Fatty acylation of phospholipase D1 on cysteine residues 240 and 241 determines localization on intracellular membranes

We have reported previously that phospholipase D1 (PLD1) is labeled specifi cally with [H-3]palmitate following transient expression and immunoprecipit ation and that this modification appeared important both for membrane local ization and catalytic activity, In this work we identify by mutagenesis tha t the acylation sites on PLD1 are cysteine residues 240 and 241, with the c ysteine at position 241 accounting for most but not all of the modification , Replacement of both cysteine residues with either serines or alanines res ulted in a mutant protein that contained undetectable [H-3]palmitate. In co mparison with the wild type protein, the double mutant showed reduced catal ytic activity in vivo, whereas its activity in vitro was unchanged. In addi tion, the localization of the double mutant was altered in comparison with the wild type protein, whereas wild type PLD1 is primarily on intracellular membranes and on punctate structures, the double mutant was on plasma memb rane. Because cysteines 240 and 241 lie within a putative pleckstrin homolo gy domain of PLD1, it is likely that fatty acylation on these residues modu lates the function of the PLD1 pleckstrin homology domain.