The C-terminal region of mitochondrial glycerol-3-phosphate acyltransferase-1 interacts with the active site region and is required for activity.
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 the mitochondrial GPAT1 isoform (mtGPAT1, 828 amino acids). mtGPAT1 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 mtGPAT1 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, none of them conferred GPAT activity. The loss of activity was not due to the miss-targeting of the proteins since immunofluorescence experiments demonstrated their mitochondrial localization. Instead, chemical crosslinking and protein cleavage studies demonstrated that the N- and C-termini of the protein interact. These results suggest that the C-terminal domain is necessary for mtGPAT1 activity, and probably contributes to catalysis or substrate binding.
Pellon-Maison
Magalí
M
Instituto de Investigaciones Bioquímicas de La Plata, UNLP, La Plata, Argentina.
Coleman
Rosalind A
RA
Gonzalez-Baró
María R
MR
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000235:genetics / Q000502:physiology*; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000502:physiology
D011993:Recombinant Fusion Proteins; D005992:Glycerol-3-Phosphate O-Acyltransferase