Cloning and functional expression of glycosyltransferases from parasitic protozoans by heterologous complementation in yeast: the dolichol phosphate mannose synthase from Trypanosoma brucei brucei.
The gene for the enzyme dolichol phosphate mannose (Dol-P-Man) synthase from the parasitic protozoan Trypanosoma brucei brucei (T. brucei) was cloned by screening a T. brucei cDNA library and then sequenced. The library was constructed in a yeast expression vector and the positive clone was identified by complementation of a temperature-sensitive defect in the yeast strain DPM 1-6 [Orlean, Albright and Robbins (1988) J. Biol. Chem. 263, 17499-17507]. The insert of this clone displayed an open reading frame of 801 nucleotides coding for a putative protein of 267 amino acids. The deduced protein sequence showed an identity of 49% and a similarity of 69% with the published yeast sequence. Additional features of the T. brucei sequence are the presence of a putative signal sequence, a C-terminal transmembrane domain, a consensus sequence for phosphorylation by cAMP-dependent protein kinase and a stretch of five nucleotides immediately upstream from the putative initiation codon that could function as a prokaryotic ribosome binding site. A consensus sequence for dolichol binding (FI/VXF/YXXIPFXF/Y) found in the yeast protein could not be detected in the putative transmembrane domain of the T. brucei sequence. Biochemical characterization of the recombinant protein showed that it is functionally expressed in the yeast strain DPM 1-6 and Escherichia coli. In both constructs Dol-P-Man synthesis was shown in a cell-free system. Synthesis was stimulated by exogenous dolichol phosphate and inhibited by amphomycin. These results confirm that we have cloned the T. brucei Dol-P-Man synthase by heterologous complementation in yeast, an approach that might be applicable for other glycosyltransferases from various sources.
Mazhari-Tabrizi
R
R
Medizinisches Zentrum für Hygiene und Medizinische Mikrobiologie, Marburg, Federal Republic of Germany.
Eckert
V
V
Blank
M
M
Müller
R
R
Mumberg
D
D
Funk
M
M
Schwarz
R T
RT
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular / Q000379:methods; D016384:Consensus Sequence; D004286:Dolichols / Q000378:metabolism; D004926:Escherichia coli; D015870:Gene Expression; D017125:Genes, Protozoan; D005816:Genetic Complementation Test; D007700:Kinetics; D008364:Mannosyltransferases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics
D004286:Dolichols; D011994:Recombinant Proteins; D008364:Mannosyltransferases; C022755:dolichyl-phosphate beta-D-mannosyltransferase