Many peripheral proteins involved in cell signaling translocate to different cell membranes in response to specific cell stimuli. Because cellular functions and regulation of these proteins depend on their specific subcellular localization (1), understanding the mechanisms of membrane targeting is of great importance. The membrane targeting of diverse peripheral proteins is mediated by a limited number of membrane-targeting domains, including protein kinase C (PKC) 1 conserved 1 (C1), PKC conserved 2 (C2), and pleckstrin homology domains. Recent structural and functional studies of individual membrane targeting domains as well as the peripheral proteins harboring these domains have provided new insights into the molecular mechanisms underlying the specific subcellular targeting and activation of peripheral proteins. This review summarizes the recent progress in our understanding of the mechanisms of C1 and C2 domain-mediated membrane targeting, with an emphasis on the correlation between the membrane binding properties of the C1 and C2 domains and the peripheral proteins containing these domains and their subcellular targeting behaviors. There are several excellent reviews (2-6) that contain more exhaustive surveys on the membrane targeting domains. Membrane-Protein InteractionsThe membrane binding of peripheral proteins involves different types of interactions ( Fig. 1) that depend upon the physicochemical properties of both membrane and protein. Membranes of different cellular compartments have different compositions of bulk lipids that can modulate membrane targeting of proteins either by providing unique microenvironments or by producing specific lipid metabolites, such as diacylglycerol (DAG) and phosphoinositides, that function as second messengers. Extensive structural and mutational studies of phospholipases A 2 (PLA 2 ) have shown that their membrane binding surfaces are composed of cationic, aliphatic, and aromatic residues (7). A recent study by surface plasmon resonance analysis indicated that cationic residues primarily accelerate the association of protein to anionic membrane surfaces, whereas aliphatic residues mainly slow the membrane dissociation by penetrating into the hydrophobic core of the membrane (8). Aromatic residues, particularly Trp, which has a preference for the waterlipid interface (9), play a pivotal role in binding to zwitterionic PC membranes (7, 10) by affecting both membrane association and dissociation steps (8). A priori, the physicochemical principles learned from these in vitro membrane binding studies should allow the prediction of the subcellular targeting behaviors of peripheral proteins, provided that the subcellular targeting is driven mainly by membrane-protein interactions. Structure, Function, and Occurrence of C1 DomainsThe C1 domain (ϳ50 amino acids) is a cysteine-rich compact structure that contains five short ␤ strands, a short ␣-helix, and two zinc ions (Fig. 2) (11, 12). The C1 domain was first identified as the interaction site for DAG and phorbol ester in PK...

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Recent genetic and structural studies have shed considerable light on the mechanism by which secretory phospholipases A 2 interact with substrate aggregates. Electrostatic forces play an essential role in optimizing interfacial catalysis. Efficient and productive adsorption of the Class I bovine pancreatic phospholipase A 2 to anionic interfaces is dependent upon the presence of two nonconserved lysine residues at sequence positions 56 and 116, implying that critical components of the adsorption surface differ among enzyme species (Dua, R., Wu, S.-K., and Cho, W. (1995) J. Biol. Chem. 270, 263-268). In an effort to further characterize the protein residues involved in interfacial catalysis, we have determined the high resolution (1.7 Å) x-ray structure of the Class II Asp-49 phospholipase A 2 from the venom of Agkistrodon piscivorus piscivorus. Correlation of the three-dimensional coordinates with kinetic data derived from sitedirected mutations near the amino terminus (E6R, K7E, K10E, K11E, and K16E) and the active site (K54E and K69Y) defines much of the interface topography. Lysine residues at sequence positions 7 and 10 mediate the adsorption of A. p. piscivorus phospholipase A 2 to anionic interfaces but play little role in the enzyme's interaction with electrically neutral surfaces or in substrate binding. Compared to the native enzyme, the mutant proteins K7E and K10E demonstrate comparable (20-fold) decreases in affinity and catalysis on polymerized mixed liposomes of 1 -hexadecanoyl-2-(1-pyrenedecanoyl) -sn-glycero-3-phosphocholine and 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphoglycerol, while the double mutant, K7E/ K10E, shows a more dramatic 500-fold decrease in catalysis and interfacial adsorption. The calculated contributions of Lys-7 and Lys-10 to the free energy of binding of A. p. piscivorus phospholipase A 2 to anionic liposomes (؊1.8 kcal/mol at 25°C per lysine) are additive (i.e. ؊3.7 kcal/mol) and together represent nearly half of the total binding energy. Although both lysine side chains lie exposed at the edge of the proposed interfacial adsorption surface, they are geographically remote from the corresponding interfacial determinants for the bovine enzyme. Our results confirm that interfacial adsorption is largely driven by electrostatic forces and demonstrate that the arrangement of the critical charges (e.g. lysines) is species-specific. This variability in the topography of the adsorption surface suggests a corresponding flexibility in the orientation of the active enzyme at the substrate interface.Phospholipases A 2 (PLA 2 ; EC 3.1. 1.4) 1 catalyze the hydrolysis of the fatty acid ester in the 2-position of 3-sn-phospholipids and are found both in intracellular and secreted forms (for recent reviews, see Refs. 1-4). The enzymes act at the lipidwater interface with a preference for organized lipids (micelles and vesicles) that is often orders of magnitudes greater than that shown for dispersed substrate. Calcium-mediated catalysis appears to involve two kinetically and struc...

Mammalian secretory class V phospholipase A2 (PLA2) is a newly discovered PLA2 that is implicated in eicosanoid formation in inflammatory cells. As a first step towards understanding the structure, function and regulation of this PLA2, we constructed a bacterial expression vector for human secretory class V PLA2 (hV-PLA2), over-expressed and purified the protein, and determined its physical and kinetic properties. When compared with human class IIa enzyme (hIIa-PLA2), hV-PLA2 has several distinct properties. First, hV-PLA2 can catalyse the hydrolysis of phosphatidylcholine more effectively than hIIa-PLA2 by two orders of magnitude. Secondly, hV-PLA2 has much higher binding affinity and activity for compactly packed phosphatidylcholine bilayers than hIIa-PLA2. Finally, hV-PLA2 has much reduced thermal stability compared with hIIa-PLA2. These data suggest that hV-PLA2 is better suited than hIIa-PLA2 for acting on the outer cellular membrane and liberating arachidonic acid from membrane phospholipids. Also, the unusually low thermal stability of hV-PLA2 might contribute to tighter regulation of its activities in extracellular media.