The active site of yeast phosphatidylinositol synthase Pis1 is facing the cytosol.
Five yeast enzymes synthesizing various glycerophospholipids belong to the CDP-alcohol phosphatidyltransferase (CAPT) superfamily. They only share the so-called CAPT motif, which forms the active site of all these enzymes. Bioinformatic tools predict the CAPT motif of phosphatidylinositol synthase Pis1 as either ER luminal or cytosolic. To investigate the membrane topology of Pis1, unique cysteine residues were introduced into either native or a Cys-free form of Pis1 and their accessibility to the small, membrane permeating alkylating reagent N-ethylmaleimide (NEM) and mass tagged, non-permeating maleimides, in the presence and absence of non-denaturing detergents, was monitored. The results clearly point to a cytosolic location of the CAPT motif. Pis1 is highly sensitive to non-denaturing detergent, and low concentrations (0.05%) of dodecylmaltoside change the accessibility of single substituted Cys in the active site of an otherwise cysteine free version of Pis1. Slightly higher detergent concentrations inactivate the enzyme. Removal of the ER retrieval sequence from (wt) Pis1 enhances its activity, again suggesting an influence of the lipid environment. The central 84% of the Pis1 sequence can be aligned and fitted onto the 6 transmembrane helices of two recently crystallized archaeal members of the CAPT family. Results delineate the accessibility of different parts of Pis1 in their natural context and allow to critically evaluate the performance of different cysteine accessibility methods. Overall the results show that cytosolically made inositol and CDP-diacylglycerol can access the active site of the yeast PI synthase Pis1 from the cytosolic side and that Pis1 structure is strongly affected by mild detergents.
['Bochud|Arlette|A|', 'Conzelmann|Andreas|A|']
D000465:Algorithms D051607:CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase D020134:Catalytic Domain D019295:Computational Biology D003545:Cysteine D003567:Cytidine Diphosphate Diglycerides D003600:Cytosol D003902:Detergents D004789:Enzyme Activation D004795:Enzyme Stability D007294:Inositol D008958:Models, Molecular D016297:Mutagenesis, Site-Directed D009154:Mutation D011487:Protein Conformation D011489:Protein Denaturation D017434:Protein Structure, Tertiary D012441:Saccharomyces cerevisiae D013329:Structure-Activity Relationship D013379:Substrate Specificity D013997:Time Factors D017855:Transferases (Other Substituted Phosphate Groups)
Phosphatidylinositol CDP-alcohol phosphatidyltransferases Topology Lipid environment Cysteine accessibility Membrane protein