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 thos e of Saccharomyces cerevisiae except that they lack mannose phosphate and h ave terminal alpha(1-->2)-linked N-acetylglucosamine. Previously, Smith ct 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 ter minal N-acetylglucosamine in their mannoproteins. The former mutant lacks t he Golgi N-acetylglucosaminyl-transferase activity, whereas the latter one was recently found to be deficient in the Golgi UDP-GlcNAc transporter acti vity. Analysis of extensive crossings between the two mutants led Ballou an d co-workers (reference cited above) to conclude that these genes were alle lic or tightly linked. We have now cloned the gene encoding the K. lactis Gels membrane N-acetylgl ucosaminyltransferase by complementation of the mnn2-1 mutation and named i t 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 trans porter; transformants were isolated, and phenotypic correction was monitore d after cell surface labeling with fluorescein isothiocyanate-conjugated Gr iffonia simplicifolia II lectin, which binds terminal N-acetylglucosamine, and a fluorescence-activated cell sorter. The above 9.5-kb DNA fragment res tored the wild-type lectin binding phenotype of the transferase mutant; fur ther subcloning of this fragment yielded a smaller one containing an openin g reading frame of 1,383 bases encoding a protein of 460 amino acids with a n estimated molecular mass of 53 kDa, which also restored the wild-type phe notype, Transformants had also regained the ability to transfer N-acetylglu cosamine to 3-O-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 transport er. Each gene can be transcribed independently by their own promoter. To ou r knowledge this is the first demonstration of two Gels apparatus functiona lly related genes being contiguous in a genome.