Tetramerization of glycosylphosphatidylinositol-specific phospholipase C from Trypanosoma brucei.
Glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) is an integral membrane protein in the protozoan parasite Trypanosoma brucei. Enzyme activity appears to be suppressed in T. brucei, although the polypeptide is readily detectable. The basis for the apparent quiescence of GPI-PLC is not known. Protein oligomerization was investigated as a possible mechanism for post-translational regulation of GPI-PLC activity. An equilibrium between monomers, dimers, and tetramers of purified GPI-PLC was detected by molecular sieving and shown to be perturbed with specific detergents. Homotetramers dominated in Nonidet P-40, and dimers and monomers of GPI-PLC were the major species in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The detergents were exploited as tools to study the effect of oligomerization on enzyme activity. Tetrameric GPI-PLC was 3. 6-20-fold more active than the monomeric enzyme. Tetramer existence was confirmed by chemical cross-linking. In vivo cross-linking revealed the oligomeric state of GPI-PLC during latency and after enzyme activation. During quiescence, monomers were the predominant species in T. brucei. Assembly of tetrameric GPI-PLC occurred when parasites were subjected to conditions known to activate the enzyme. In Leishmania where heterologous expression of GPI-PLC causes a GPI deficiency, the enzyme existed as a tetramer. Hence, oligomerization of GPI-PLC is associated with high enzyme activity both in vivo and in vitro.
Armah
D A
DA
Department of Cellular Biology, the University of Georgia, Athens, Georgia 30602, USA.
Mensa-Wilmot
K
K
D000818:Animals; D001704:Biopolymers; D002793:Cholic Acids; D003432:Cross-Linking Reagents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D054800:Glycosylphosphatidylinositol Diacylglycerol-Lyase; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D014346:Trypanosoma brucei brucei / Q000201:enzymology*; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism*
D001704:Biopolymers; D002793:Cholic Acids; D003432:Cross-Linking Reagents; D010738:Type C Phospholipases; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054800:Glycosylphosphatidylinositol Diacylglycerol-Lyase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate