Mapping the phospholipid-binding surface and translocation determinants of the C2 domain from cytosolic phospholipase A2

Cytosolic phospholipase A(2) (cPLA(2)) plays a key role in the generation of arachidonic acid, a precursor of potent inflammatory mediators, Intact cPLA(2) is known to translocate in a calcium-dependent manner from the cytosol to the nuclear envelope and endoplasmic reticulum, We show here that the C2 domain of cPLA(2) alone is sufficient for this calcium-dependent translocation in living cells, We have identified sets of exposed hydrophobic residues in loops known as calcium-binding region (CBR) 1 and CBR3, which surround the C2 domain calcium-binding sites, whose mutation dramatically decreased phospholipid binding in vitro without significantly affecting calcium binding. Mutation of a residue that binds calcium ions (D43N) also eliminated phospholipid binding. The same mutations that prevent phospholipid binding of the isolated C2 domain in vitro abolished the calcium-dependent translocation of cPLA(2) to internal membranes in vivo, suggesting that the membrane targeting is driven largely by direct interactions with the phospholipid bilayer, Using fluorescence quenching by spin-labeled phospholipids for a series of mutants containing a single tryptophan residue at various positions in the cPLA(2) C2 domain, we show that two of the calcium-binding loops, CBR1 and CBR3, penetrate in a calcium-dependent manner into the hydrophobic core of the phospholipid bilayer, establishing an anchor for docking the domain onto the membrane.