Synthesis of phospholipids, sterols and sphingolipids is thought to occur at contact sites between the endoplasmic reticulum (ER) and other organelles because many lipid-synthesizing enzymes are enriched in these contacts. In only a few cases have the enzymes been localized to contacts in vivo and in no instances have the contacts been demonstrated to be required for enzyme function. Here, we show that plasma membrane (PM)-ER contact sites in yeast are required for phosphatidylcholine synthesis and regulate the activity of the phosphatidylethanolamine N-methyltransferase enzyme, Opi3. Opi3 activity requires Osh3, which localizes to PM-ER contacts where it might facilitate in trans catalysis by Opi3. Thus, membrane contact sites provide a structural mechanism to regulate lipid synthesis.

Escherichia coli outer membrane protease T (OmpT) is an endopeptidase that specifically cleaves between two consecutive basic residues. In this study we have investigated the substrate specificity of OmpT using spatially addressed SPOT peptide libraries. The peptide acetyl-Dap(dnp)-Ala-Arg/Arg-Ala-Lys(Abz)-Gly was synthesized directly onto cellulose membrane. The peptide contained the aminobenzoyl (Abz) fluorophore, which was internally quenched by the dinitrophenyl (dnp) moiety. Treatment of the SPOT membrane with the small, water-soluble protease trypsin resulted in highly fluorescent peptide SPOTs. However, no peptide cleavage was observed after incubation with detergent-solubilized OmpT, a macromolecular complex with an estimated molecular mass of 180 kDa. This problem could be solved by the introduction of a long, polar polyoxyethylene glycol linker between the membrane support and the peptide. Peptide libraries for the P(2), P(1), P(1)', and P(2)' positions in the substrate were screened with OmpT, and peptides of positive SPOTs were resynthesized and subjected to kinetic measurements in solution. The best substrate Abz-Ala-Lys-Lys-Ala-Dap(dnp)-Gly had a turnover number k(cat) of 40 s(-)(1), which is 12-fold higher than the starting substrate. Peptides containing an acidic residue at P(2) or P(2)' were not substrates for OmpT, suggesting that long-range electrostatic interactions are important for the formation of the enzyme-substrate complex. OmpT was highly selective toward L-amino acids at P(1) but was less so at P(1)' where a peptide with D-Arg at P(1)' was a competitive inhibitor (K(i) of 19 microM). An affinity chromatography resin based on these findings was developed, which allowed for the one-step purification of OmpT from a bacterial lysate. The implications of the determined consensus substrate sequence (Arg/Lys)/(Arg/Lys)-Ala for the proposed biological function of OmpT in defense against antimicrobial peptides are discussed.

The genes coding for the mature part of the lipases from Sfaphy1ococc~u.s uureuks NCTC8530 and Stup1iylococcu.s hyicus have been cloned and overexpressed in Escherichiu coli as fusion proteins with an N-terminal hexa-histidine tag. The enzymes accumulated in the cytoplasm and were purified using sequential precipitation with protamine sulphate and ammonium sulphate, followed by metal-affinity and hydroxyapatite chromatography. The yield of pure lipase was 4.5 mg/g wet cells for S. uureus lipase and 13 mglg for S. hyicus lipase. The purified enzymes need calcium for activity, albeit with different affinities, and a low residual activity was found in the absence of calcium. In contrast to S. hyicus lipase, not only strontium but also barium can replace calcium with full retention of activity of S. uureus lipase. Whereas S. hyicu.s lipase is optimally active at pH 8.5, the optimum pH for enzymatic activity for S. uureus lipase was found to be pH 6.5. The S. uureMs lipase has a narrow substrate specificity: short-chain triacylglycerols and acyl esters of both p-nitrophenol and umbelliferone are readily degraded, whereas medium-and long-chain lipids, as well as phospholipids, are poor substrates. In contrast, S. hyicus lipase prefers phospholipids as substrate and hydrolyses neutral lipids irrespective of their chain length. The results are discussed in view of the large sequence similarity between both lipases.Keywords: Stuphylococcus u u e u s lipase ; Sfaplzylococcw hyicus lipase ; overexpression ; purification ; substrate specificity.Lipases (glycerol ester hydrolase) are active at a lipid-water interface where they degrade water-insoluble triacylglycerols. These enzymes usually have a broad substrate specificity and also degrade acyl p-nitrophenyl esters, Tweens and phospholipids often with positional, stereo-and chain-length selectivity (Jaeger et al., 1994, and references therein).All lipases of known three-dimensional structure belong to the class of serine esterases (Brady et al., 1990; Grochulski et al., 1993: Schrag et al., 1991Winkler et al., 1990). The active site of these enzymes contains a catalytic triad consisting of Ser, His and an acidic residue which in the case of lipases is either Asp or Glu. This catalytic machinery is covered by at1 amphipathic surface loop, the so-called lid. The structures of a lipaseinhibitor complex and a lipase-micelle complex (Brzozowski et al., 1991 ; van Tilbeurgh et al., 1993) showed that the lid moves away, presumably due to the interaction with the substrate interCorr-[,sl)ondenc,r ro H. M. Verheij,

A promising tool in membrane research is the use of the styrene–maleic acid (SMA) copolymer to solubilize membranes in the form of nanodiscs. Since membranes are heterogeneous in composition, it is important to know whether SMA thereby has a preference for solubilization of either specific types of lipids or specific bilayer phases. Here, we investigated this by performing partial solubilization of model membranes and analyzing the lipid composition of the solubilized fraction. We found that SMA displays no significant lipid preference in homogeneous binary lipid mixtures in the fluid phase, even when using lipids that by themselves show very different solubilization kinetics. By contrast, in heterogeneous phase-separated bilayers, SMA was found to have a strong preference for solubilization of lipids in the fluid phase as compared to those in either a gel phase or a liquid-ordered phase. Together the results suggest that (1) SMA is a reliable tool to characterize native interactions between membrane constituents, (2) any solubilization preference of SMA is not due to properties of individual lipids but rather due to properties of the membrane or membrane domains in which these lipids reside and (3) exploiting SMA resistance rather than detergent resistance may be an attractive approach for the isolation of ordered domains from biological membranes.Electronic supplementary materialThe online version of this article (doi:10.1007/s00249-016-1181-7) contains supplementary material, which is available to authorized users.

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