056:Golgi Apparatus / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007716:Kluyveromyces / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D008969:Molecular Sequence Data; D009154:Mutation; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics*; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics*; D016415:Sequence Alignment
D017351:N-Acetylglucosaminyltransferases; C037649:N-acetyllactosaminide beta-1,6-N-acetylglucosaminyltransferase; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C081515:acetylglucosamine transporter