The organization and acetylation of nascent histones prior to their stable incorporation into chromatin were examined. Through sedimentation and immunoprecipitation analyses of HeLa cytosolic extracts, two somatic non-nucleosomal histone complexes were detected: one containing nascent H3 and H4, and a second containing H2A (and probably H2B) in association with the nonhistone protein NAP-1. The H3/H4 complex has a sedimentation coefficient of 5-6S, consistent with the presence of one or more escort proteins. H4 in the cytosolic H3/H4 complex is diacetylated, fully in accord with the acetylation state of newly synthesized H4 in chromatin. The diacetylation of nascent human H4 is therefore completed prior to nucleosome assembly. As part of our studies of the nascent H3/H4 complex, the cytoplasmic histone acetyltransferase most likely responsible for acetylating newly synthesized H4 was also investigated. HeLa histone acetyltransferase B (HAT B) acetylates H4 but not H3 in vitro, and maximally diacetylates H4 even in the presence of sodium butyrate. Human HAT B acetylates H4 exclusively on the lysine residues at positions 5 and 12, in complete agreement with the highly conserved acetylation pattern of nascent nucleosomal H4 (Sobel et al., 1995), and has a native molecular weight of approximately 100 kDa. Based on our findings a model is presented for the involvement of histone acetylation and NAP-1 in H2A/H2B deposition and exchange, during nucleosome assembly and chromatin remodeling in vivo.

RGS4, a mammalian GTPase-activating protein for G protein ␣ subunits, requires its N-terminal 33 amino acids for plasma membrane localization and biological activity (Srinivasa, S. P., Bernstein, L. S., Blumer, K. J., and Linder, M. E. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 5584 -5589). In this study, we tested the hypothesis that the N-terminal domain mediates membrane binding by forming an amphipathic ␣-helix. RGS4 bound to liposomes containing anionic phospholipids in a manner dependent on the first 33 amino acids. Circular dichroism spectroscopy of a peptide corresponding to amino acids 1-31 of RGS4 revealed that the peptide adopted an ␣-helical conformation in the presence of anionic phospholipids. Point mutations that either neutralized positive charges on the hydrophilic face or substituted polar residues on the hydrophobic face of the model helix disrupted plasma membrane targeting and biological activity of RGS4 expressed in yeast. Recombinant mutant proteins were active as GTPase-activating proteins in solution but exhibited diminished binding to anionic liposomes. Peptides corresponding to mutants with the most pronounced phenotypes were also defective in forming an ␣-helix as measured by circular dichroism spectroscopy. These results support a model for direct interaction of RGS4 with membranes through hydrophobic and electrostatic interactions of an N-terminal ␣-helix. Regulators of G protein signaling (RGS proteins)1 are a recently appreciated family of proteins that participate as negative regulators or effectors in G protein pathways (reviewed in Refs. 1 and 2). RGS proteins catalytically accelerate GTP hydrolysis on ␣ subunits, resulting in faster termination of G protein signaling. The GAP activity of RGS proteins may account for discrepancies between the measured intrinsic rates of GTP hydrolysis of the ␣ subunit and the deactivation rate of physiological effectors. In addition to their functions as GAPs, some RGS proteins may also regulate G protein pathways by serving as effector antagonists (3, 4). As new RGS family members are identified and characterized, it has become clear that RGS proteins can act as effectors, as well as inhibitors, of G protein pathways (5).More than 20 mammalian RGS proteins have been identified to date (1). All RGS family members share sequence similarity that extends over approximately 120 amino acids. In many RGS proteins, this so-called "RGS box" or core domain is sufficient to bind G protein ␣ subunits and catalyze GTPase activity in vitro (6 -9). However in a cellular context, regions outside the RGS domain are necessary for biological activity of the protein (8, 10). Thus, important regulatory information is likely to be contained within these highly divergent flanking regions of RGS proteins.One way in which these RGS flanking regions can modulate protein activity is by determining subcellular localization. For several RGS proteins, regions near the N terminus are responsible for targeting the proteins to particular cellular locations. RGS-GAIP and RGSZ1...

Membrane trafficking is a fundamental cellular process that must operate with high fidelity to maintain organelle identity, cell function, and viability. Integral membrane proteins referred to as SNAREs 1 (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are important elements in this process, participating in the docking and fusion of vesicles with target membranes (1). The hallmark of SNARE proteins is the presence of one or more ␣-helices in the cytoplasmic domain, which have a propensity to form coiled-coils (reviewed in Refs. 2 and 3). Pairing between cognate SNAREs on opposing membranes results in the formation of a parallel four-helix bundle that brings the membranes together to allow fusion (4, 5). At the neuronal synapse, the SNARE complex is formed between SNAP-25 and syntaxin, localized on the presynaptic membrane, and synaptobrevin/VAMP, localized on the vesicle membrane (1). The central role that SNAP-25 plays in this complex is evident from the impairment of neuronal secretion by botulinum neurotoxin A/E, a toxin that proteolytically inactivates SNAP-25 (6). Moreover, loss of SNAP-25 expression in mice results in embryonic lethality late in gestation with evoked neurotransmitter release absent at the neuromuscular junction and central synapses (7).Each membrane compartment appears to be associated with a unique set of SNAREs, thereby contributing to the specificity of membrane fusion. The mechanisms by which SNAREs are targeted to their resident membranes are only beginning to be understood. Most syntaxin and synaptobrevin/VAMP family members are classified as tail-anchored proteins based on the presence of a transmembrane segment at the C terminus. Studies of synaptobrevin and other vesicle-associated SNAREs (vSNAREs) demonstrate that these proteins are initially targeted to the endoplasmic reticulum and then sorted to their ultimate destinations (8, 9).The SNAP-25 family of SNAREs, SNAP-25a and b, SNAP-23, and SNAP-29, is structurally distinct from the tail-anchored SNAREs (3) and thus uses different mechanisms for membrane localization. The tail-anchored SNARES have a single cytoplasmic coiled-coil domain or SNARE motif, whereas SNAP-25 family members have two SNARE motifs that are connected by an interhelical domain (5). SNAP-25 and SNAP-23 are palmitoylated at a cluster of cysteine residues in the interhelical domain (10 -12). Many soluble proteins rely on fatty acylation for membrane association (13,14), and there is evidence to support a similar functional role for SNAP-25 palmitoylation. Our study of the native protein in PC12 cells revealed that palmitoylation of newly synthesized SNAP-25 coincides temporally with its membrane association (15). Moreover, palmitoylation and membrane association of the newly synthesized protein are sensitive to Brefeldin A, suggesting that these events are coupled and depend on an intact secretory pathway (15). Palmitoylation-defective mutants of SNAP-25 and SNAP-23 are found predominately in the cytoplasm of transfected cells (11,16,1...