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 O-mannosylation 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-P-Man 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.

Figure 1

Alignment of the amino acid sequences of Dol-P-Man synthases. Sequences were aligned using the clustal v program (21). Amino acids identical in at least four of the six sequences are highlighted in black and similar residues are highlighted in gray. The aspartic acid residues marked ∗ are conserved in β-glycosyltransferases that transfer a single sugar residue and which may be involved in catalysis (34). The serine marked + and the amino acids NH₂-terminal to it meet certain criteria for a consensus site for phosphorylation by cAMP-dependent kinase. Abbreviations and references to published sequences are as follows: Sc, S. cerevisiae (11); Um, U. maydis (12); Tb, T. brucei (13); Hs, human; Cb, C. briggsiae; Sp, S. pombe.

Figure 2

Hydropathy plots of Dol-P-Man synthases. Plots were generated according to Kyte and Doolittle (22) with a window of 11, using the dna strider 1.1 program (20). Plots are aligned to the Gly-Thr-Arg-Tyr (GTRY) sequence conserved in all Dol-P-Man synthases. Stretches of amino acids with hydropathies above 1.6 (---) may form transmembrane domains. Transmembrane sequences predicted by the program of Rost et al. (23) are shaded in black. (Bar = 50 amino acids.)

Figure 3

S. cerevisiae and human Dol-P-Man synthase genes allow growth of haploid S. pombe dpm1⁺::his7⁺ cells. Heterozygous dpm1⁺::his7⁺/dpm1⁺ diploids harboring pScDPM1, pHsDPM1, pSpdpm1⁺, or the vector pREP2 were allowed to sporulate, and the meiotic progeny were tested for growth on medium selective for histidine and uracil prototrophy. Segregants from the dpm1⁺::his7⁺/dpm1⁺ diploid harboring pREP2 were also plated on medium containing histidine to select for wild-type haploids containing pREP2.