Human and Saccharomyces cerevisiae dolichol phosphate mannose synthases represent two classes of the enzyme, but both function in Schizosaccharomyces pombe

Abstract

Dolichol phosphate mannose (Dol-P-Man), formed upon transfer of Man from GDPMan to Dol-P, is a mannosyl donor in pathways leading to N-glycosylation, glycosyl phosphatidylinositol membrane anchoring, and Omannosylation of protein. Dol-P-Man synthase is an essential protein in Saccharomyces cerevisiae. We have cloned cDNAs encoding human and Schizosaccharomyces pombe proteins that resemble S. cerevisiae Dol-P-Man synthase. Disruption of the gene for the S. pombe Dol-P-Man synthase homolog, dpm1 ؉ , is lethal. The known Dol-P-Man synthase sequences can be divided into two classes. One contains the S. cerevisiae, Ustilago maydis, and Trypanosoma brucei enzymes, which have a COOH-terminal hydrophobic domain, and the other contains the human, S. pombe, and Caenorhabditis synthases, which lack a hydrophobic COOH-terminal domain. The two classes of synthase are functionally equivalent, because S. cerevisiae DPM1 and its human counterpart both complement the lethal null mutation in S. pombe dpm1 ؉ . The findings that Dol-PMan synthase is essential in yeast and that the Ustilago and Trypanosoma synthases are in a different class from the human enzyme raise the possibility that Dol-P-Man synthase could be exploited as a target for inhibitors of pathogenic eukaryotic microbes.Dolichol phosphate mannose (Dol-P-Man), which is formed by transfer of mannose from GDPMan to the polyisoprenoid Dol-P, is a mannosyl donor in the assembly of the precursor oligosaccharide in N-glycosylation, in the synthesis of glycosylphosphatidylinositol anchors, and in O-mannosylation of fungal protein (1-3). Dol-P-Man seems to be synthesized at the cytoplasmic face of the membrane of the endoplasmic reticulum (ER) (4, 5), yet the Dol-P-Man-requiring steps in glycosylation are believed to take place in the ER lumen (5). Thus, Dol-P-Man supply may require not only a synthase but also proteins that translocate it into the ER lumen. Indeed, at least three genes may be involved in Dol-P-Man supply in mammals. Candidates for the synthase gene itself are the loci defective in the Thy-1 class E murine lymphoma mutant, the Chinese hamster ovary cell B4-2-1͞Lec15 mutant, and a murine T cell hybridoma mutant, which are all defective in Dol-P-Man synthesis (6-8). The Lec35 gene may encode a protein that supplies Dol-P-Man to the ER lumen. Chinese hamster ovary cell Lec35 mutants make normal amounts of Dol-P-Man in vivo and in vitro but are unable to utilize it efficiently in vivo for glycosylation reactions, consistent with a defect in Dol-P-Man translocation (9). A third protein, SL15, may also be involved in Dol-P-Man supply, for the SL15 gene suppresses the mutations in both Dol-P-Man synthasedeficient Lec15 and Lec35 cells (10).The Saccharomyces cerevisiae gene for Dol-P-Man synthase, DPM1, has been cloned and shown indeed to be the structural gene for the enzyme because it confers Dol-P-Man synthase activity on Escherichia coli when expressed in the bacteria (11). Dol-P-Man synthase genes from Trypanosoma brucei (12) and from Ustilago maydis (13...

Leishmania mexicanaMutants Lacking Glycosylphosphatidylinositol (GPI):Protein Transamidase Provide Insights into the Biosynthesis and Functions of GPI-anchored Proteins

“…The human and fission yeast enzymes lack a C-terminal hydrophobic domain, whereas the trypanosome and budding yeast enzymes contain one. The two classes of this enzyme, however, are functionally equivalent, because both the human and S. cerevisiae genes can complement a lethal null mutant in fission yeast (Colussi et al, 1997).…”

“…This type of variation between ER isoenzymes involved in GPI biosynthesis is not unprecedented. Two classes of dolicholphosphate-mannose synthase have been identified in differ- ent species (Colussi et al, 1997). The human and fission yeast enzymes lack a C-terminal hydrophobic domain, whereas the trypanosome and budding yeast enzymes contain one.…”

In Vivo Interaction between the Polyprenol Phosphate Mannose Synthase Ppm1 and the Integral Membrane Protein Ppm2 from Mycobacterium smegmatis Revealed by a Bacterial Two-hybrid System

“…Thus, the MsPpm1-MsPpm2 interaction is reminiscent of that of the mammalian Dpm1 with Dpm3 (8,9). In contrast, MsPpm1 is functionally active when expressed in Escherichia coli, similarly to the S. cerevisiae group of Dol-P-Man synthases (7,17). As a consequence, MsPpm1 may constitute a new intermediate group of Polyprenol-P-Man synthases.…”

In Vitro Phosphorylation by cAMP-dependent Protein Kinase Up-regulates Recombinant Saccharomyces cerevisiae Mannosylphosphodolichol Synthase

“…Sequence analysis has suggested that the Dol-P-Man synthase can now be divided into two classes: one includes the enzymes from S. cerevisiae, U. maydis, Trypanosoma brucei, and Leishmania mexicana (29,30). They share 50 -60% amino acid identity and have a stretch of hydrophobic amino acid residues near the COOH terminus constituting a transmembrane domain.…”

“…Dol-P-Man synthase has now been cloned from a number of species such as Trypanosoma brucei, Ustilago maydis, Schizosaccharomyces pombe, Caenorhabditis briggsiae as well as from humans by complementation of a temperature-sensitive S. cerevisiae DPM1 mutant, or by cDNA cloning (27)(28)(29). Sequence analysis has suggested that the Dol-P-Man synthase can now be divided into two classes: one includes the enzymes from S. cerevisiae, U. maydis, Trypanosoma brucei, and Leishmania mexicana (29,30).…”