Mapping the phospholipid-binding surface and translocation determinants ofthe C2 domain from cytosolic phospholipase A(2)

Cytosolic phospholipase A(2) (cPLA(2)) plays a key role in the generation o f arachidonic acid, a precursor of potent inflammatory mediators, Intact cP LA(2) is known to translocate in a calcium-dependent manner from the cytoso l to the nuclear envelope and endoplasmic reticulum, We show here that the C2 domain of cPLA(2) alone is sufficient for this calcium-dependent translo cation in living cells, We have identified sets of exposed hydrophobic resi dues in loops known as calcium-binding region (CBR) 1 and CBR3, which surro und the C2 domain calcium-binding sites, whose mutation dramatically decrea sed phospholipid binding in vitro without significantly affecting calcium b inding. Mutation of a residue that binds calcium ions (D43N) also eliminate d phospholipid binding. The same mutations that prevent phospholipid bindin g of the isolated C2 domain in vitro abolished the calcium-dependent transl ocation of cPLA(2) to internal membranes in vivo, suggesting that the membr ane targeting is driven largely by direct interactions with the phospholipi d bilayer, Using fluorescence quenching by spin-labeled phospholipids for a series of mutants containing a single tryptophan residue at various positi ons 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 hydrophob ic core of the phospholipid bilayer, establishing an anchor for docking the domain onto the membrane.