The c-terminal domain of glycerol-3-phosphate acyltransferase is required for enzymatic activity

GONZALEZ-BARO, M.R.; COLEMAN, R.A.

Villa Carlos Paz, Cordoba

Congreso; XXXVIII Reunión Anual de la Sociedad Argentina de Investigación Bioquímica (SAIB); 2002

SAIB

                Mitochondrial glycerol-3-phosphate acyltransferase (GPAT, 828 aa) catalyses the first step in glycerolipid synthesis. It is inserted in the outer membrane by two transmembrane domains (TMDs), having its active site located in the N-terminal domain. Other acyltransferases whose substrates and products are structurally similar than GPAT have homologous active sites but lack GPAT’s TMDs and C-terminal domain (CTD), suggesting that GPAT’s CTD is not involved in catalysis. Nevertheless, truncation of GPAT before the second TMD resulted in an inactive enzyme. So our aim is to determine if GPAT activity is dependent on the presence of the entire CTD. Based on the presence of possible regulatory consensus sequences in the CTD, two truncated GPATs at aa 626 and 742 with a Flag epitope in the C-terminus were transiently expressed in CHO cells. The overexpression of both constructs (confirmed by immunodetection with the anti-Flag antibody) yielded inactive enzymes. To determine whether the inactivation was due to the Flag epitope or the truncations themselves, an active GPAT with a HA epitope in the N-terminus was truncated after aa 626, 742 and 751, overexpressed in CHO cells and immunodetected with anti-HA antibody. The protein truncated after aa 742 showed no enzymatic activity; the GPAT truncations after aa 626 and 751 had activities only 15% of the full-length transfected enzyme. We postulate that the CTD is required for activity; it may contribute to the proper folding of the enzyme or it may be involved in supramolecular regulatory interactions.