Mannan chains of Kluyveromyces lactis mannoproteins are similar to those of Saccharomyces cerevisiae except that they have terminal 12-linked N-acetylglucosamine and lack mannose phosphate. In a previous study, Douglas and Ballou (Douglas, R. K., and Ballou, C. E. (1982) Biochemistry 21, 1561-1570) characterized a mutant, mnn2-2, which lacked terminal N-acetylglucosamine in its mannoproteins. The mutant had normal levels of N-acetylglucosaminyltransferase activity, and the partially purified enzyme from wild-type and mutant cells had the same apparent size, heat stability, affinity for substrates, metal requirement, and subcellular location. No qualitative or quantitative differences were found between mutant and wild-type cells in endogenous mannan acceptors and pools of UDP-GlcNAc. Chitin was synthesized at similar rates in wild-type and mutant cells, and the latter did not have a soluble inhibitor of the N-acetylglucosaminyltransferase or a hexosaminidase that could remove N-acetylglucosamine from mannoproteins. Together, the above observations led Douglas and Ballou ((1982) Biochemistry 21, 1561-1570) to postulate that the mutant might have a defect in compartmentation of substrates involved in the biosynthesis of mannoproteins.

We determined whether the above mutant phenotype is the result of defective transport of UDP-GlcNAc into Golgi vesicles from K. lactis. Golgi vesicles which were sealed and of the same membrane topographical orientation as in vivo were isolated from wild-type and mnn2-2 mutant cells and incubated with UDP-GlcNAc in an assay in vitro. The initial rate of transport of UDP-GlcNAc into Golgi vesicles from wild-type cells was temperature dependent, saturable with an apparent K of 5.5 µM and a V of 8.2 pmol/mg of protein/3 min. No transport of UDP-GlcNAc was detected into Golgi vesicles from mutant cells. However, Golgi vesicles from both cells translocated GDP-mannose at comparable velocities, indicating that the above transport defect is specific. In addition to the above defect in mannoproteins, mutant cells were also deficient in the biosynthesis of glucosamine containing lipids.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				C. E. Caffaro, C. B. Hirschberg, and P. M. Berninsone Independent and simultaneous translocation of two substrates by a nucleotide sugar transporter PNAS, October 31, 2006; 103(44): 16176 - 16181. [Abstract] [Full Text] [PDF]

				J. Tiralongo, A. Ashikov, F. Routier, M. Eckhardt, H. Bakker, R. Gerardy-Schahn, and M. von Itzstein Functional expression of the CMP-sialic acid transporter in Escherichia coli and its identification as a simple mobile carrier Glycobiology, January 1, 2006; 16(1): 73 - 81. [Abstract] [Full Text] [PDF]

				J. Hoflich, P. Berninsone, C. Gobel, M. J. Gravato-Nobre, B. J. Libby, C. Darby, S. M. Politz, J. Hodgkin, C. B. Hirschberg, and R. Baumeister Loss of srf-3-encoded Nucleotide Sugar Transporter Activity in Caenorhabditis elegans Alters Surface Antigenicity and Prevents Bacterial Adherence J. Biol. Chem., July 16, 2004; 279(29): 30440 - 30448. [Abstract] [Full Text] [PDF]

				M. D. Lopez-Avalos, D. Uccelletti, C. Abeijon, and C. B. Hirschberg The UDPase activity of the Kluyveromyces lactis Golgi GDPase has a role in uridine nucleotide sugar transport into Golgi vesicles Glycobiology, May 1, 2001; 11(5): 413 - 422. [Abstract] [Full Text] [PDF]

				P. Berninsone, H.-Y. Hwang, I. Zemtseva, H. R. Horvitz, and C. B. Hirschberg SQV-7, a protein involved in Caenorhabditis elegans epithelial invagination and early embryogenesis, transports UDP-glucuronic acid, UDP-N- acetylgalactosamine, and UDP-galactose PNAS, March 16, 2001; (2001) 61593098. [Abstract] [Full Text]

				E. Guillen, C. Abeijon, and C. B. Hirschberg The Genes for the Golgi Apparatus N-Acetylglucosaminyltransferase and the UDP-N-acetylglucosamine Transporter Are Contiguous in Kluyveromyces lactis J. Biol. Chem., March 5, 1999; 274(10): 6641 - 6646. [Abstract] [Full Text] [PDF]

				E. Guillen, C. Abeijon, and C. B. Hirschberg Mammalian Golgi apparatus UDP-N-acetylglucosamine transporter: Molecular cloning by phenotypic correction of a yeast mutant PNAS, July 7, 1998; 95(14): 7888 - 7892. [Abstract] [Full Text] [PDF]

				N. Dean, Y. B. Zhang, and J. B. Poster The VRG4 Gene Is Required for GDP-mannose Transport into the Lumen of the Golgi in the Yeast, Saccharomyces cerevisiae J. Biol. Chem., December 12, 1997; 272(50): 31908 - 31914. [Abstract] [Full Text] [PDF]

				D. Ma, D. G. Russell, S. M. Beverley, and S. J. Turco Golgi GDP-mannose Uptake Requires Leishmania LPG2. A MEMBER OF A EUKARYOTIC FAMILY OF PUTATIVE NUCLEOTIDE-SUGAR TRANSPORTERS J. Biol. Chem., February 7, 1997; 272(6): 3799 - 3805. [Abstract] [Full Text] [PDF]

				X.-D. Gao, A. Nishikawa, and N. Dean Identification of a Conserved Motif in the Yeast Golgi GDP-mannose Transporter Required for Binding to Nucleotide Sugar J. Biol. Chem., February 2, 2001; 276(6): 4424 - 4432. [Abstract] [Full Text] [PDF]

				P. Berninsone, H.-Y. Hwang, I. Zemtseva, H. R. Horvitz, and C. B. Hirschberg SQV-7, a protein involved in Caenorhabditis elegans epithelial invagination and early embryogenesis, transports UDP-glucuronic acid, UDP-N- acetylgalactosamine, and UDP-galactose PNAS, March 27, 2001; 98(7): 3738 - 3743. [Abstract] [Full Text] [PDF]