Dimannosyldiacylglycerol serves as a lipid anchor precursor in the assembly of the membrane-associated lipomannan in Micrococcus luteus.
Based on recent analytical and enzymological studies, a topological model for the role of alpha-D-mannosyl-(1-->3)-alpha-D-mannosyl-(1-->3)-diacylglycerol (Man(2)-DAG) as a lipid anchor precursor and mannosylphosphorylundecaprenol (Man-P-Und) as a mannosyl donor in the assembly of a membrane-associated lipomannan (LM) in Micrococcus luteus has been proposed. In this study, a [(3)H]mannose-suicide selection procedure has been used to identify temperature-sensitive (ts) mutants defective in LM assembly. Two micrococcal mutants with abnormal levels of Man(2)-DAG and LM at the nonpermissive temperature (37 degrees C), mms1 and mms2, have been isolated and characterized. In vivo and in vitro biochemical assays indicate that mms1 cells have a defect in the mannosyltransferase catalyzing the conversion of Man-DAG to Man(2)-DAG, and mms2 has a temperature-sensitive defect in the synthesis of Man-P-Und. Because mms1 cells are depleted of endogenous Man(2)-DAG, membranes from this mutant efficiently converted purified, exogenous [(3)H]Man(2)-DAG to [(3)H]LM by a Man-P-Und-dependent process. An obligatory role for Man-P-Und as a mannosyl donor in the elongation process was also demonstrated by showing that the conversion of exogenous [(3)H]Man(2)-DAG to [(3)H]LM by membranes from mms1 cells in the presence of GDP-Man was inhibited by amphomycin. In addition, consistent with Man(2)-DAG serving as a lipid anchor precursor for LM assembly, endogenous, prelabeled [(3)H]Man(2)-DAG was converted to [(3)H]LM when membranes from mms2 cells were incubated with purified, exogenous Man-P-Und. These studies provide the first direct proof for the role of Man(2)-DAG as the lipid anchor precursor for LM, and suggest that Man(2)-DAG may be essential for the normal growth of M. luteus cells.
Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY 40536, USA.
Leroy S
LS
Pakkiri
Charles J
CJ
Waechter
D002462:Cell Membrane / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D003208:Concanavalin A; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives*; D006017:Glycolipids / Q000378:metabolism*; D050356:Lipid Metabolism; D008070:Lipopolysaccharides / Q000378:metabolism*; D008358:Mannose / Q000378:metabolism; D008364:Mannosyltransferases / Q000235:genetics / Q000378:metabolism; D016982:Micrococcus luteus / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D010743:Phospholipids / Q000378:metabolism; D011523:Protoplasts / Q000166:cytology / Q000378:metabolism; D013696:Temperature
D006017:Glycolipids; D008070:Lipopolysaccharides; D010743:Phospholipids; C031647:dimannosyl diacylglycerol; C042784:fluorescein isothiocyanate-concanavalin A; C064351:lipomannan; D003208:Concanavalin A; D008364:Mannosyltransferases; D016650:Fluorescein-5-isothiocyanate; D008358:Mannose