Triggering of mannosyltransferase activity in inner mitochondrial membranes by dolichyl‐monophosphate incorporation mediated through phospholipids or fatty acids

Abstract

The activity of CDPmannose : dolichyl monophosphate mannosyltransferase in inner mitochondrial membranes can be triggered by dolichyl-monophosphate incorporation mediated through phospholipids or fatty acids. The efficiency of this incorporation and the efficiency of the enzyme activity are not equivalent. Among a variety of amphiphiles which were tested, the highest mannosyltransferase activity was obtained with the mixture of lipids extracted from the outer mitochondrial membranes. The results presented here appear consistent only with a mechanism involving collisional contacts of the phospholipid vesicles and fusion with the membranes. ESR spectroscopy confirms that (a) the incorporation process is followed by solubilization of dolichyl monophosphate molecules in the lipid phase and (b) the general organization of the inner mitochondrial membranes is not perturbed by the addition of dolichyl monophosphate.The biosynthesis of asparagine-linked oligosaccharides of glycoproteins is initiated by the assembly of a dolichyl-diphospho-[(Glc)~(Man),(GlcNAc),-PP-Dol]-oligosaccharide intermediate with the participation of specific membranous glycosyltransferases [l -31. The four mannosyl transfer reactions that occur during the latter stages of oligosaccharidelipid formation require Dol-P-Man as a glycosyl donor. A Dol-P-Man synthetase which catalyzes the synthesis of Dol-P-Man from GDPmannose into exogenous dolichyl monophosphate has been described in purified inner mitochondrial membranes [4]. This enzyme did not catalyze mannosyl transfer to the polyprenol acceptor in the presence of detergents (e.g. Triton X-100, Cutscum), but mannosyl transfer was markedly stimulated when the hydrophobic acceptor was incorporated into a lipid matrix [4]. Abbreviations. PtdCho, phosphatidylcholine; Dol-P, dolichyl monophosphate; PtdEtn, phosphatidylethanolamine; (rn,n)Ste, spinlabel derivatives of stearic acid of the general formula CH-1-(CH2),-C-(CH2),-C02-Enzymes. GDPmannose : dolichyl-monophosphate mannosyltransferase (EC 2.4.1.83); cytochrome c oxidase (EC 1.9.3.1); monoamine oxidase (EC 1.4.3.4); glucose-6-phosphatase (EC 3.1.3.9). slow exchange between the layers and a monomolecular dispersion of dolichyl phosphate in phosphatidylcholine vesicles. NMR studies of the headgroup 2H-labeled dolichyl phosphate [S] suggest an unusual conformation of the long poly(cisprenyl) chains.In this paper, we describe the effects of the incorporation of dolichyl monophosphate into inner mitochondrial membranes on the triggering of mannosyltransferase activity. Inner membranes are known to contain a low concentration of dolichyl monophosphate [9] and the incorporation of exogenous dolichyl monophosphate would be expected, as in model membrane systems, to disturb lipid packing or alter local membrane architecture.The results presented here show that dolichyl monophosphate can be incorporated by several amphiphiles into inner mitochondrial membranes. This incorporation triggers mannosyltransferase activity in the absence of detectable struct...

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