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Glycobiology Advance Access published online on October 13, 2004
Glycobiology, doi:10.1093/glycob/cwi003
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Received September 8, 2004
Accepted October 6, 2004

ORIGINAL ARTICLES

Dimannosyldiacylglycerol serves as a lipid anchor precursor in the assembly of the membrane-associated lipomannan in Micrococcus luteus1

Leroy S. Pakkiri 1 and Charles J. Waechter 1*

1 Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky 40536

* To whom correspondence should be addressed. E-mail: waechte{at}pop.uky.edu.


  Abstract

Based on recent analytical and enzymological studies, a topological model for the role of {alpha}-Dmannosyl-(1->3)-{alpha}-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 M. 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 abnormally low levels of Man2-DAG and LM at the non-permissive 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. Since 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, pre-labeled [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.

Keywords: dimannosyldiacylglycerol; lipomannan; [3H]mannose-suicide; temperature-sensitive.

1 This research was supported by National Institutes of Health Grant GM36065 awarded to C.J.W.


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