Mitochondrial glycerol-3-phosphate acyltransferase activity requires the integrity of the C-terminal domain

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

Hannover, NH, USA

Conferencia; Gordon Research Conference. Molecular and Cellular Biology of Lipids; 2003

GRC

Glycerol phosphate acyltransferase (GPAT) catalyzes the formation of 1-acyl-sn-glycerol-3-phosphate from glycerol-3-phosphate and long chain fatty acyl-CoA substrates. We previously determined the topography of mitochondrial GPAT isoform. (828 amino acids). It has two transmembrane domains (TMDs) (aa 472-493 and aa 576-592) with both the N- and C-termini facing the cytosol and a loop (aa 494-575) facing the intermembrane space. Alignment of amino acid sequences from GPAT and other acyltransferases and site directed mutagenesis studies have demonstrated that the active site of the enzyme resides in the N-terminal domain of the protein.  In this study we sequentially truncated the C-terminal domain and characterized the properties of the resulting mutants expressed in CHO cells. Although the mutants were overexpressed, neither of them conferred GPAT activity. The loss of activity was not due to the miss- targeting of the proteins since the immunofluoresence experiments demonstrated their mitochondrial localization. Experiments of chemical crosslinking and protein cleavage demonstrated a possible interaction between the  N- and C-termini of the protein. These results suggest  that the C-terminal domain is absolutely necessary for GPAT activity, and probably contributes to catalysis or substrate binding. To date, no studies have assessed the quaternary structure of the enzyme. It was therefore another aim of the present study to provide evidence for the existence of oligomeric mtGPAT complexes, using recombinant GPAT overexpressed in different eukaryotic systems. Using chemical crosslinking and SDS-PAGE dimeric, trimeric, tetrameric and a higher molecular mass complex were found. At inhibitory palmitoylCoA concentration the complex could also be observed. Using Triton X-100 combined with non-denaturing gel, mtGPAT was mainly found as a tetramer. mt GPATs trucated at the C-terminal domain were found to oligomerize normally. These results demonstrate that: 1) mtGPAT exists as oligomeric complexes. 2) high palmitoylCoA concentrations did not alter the oligomerization  3) The C-terminal domain of mtGPAT is not involved in the  arrangement of the quaternary structure and 4) the possibility of mtGPAT interacting with other mitochondrial protein/s can not be overlooked.