The topology of the ER-resident phospholipid methyltransferase Opi3 of Saccharomyces cerevisiae is consistent with in trans catalysis.

Phospholipid N-methyltransferases (PLMTs) synthesize phosphatidylcholine by methylating phosphatidylethanolamine using S-adenosylmethionine as a methyl donor. Eukaryotic PLMTs are integral membrane enzymes located in the endoplasmic reticulum (ER). Recently Opi3, a PLMT of the yeast Saccharomyces cerevisiae was proposed to perform in trans catalysis, i.e. while localized in the ER, Opi3 would methylate lipid substrates located in the plasma membrane at membrane contact sites. Here, we tested whether the Opi3 active site is located at the cytosolic side of the ER membrane, which is a prerequisite for in trans catalysis. The membrane topology of Opi3 (and its human counterpart, phosphatidylethanolamine N-methyltransferase, expressed in yeast) was addressed by topology prediction algorithms and by the substituted cysteine accessibility method. The results of these analyses indicated that Opi3 (as well as phosphatidylethanolamine N-methyltransferase) has an N-out C-in topology and contains four transmembrane domains, with the fourth forming a re-entrant loop. On the basis of the sequence conservation between the C-terminal half of Opi3 and isoprenyl cysteine carboxyl methyltransferases with a solved crystal structure, we identified amino acids critical for Opi3 activity by site-directed mutagenesis. Modeling of the structure of the C-terminal part of Opi3 was consistent with the topology obtained by the substituted cysteine accessibility method and revealed that the active site faces the cytosol. In conclusion, the location of the Opi3 active site identified here is consistent with the proposed mechanism of in trans catalysis, as well as with conventional catalysis in cis.

Lipid Conversion by Cell-Free Synthesized Phospholipid Methyltransferase Opi3 in Defined Nanodisc Membranes Supports an in Trans Mechanism.

Biomembranes composed of lipids and proteins play central roles in physiological processes, and the precise balance between different lipid species is crucial for maintaining membrane function. One pathway for the biosynthesis of the abundant lipid phosphatidylcholine in eukaryotes involves a membrane-integrated phospholipid methyltransferase named Opi3 in yeast. A still unanswered question is whether Opi3 can catalyze phosphatidylcholine synthesis in trans, at membrane contact sites. While evidence for this activity was obtained from studies with complex in vitro-reconstituted systems based on endoplasmic reticulum membranes, isolated and purified Opi3 could not be analyzed. We present new insights into Opi3 activity by characterizing the in vitro-synthesized enzyme in defined hydrophobic environments. Saccharomyces cerevisiae Opi3 was cell-free synthesized and either solubilized in detergent micelles or co-translationally inserted into preformed nanodisc membranes of different lipid compositions. While detergent-solubilized Opi3 was inactive, the enzyme inserted into nanodisc membranes showed activity and stayed monomeric as revealed by native mass spectrometry. The methylation of its lipid substrate dioleoylphosphatidylmonomethylethanolamine to phosphatidylcholine was monitored by one-dimensional 31P nuclear magnetic resonance. Phosphatidylcholine formation was observed not only in nanodiscs containing inserted Opi3 but also in nanodiscs devoid of the enzyme containing the lipid substrate. This result gives a clear indication for in trans catalysis by Opi3; i.e., it acts on the substrate in juxtaposed membranes, while in cis lipid conversion may also contribute. Our established system for the characterization of pure Opi3 in defined lipid environments may be applicable to other lipid biosynthetic enzymes and help in understanding the subcellular organization of lipid synthesis.

Progesterone modulation of transmembrane helix-helix interactions between the alpha-subunit of Na/K-ATPase and phospholipid N-methyltransferase in the oocyte plasma membrane.

BACKGROUND: Progesterone binding to the surface of the amphibian oocyte initiates the meiotic divisions. Our previous studies with Rana pipiens oocytes indicate that progesterone binds to a plasma membrane site within the external loop between the M1 and M2 helices of the alpha-subunit of Na/K-ATPase, triggering a cascade of lipid second messengers and the release of the block at meiotic prophase. We have characterized this site, using a low affinity ouabain binding isoform of the alpha1-subunit. RESULTS: Preparations of isolated plasma membranes from Rana oocytes demonstrate that physiological levels of progesterone (or the non-metabolizable progestin R5020) successively activate phosphatidylethanolamine-N-methyltransferase (PE-NMT) and sphingomyelin synthase within seconds. Inhibition of PE-NMT blocks the progesterone induction of meiosis in intact oocytes, whereas its initial product, phosphatidylmonomethylethanolamine (PME), can itself initiate meiosis in the presence of the inhibitor. Published X-ray crystallographic data on Na/K-ATPase, computer-generated 3D projections, heptad repeat analysis and hydrophobic cluster analysis of the transmembrane helices predict that hydrophobic residues L, V, V, I, F and Y of helix M2 of the alpha1-subunit interact with F, L, G, L, L and F, respectively, of helix M3 of PE-NMT. CONCLUSION: We propose that progesterone binding to the first external loop of the alpha1-subunit facilitates specific helix-helix interactions between integral membrane proteins to up-regulate PE-NMT, and, that successive interactions between two or more integral plasma membrane proteins induce the signaling cascades which result in completion of the meiotic divisions.