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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...

Human apolipoprotein C-I (apo C-I) in solution, in monomeric and oligomeric form, and in micellar complexes with dimyristoylphosphatidylcholine (DMPC), below and above the phase transition temperature of DMPC, was investigated with steady-state and time-resolved fluorescence methods. The environment of the Trp residue of apo C-I, in each physical state, was evaluated from fluorescence spectra and their changes upon KI quenching. Rotational correlation times of Trp residues were obtained from fluorescence anisotropy decay measurements. Static fluorescence anisotropy was determined as a function of temperature for the Trp residues of apo C-I in all physical states and for diphenylhexatriene dissolved in apo C-I X DMPC complexes. It was found that the Trp residues of apo C-I in solution are exposed from 75 to 88% to the aqueous medium, depending on the state of self-association. On the other hand, the Trp residues in apo C-I X DMPC complexes are only 42-45% exposed to KI quenching through an environment distinct from water. Apolipoprotein C-I in all its physical forms had two rotational correlation times associated with Trp motions: a longer one dependent on the size and flexibility of the entire particle and a very short one in the range from 0.2 to 0.4 ns. The later correlation times correspond to local Trp residue motions. These Trp motions were not significantly affected by a transition from the gel to the liquid-crystalline state of the lipid in apo C-I X DMPC complexes, suggesting that there is no coupling between the local motions of lipids and those of Trp side chains of apo C-I.