259:	Biosynthesis of membrane lipids in Chlamydomonas reinhardtii: intracellular compartmentalization and characteristics of diacyglyceroltrimethylhomoserine formation.

Authors:	Moore, Thomas, S.(A)Du, Zhirong(A)Chen, Zhi(A)
Affiliations:	(A): Department of Biological Sciences, Louisiana State University
Presenter:	Moore, Thomas S., btmoor@unix1.sncc.lsu.edu

Biosynthesis of diacylglyceroltrimethylhomoserine (DGTS) has been examined in membrane fractions from Chlamydomonas reinhardtii cells. This synthesis was found in two fractions, one being a microsomal fraction and co-sedimenting with phosphatidylinositol, the other being a 760xg fraction enriched in chlorophyll and RuBisCo activity. This latter activity probably results from partially broken cells, since attempts to identify DGTS synthesis in plastid envelope and thylakoid subfractions were not successful. There was no significant correlation between DGTS synthesis and either cytochrome c oxidase or PEP carboxylase sedimentation profiles (mitochondrial and cytoplasmic markers, respectively). Synthesis of this complex lipid by the microsomal fraction utilized S-adenosyl-L-methionine (SAM) as the water-soluble substrate. Two radiolabeled spots following TLC were found when S-adenosyl-L-[carboxyl-14C]methionine was utilized, one of which was identified as DGTS. The second compound appears to be an intermediate in DGTS synthesis. Pulse-chase experiments resulted in the incorporation of SAM into the second compound prior to its appearance in DGTS. The multi-step synthesis, measured by incorporation of radiolabeled SAM into the DGTS fraction, was maximal at pH 7.5-8.0 and 30°C. The Km for SAM was 74µM. Additions of the putative lipid substrate, DAG, at concentrations as low as 25 µM were inhibitory. There appeared to be no divalent cation requirement. We conclude that DGTS synthesis occurs in the microsomes, probably the ER, and occurs by addition of a homoserine group from SAM followed by three methylations, also using SAM. Supported by NSF grant number MCB-9603626 to TSM.