Membrane targeting of C2 domains of phospholipase C-delta isoforms.
The C2 domain is a Ca(2+)-dependent membrane-targeting module found in many cellular proteins involved in signal transduction or membrane trafficking. To understand the mechanisms by which the C2 domain mediates the membrane targeting of PLC-delta isoforms, we measured the in vitro membrane binding of the C2 domains of PLC-delta1, -delta3, and -delta4 by surface plasmon resonance and monolayer techniques and their subcellular localization by time-lapse confocal microscopy. The membrane binding of the PLC-delta1-C2 is driven by nonspecific electrostatic interactions between the Ca(2+)-induced cationic surface of protein and the anionic membrane and specific interactions involving Ca(2+), Asn(647), and phosphatidylserine (PS). The PS selectivity of PLC-delta1-C2 governs its specific Ca(2+)-dependent subcellular targeting to the plasma membrane. The membrane binding of the PLC-delta3-C2 also involves Ca(2+)-induced nonspecific electrostatic interactions and PS coordination, and the latter leads to specific subcellular targeting to the plasma membrane. In contrast to PLC-delta1-C2 and PLC-delta3-C2, PLC-delta4-C2 has significant Ca(2+)-independent membrane affinity and no PS selectivity due to the presence of cationic residues in the Ca(2+)-binding loops and the substitution of Ser for the Ca(2+)-coordinating Asp in position 717. Consequently, PLC-delta4-C2 exhibits unique pre-localization to the plasma membrane prior to Ca(2+) import and non-selective Ca(2+)-mediated targeting to various cellular membranes, suggesting that PLC-delta4 might have a novel regulatory mechanism. Together, these results establish the C2 domains of PLC-delta isoforms as Ca(2+)-dependent membrane targeting domains that have distinct membrane binding properties that control their subcellular localization behaviors.
Ananthanarayanan
Bharath
B
Department of Chemistry, University of Illinois, Chicago 60607, USA.
Das
Sudipto
S
Rhee
Sue Goo
SG
Murray
Diana
D
Cho
Wonhwa
W
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D020134:Catalytic Domain; D002460:Cell Line; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010713:Phosphatidylcholines / Q000378:metabolism; D054803:Phospholipase C delta; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000201:enzymology; D020349:Surface Plasmon Resonance; D014162:Transfection; D010738:Type C Phospholipases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D007527:Isoenzymes; D010713:Phosphatidylcholines; D020033:Protein Isoforms; D011994:Recombinant Proteins; D001224:Aspartic Acid; D005982:Glutathione Transferase; D010738:Type C Phospholipases; C518024:PLCD1 protein, human; C518028:PLCD3 protein, human; C518030:PLCD4 protein, human; D054803:Phospholipase C delta; D002118:Calcium