Dimannosyldiacylglycerol serves as a lipid anchor precursor in the assembly of the membrane-associated lipomannan in Micrococcus luteus

Abstract

Based on recent analytical and enzymological studies, a topological model for the role of α-D-mannosyl-(1→3)-α-D-mannosyl- (1→3)-diacylglycerol (Man2-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 [3H]mannose-suicide selection procedure has been used to identify temperature-sensitive (ts) mutants defective in LM assembly. Two micrococcal mutants with abnormal levels of Man2-DAG and LM at the nonpermissive temperature (37°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 Man2-DAG, and mms2 has a temperature-sensitive defect in the synthesis of Man-P-Und. Because mms1 cells are depleted of endogenous Man2-DAG, membranes from this mutant efficiently converted purified, exogenous [3H]Man2-DAG to [3H]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 [3H]Man2-DAG to [3H]LM by membranes from mms1 cells in the presence of GDP-Man was inhibited by amphomycin. In addition, consistent with Man2-DAG serving as a lipid anchor precursor for LM assembly, endogenous, prelabeled [3H]Man2-DAG was converted to [3H]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 Man2-DAG as the lipid anchor precursor for LM, and suggest that Man2-DAG may be essential for the normal growth of M. luteus cells. © Oxford University Press 2005; all rights reserved.