Hierarchy of membrane-targeting signals of phospholipase D1 involving lipid modification of a pleckstrin homology domain.
The amino terminus of phospholipase D1 (PLD1) contains three potential membrane-interacting determinants: a phox homology (PX) domain, a pleckstrin homology (PH) domain and two adjacent cysteines at positions 240 and 241 within the PH domain that are fatty acylated in vivo. To understand how these determinants contribute to membrane localization, we have mutagenized critical residues of the PLD1 PH domain in the wild type or palmitate-free background in the intact protein, in a fragment that deletes the first 210 amino acids including the PX domain, and in the isolated PH domain. Mutants were expressed in COS-7 cells and examined for membrane residence, intracellular localization, palmitoylation, and catalytic activity. Our results are as follows. 1) Mutagenesis of critical residues of the PH domain results in redistribution of PLD1 from membranes to cytosol, independently of fatty acylation sites. Importantly, PH domain mutants in the wild type background showed greatly reduced fatty acylation, despite the presence of all relevant cysteines. 2) The isolated PH domain did not co-localize with PLD1 and was not palmitoylated. 3) The PX deletion mutant showed similar distribution and palmitoylation to the intact protein. Interestingly, PH domain mutants in this background showed significant palmitoylation and incomplete cytosolic redistribution. 4) PH domain mutants in the wild type or palmitate-free background maintained catalytic activity. We propose that membrane targeting of PLD1 involves a hierarchy of signals with a functional PH domain allowing fatty acylation leading to strong membrane binding. The PX domain may modulate function of the PH domain.
Sugars
Jane M
JM
Department of Signalling, Babraham Institute, Cambridge CB2 4AT, United Kingdom.
Cellek
Selim
S
Manifava
Maria
M
Coadwell
John
J
Ktistakis
Nicholas T
NT
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002462:Cell Membrane / Q000201:enzymology* / Q000378:metabolism; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D008024:Ligands; D050356:Lipid Metabolism; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010169:Palmitic Acids / Q000378:metabolism; D010739:Phospholipase D / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D014162:Transfection
D003902:Detergents; D008024:Ligands; D010169:Palmitic Acids; D010739:Phospholipase D; C106801:phospholipase D1