The presynaptic protein ␣-synuclein (␣-syn), particularly in its amyloid form, is widely recognized for its involvement in Parkinson disease (PD). Recent genetic studies reveal that mutations in the gene GBA are the most widespread genetic risk factor for parkinsonism identified to date. GBA encodes for glucocerebrosidase (GCase), the enzyme deficient in the lysosomal storage disorder, Gaucher disease (GD). In this work, we investigated the possibility of a physical linkage between ␣-syn and GCase, examining both wild type and the GD-related N370S mutant enzyme. Using fluorescence and nuclear magnetic resonance spectroscopy, we determined that ␣-syn and GCase interact selectively under lysosomal solution conditions (pH 5.5) and mapped the interaction site to the ␣-syn C-terminal residues, 118 -137. This ␣-syn-GCase complex does not form at pH 7.4 and is stabilized by electrostatics, with dissociation constants ranging from 1.2 to 22 M in the presence of 25 to 100 mM NaCl. Intriguingly, the N370S mutant form of GCase has a reduced affinity for ␣-syn, as does the inhibitor conduritol-␤-epoxidebound enzyme. Immunoprecipitation and immunofluorescence studies verified this interaction in human tissue and neuronal cell culture, respectively. Although our data do not preclude protein-protein interactions in other cellular milieux, we suggest that the ␣-syn-GCase association is favored in the lysosome, and that this noncovalent interaction provides the groundwork to explore molecular mechanisms linking PD with mutant GBA alleles.

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...

Histone tails and their post-translational modifications (PTM) play important roles in regulating the structure and dynamics of chromatin. For histone H4, the basic patch K16R17H18R19 in the N-terminal tail modulates chromatin compaction and nucleosome sliding catalyzed by ATP-dependent ISWI chromatin remodeling enzymes while acetylation of H4 K16 affects both functions. The structural basis for the effects of this acetylation is unknown. Here we investigated the conformation of histone tails in the nucleosome by solution NMR. We found that backbone amides of the N-terminal tails of histones H2A, H2B, and H3 are largely observable due to their conformational disorder. However, only residues 1–15 in H4 can be detected, indicating that residues 16–22 in the tails of both H4 histones fold onto the nucleosome core. Surprisingly, we found that K16Q mutation in H4, a mimic of K16 acetylation, leads to a structural disorder of the basic patch. Thus, our study suggests that the folded structure of the H4 basic patch in the nucleosome is important for chromatin compaction and nucleosome remodeling by ISWI enzymes while K16 acetylation affects both functions by causing structural disorder of the basic patch, K16R17H18R19.

SUMMARY We have defined the molecular basis for association of the PH domain of the Arf GAP ASAP1 with phospholipid bilayers. Structures of the unliganded and dibutyryl PtdIns(4,5)P2-bound PH domain were solved. PtdIns(4,5)P2 made contact with both a canonical site (C site) and an atypical site (A site). We hypothesized cooperative binding of PtdIns(4,5)P2 to the C site and a nonspecific anionic phospholipid to the A site. PtdIns(4,5)P2 dependence of binding to large unilamellar vesicles and GAP activity was sigmoidal, consistent with cooperative sites. In contrast, PtdIns(4,5) P2 binding to the PH domain of PLC δ1 was hyperbolic. Mutation of amino acids in either the C or A site resulted in decreased PtdIns(4,5)P2-dependent binding to vesicles and decreased GAP activity. The results support the idea of cooperative phospholipid binding to the C and A sites of the PH domain of ASAP1. We propose that the mechanism underlies rapid switching between active and inactive ASAP1.