Skip Navigation

This Article
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (3)
Right arrowRequest Permissions
Right arrow Disclaimer
Citing Articles
Right arrowScopus Links
Google Scholar
Right arrow Articles by Scocca, J. R.
Right arrow Articles by Krag, S. S.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Scocca, J. R.
Right arrow Articles by Krag, S. S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Glycobiology vol 7 no 8 pp. 1181-1191, 1997
© 1997

Aspartic acid 252 and asparagine 185 are essential for activity of lipid N-acetylglucosaminylphosphate transferase

Jane R. Scocca and Sharon S. Krag1

Department of Biochemistry, School of Hygiene and Public Health, Johns Hopkins University 615 North Wolfe Street, Baltimore, MD 21205-2179, USA

1To whom correspondence should be addressed

Received on March 13, 1997; revised on April 25, 1997; accepted on April 28, 1997

A key step in the assembly of oligosaccharide-lipid intermediates in N-linked glycosylation is the transfer of N-acetylglucosamine 1-phosphate to dolichyl phosphate, catalyzed by the enzyme UDP-N-acetylglucosaminyl:dolichyl phosphate N-acetylglucosaminyl phosphoryl transferase (L-G1PT). Comparison of the amino acid sequences of L-G1PT from five diverse species showed 75 amino acids identical in all five proteins. Using site-directed mutagen-esis, we analyzed the importance of a number of these conserved residues to the enzymatic activity of L-G1PT using a plasmid shuffling procedure in Schizosaccharomyces pombe. S.pombe cells containing a chromosomal deletion of the essential gpt+ gene are rescued by a plasmid containing the S.pombe gpt open reading frame. Replacement of that plasmid by a plasmid encoding a mutated hamster L-G1PT cDNA sequence indicated that the mutated protein provided sufficient enzyme activity to permit cell growth. Mutations of aspartic acid 252 and asparagine 185 did not allow plasmid shuffling, indicating these residues were essential for activity. A combination of mutations at asparagine 182 and tryptophan 122 did not allow plasmid shuffling, although the single mutations did. Overexpression of the mutant proteins in S.pombe conferred tunicamycin (TM) resistance, indicating that the mutant proteins had a conformation necessary for binding TM, a substrate analog. The mutant proteins were also detected in Western blots and were correctly localized to the membrane fractions. However, the overexpressed proteins did not increase the endogenous level of enzymatic activity in these cells, indicating they were enzymatically inactive.

N-acetylglucosaminylphosphate transferase mutagenesis plasmid shuffling Schizosaccharomyces pombe tunicamycin


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 GlycobiologyHome page
N. P. Price and F. A. Momany
Modeling bacterial UDP-HexNAc: polyprenol-P HexNAc-1-P transferases
Glycobiology, September 1, 2005; 15(9): 29R - 42R.
[Abstract] [Full Text] [PDF]


Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.