Hamster liver post-nuclear membranes catalyze the transfer of
mannose from
GDP-mannose to endogenous
dolichyl phosphate and to a second major endogenous acidic
lipid. This
mannolipid was believed to be synthesized from endogenous
retinyl phosphate and was tentatively identified as
retinyl phosphate mannose (
Ret-P-Man) (De Luca, L. M., Brugh, M. R. Silverman-Jones, C. S. and Shidoji, Y. (1982) Biochem. J. 208, 159-170). To characterize this endogenous
mannolipid in more detail, we isolated and purified the
mannolipid from incubations containing hamster liver membranes and
GDP-[14C]
mannose and compared its properties to those of authentic
Ret-P-Man. We found that the endogenous
mannolipid was separable from authentic
Ret-P-Man on a
Mono Q anion exchange column, did not exhibit the absorbance spectrum characteristic of a
retinol moiety, and was stable to mild
acid under conditions which cleave authentic
Ret-P-Man. The endogenous
mannolipid was sensitive to mild base hydrolysis and
mannose was released from the
mannolipid by
snake venom phosphodiesterase digestion. These properties were consistent with the endogenous acceptor being
phosphatidic acid. Addition of exogenous
phosphatidic acid, but not
phospholipids with a head group blocking the
phosphate moiety, to incubations containing hamster liver membranes and
GDP-[14C]
mannose resulted in the synthesis of a
mannolipid with chromatographic and physical properties identical to the endogenous
mannolipid. A double-labeled
mannolipid was synthesized in incubations containing hamster liver membranes,
GDP-[14C]
mannose, and [3H]
phosphatidic acid. Mannosyl transfer to exogenous
phosphatidic acid was saturable with increasing concentrations of
phosphatidic acid and
GDP-mannose and specific for glycosyl transfer from
GDP-mannose. Class E Thy-1-negative mutant mouse
lymphoma cell membranes, which are defective in
dolichyl phosphate mannose synthesis, also fail to transfer
mannose from
GDP-mannose to exogenous
phosphatidic acid or
retinyl phosphate.
Amphomycin, an inhibitor of
dolichyl phosphate mannose synthesis, blocked mannosyl transfer to the endogenous
lipid, and to exogenous
retinyl phosphate and
phosphatidic acid. We conclude that the same
mannosyltransferase responsible for
dolichyl phosphate mannose synthesis can also utilize in vitro exogenous
retinyl phosphate and
phosphatidic acid as well as endogenous
phosphatidic acid as mannosyl acceptors.