The ADP-ribosylation factor (Arf) family of GTP-binding proteins are regulators of membrane traffic and the actin cytoskeleton. Both negative and positive regulators of Arf, the centaurin ␤ family of Arf GTPase-activating proteins (GAPs) and Arf guanine nucleotide exchange factors, contain pleckstrin homology (PH) domains and are activated by phosphoinositides. To understand how the activities are coordinated, we have examined the role of phosphoinositide binding for Arf GAP function using ASAP1/centaurin ␤4 as a model. In contrast to Arf exchange factors, phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P 2 ) specifically activated Arf GAP. D3 phosphorylated phosphoinositides were less effective. Activation involved PtdIns-4,5-P 2 binding to the PH domain; however, in contrast to the Arf exchange factors and contrary to predictions based on the current paradigm for PH domains as independently functioning recruitment signals, we found the following: (i) the PH domain was dispensable for targeting to PDGF-induced ruffles; (ii) activation and recruitment could be uncoupled; (iii) the PH domain was necessary for activity even in the absence of phospholipids; and (iv) the Arf GAP domain influenced localization and lipid binding of the PH domain. Furthermore, PtdIns-4,5-P 2 binding to the PH domain caused a conformational change in the Arf GAP domain detected by limited proteolysis. Thus, these data demonstrate that PH domains can function as allosteric sites. In addition, differences from the published properties of the Arf exchange factors suggest a model in which feedforward and feedback loops involving lipid metabolites coordinate GTP binding and hydrolysis by Arf. Arf1 proteins are ubiquitous and essential GTP-binding proteins in eukaryotes. They were first identified as cofactors for cholera toxin-catalyzed ADP-ribosylation of G s (1). The relationship between this activity and the normal cellular function of Arf is not clear. The best described activity of Arf is the regulation of membrane traffic (2, 3). More recently, potential roles in reorganization of the actin cytoskeleton have been identified for Arf1 and Arf6 (4 -7). Neither the molecular mechanism by which Arf proteins regulate these pathways nor the means by which Arf itself is regulated have been delineated; however, phospholipids probably function in both capacities.The relationship between Arf and phospholipids is complex. Arf activates phospholipase D and phosphatidylinositol 4-phosphate 5-kinase, resulting in the production of phosphatidic acid (PA) and PtdIns-4,5-P 2 (8 -11). Phosphoinositides and phosphatidic acid also regulate Arf function. Arf itself binds PtdIns-P 2 (12). Phosphoinositides have a role in converting Arf-GTP to Arf-GDP by the centaurin ␤ family of Arf GAPs. Four members of this family have been identified 2 (13, 14), and one member of the family, ASAP1 (also called centaurin ␤4), was purified as an Arf GAP that is coordinately activated by PA and phosphoinositides (13). The Arf⅐PtdIns-P 2 complex was found to be the substrat...

The dynamic structure of detergent-resistant membranes (DRMs) isolated from RBL-2H3 cells was characterized using two different acyl chain spin-labeled phospholipids (5PC and 16PC), a headgroup labeled sphingomyelin (SM) analog (SD-Tempo) and a spin-labeled cholestane (CSL). It was shown, by comparison to dispersions of SM, dipalmitoylphosphatidylcholine (DPPC), and DPPC/cholesterol of molar ratio 1, that DRM contains a substantial amount of liquid ordered phase: 1) The rotational diffusion rates (R( perpendicular)) of 16PC in DRM between -5 degrees C and 45 degrees C are nearly the same as those in molar ratio DPPC/Chol = 1 dispersions, and they are substantially greater than R( perpendicular) in pure DPPC dispersions in the gel phase studied above 20 degrees C; 2) The order parameters (S) of 16PC in DRM at temperatures above 4 degrees C are comparable to those in DPPC/Chol = 1 dispersions, but are greater than those in DPPC dispersions in both the gel and liquid crystalline phases. 3) Similarly, R( perpendicular) for 5PC and CSL in DRM is greater than in pure SM dispersions in the gel phase, and S for these labels in DRM is greater than in the SM dispersions in both the gel and liquid crystalline phases. 4) R( perpendicular) of SD-Tempo in DRM is greater than in dispersions of SM in both gel and liquid phases, consistent with the liquid-like mobility in the acyl chain region in DRM. However, S of SD-Tempo in DRM is substantially less than that of this spin label in SM in gel and liquid crystalline phases (in absolute values), indicating that the headgroup region in DRMs is less ordered than in pure SM. These results support the hypothesis that plasma membranes contain DRM domains with a liquid ordered phase that may coexist with a liquid crystalline phase. There also appears to be a coexisting region in DRMs in which the chain labels 16PC and 5PC are found to cluster. We suggest that other biological membranes containing high concentrations of cholesterol also contain a liquid ordered phase.

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Duramycin is a polypeptide antibiotic (molecular weight 2012) obtained from culture filtrates of Streptomyces cinnamomeus forma azacoluta. In this work, we show that low concentrations of duramycin induced aggregation of lipid vesicles containing unsaturated phosphatidylethanolamine and unsaturated monogalactosyl diglyceride, and of sarcoplasmic reticulum vesicles from rabbit skeletal muscle. Furthermore, duramycin inhibited the ATP-dependent Ca2+ uptake in sarcoplasmic reticulum vesicles without affecting the hydrolysis of ATP or the permeability of Ca2+. Also, duramycin only inhibited the bacteriorhodopsin proton pump reconstituted into phospholipid vesicles containing phosphatidylethanolamine. We have isolated a duramycin-resistant strain of Bacillus subtilis and have mapped the location of duramycin resistance. In this strain, the secretion of protons and influx of calcium were resistant to duramycin, and its lipid composition was profoundly different from that of the parent strain. No phosphatidylethanolamine was detected in the resistant strain. Our findings are consistent with the idea that duramycin recognizes a particular membrane conformation determined by the presence of phosphatidylethanolamine or monogalactosyl diglyceride.

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