Glycobiology, Vol 8, 77-85, Copyright © 1998 by Society for Glycobiology
V Eckert, M Blank, R Mazhari-Tabrizi, D Mumberg, M Funk and RT Schwarz
The gene for the human dolichol cycle GlcNAc-1-P transferase (ALG7/GPT) was
cloned by screening a human lung fibroblast cDNA library. The library was
constructed in a Saccharomyces cerevisiae expression vector, and the
positive clone was identified by complementation of the conditional lethal
S.cerevisiae strain YPH-A7-GAL. This strain was constructed by replacing
the endogenous promoter of the GPT-gene by the stringently regulated
GAL1-promoter. This construct allows to specifically suppress the
endogenous enzyme activity. The insert of the positive clone displayed an
open reading frame of 1200 nucleotides, coding for a putative protein of
400 amino acids with a calculated molecular weight of 44.7 kDa. The deduced
protein sequence shows a homology of over 90% when compared with other
mammalian GPT sequences, thus resembling the close phylogenetic
relationship between mammalian species. This homology however decreases to
40-50% when compared to more distantly related organisms such as
S.cerevisiae , Schizosaccharomyces pombe , or Leishmania amazonensis .
Biochemical characterization of the recombinant protein showed that it is
functionally expressed in the S.cerevisiae strain YPH-A7-GAL. GlcNAc- and
GlcNAc2-PP-Dolichol biosynthesis could be shown with isolated S.cerevisiae
membranes from cells harboring the recombinant plasmid and grown on glucose
thus suppressing transcription of the endogenous gene. Synthesis could be
stimulated by dolicholphosphate and was inhibited by tunicamycin. These
results show that we have cloned the human GlcNAc-1- P transferase by
heterologous complementation in S. cerevisiae, a strategy that may be
useful for the cloning and characterization of glycosyltransferases from a
variety of organisms.
ORIGINAL ARTICLES
Cloning and functional expression of the human GlcNAc-1-P transferase, the enzyme for the committed step of the dolichol cycle, by heterologous complementation in Saccharomyces cerevisiae
Medizinisches Zentrum fur Hygiene und Med. Mikrobiologie, Robert Koch Strasse 17, Philipps-Universitat-Marburg, D-35037 Marburg, Germany.
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