We describe the identification and initial characterization of neurobeachin, a neuron-specific multidomain protein of 327 kDa with a high-affinity binding site (K d , 10 nM) for the type II regulatory subunit of protein kinase A (PKA RII). Neurobeachin is peripherally associated with pleomorphic tubulovesicular endomembranes near the trans sides of Golgi stacks and throughout the cell body and cell processes. It is also found in a subpopulation of synapses, where it is concentrated at the postsynaptic plasma membrane. In live cells, perinuclear neurobeachin is dispersed by brefeldin A (BFA) within 1 min, and in permeabilized cells a recruitment of neurobeachin from cytosol to Golgi-near membranes is stimulated by GTP␥S and prevented by brefeldin A. Spots of neurobeachin recruitment are close to but distinct from recruitment sites of COP-I, AP-1, and AP-3 coat proteins involved in vesicle budding. These observations indicate that neurobeachin binding to membranes close to the trans-Golgi requires an ADP-ribosylation factor-like GTPase, possibly in association with a novel type of protein coat. A neurobeachin isoform that does not bind RII, beige-like protein (BGL), is expressed in many tissues. Neurobeachin, BGL, and ϳ10 other mammalian gene products share a characteristic C-terminal BEACH-WD40 sequence module, which is also present in gene products of invertebrates, plants, protozoans, and yeasts, thus defining a new protein family. The prototype member of this family of BEACH domain proteins, lysosomal trafficking regulator (LYST), is deficient in genetic defects of protein sorting in lysosome biogenesis (the beige mouse and Chediak-Higashi syndrome). Neurobeachin's subcellular localization, its coat proteinlike membrane recruitment, and its sequence similarity to LYST suggest an involvement in neuronal post-Golgi membrane traffic, one of its functions being to recruit protein kinase A to the membranes with which it associates. Key words: AKAP; ARF; BEACH domain; BGL; coat protein; Golgi complex; LYST; membrane traffic; neurobeachin; protein kinase A; scaffolding protein; synapse; TGNThe progression of membranes and proteins through the stages and compartments of the secretory and endocytic pathways is a highly organized and regulated process. The maintenance of the overall architecture of endomembranes and of the plasma membrane requires a balance of lipid flows into and out of the various compartments, and proteins destined for diverse organelles or plasma membrane domains must be appropriately sorted and targeted, whereas resident proteins of specific pathway stages must be retained or retrieved. These events require the interplay of lipids, membrane proteins, soluble cytosolic and lumenal proteins, and cytoskeletal and motor proteins. Their internal coordination and external regulation is known to involve protein phosphorylation and small and heterotrimeric G-proteins.In neurons, the mechanisms for the trafficking of membranes and membrane proteins must be particularly active and complex. Because of their m...

Multidomain scaffolding proteins organize the molecular machinery of neurotransmitter vesicle dynamics during synaptogenesis and synaptic activity. We find that domains of five active zone proteins converge on an interaction node that centers on the N-terminal region of Munc13-1 and includes the zinc-finger domain of Rim1, the C-terminal region of Bassoon, a segment of CAST1/ELKS2, and the third coiled-coil domain (CC3) of either Aczonin/Piccolo or Bassoon. This multidomain complex may constitute a center for the physical and functional integration of the protein machinery at the active zone. An additional connection between Aczonin and Bassoon is mediated by the second coiled-coil domain of Aczonin. Recombinant Aczonin-CC3, expressed in cultured neurons as a green fluorescent protein fusion protein, is targeted to synapses and suppresses vesicle turnover, suggesting involvements in synaptic assembly as well as activity. Our findings show that Aczonin, Bassoon, CAST1, Munc13, and Rim are closely and multiply interconnected, they indicate that Aczonin-CC3 can actively participate in neurotransmitter vesicle dynamics, and they highlight the N-terminal region of Munc13-1 as a hub of protein interactions by adding three new binding partners to its mechanistic potential in the control of synaptic vesicle priming.

The protein machinery of neurotransmitter exocytosis requires efficient orchestration in space and time, for speed and precision of neurotransmission and also for synaptic ontogeny and plasticity. However, its spatial organization in situ is virtually unknown. Aczonin/Piccolo is a putative organizer protein of mammalian active zones. We determined by immunogold electron microscopy (EM) (i) the spatial arrangement (i.e., topology) of 11 segments of the Aczonin polypeptide in situ, and correlated it to (ii) the positioning of Aczonin-interacting domains of Bassoon, CAST/ELKS, Munc13, and RIM and (iii) the ultrastructurally defined presynaptic macromolecular aggregates known as dense projections and synaptic ribbons. At conventional synapses, Aczonin assumes a compact molecular topology within a layer 35 to 80 nm parallel to the plasma membrane (PM), with a "trunk" sitting on the dense projection top and a C-terminal "arm" extending down toward the PM and sideward to the dense projection periphery. At ribbon synapses, Aczonin occupies the whole ribbon area. Bassoon colocalizes with Aczonin at conventional synapses but not at ribbon synapses. At both conventional and ribbon synapses, CAST, Munc13, and RIM are segregated from Aczonin, closer to the PM, and Aczonin is positioned such that it may control the access of neurotransmitter vesicles to the fusion site.scaffolding protein | protein structure N eurotransmitter release is confined to a specialized area of the presynaptic plasma membrane (PM) known as the active zone. Calcium-dependent exocytosis at the synapse is an elaborate, multistep process that is fast and precise, yet highly restrained and subject to modulation (i.e., presynaptic plasticity) (1). A solubilization-resistant lattice of proteins associated with the inner face of the presynaptic PM, the cytomatrix at the active zone (CAZ), is apparent on electron microscopy (EM) imaging and seems to integrate and organize many proteins of the presynaptic machinery (2).When mammalian conventional synapses are stained with heavy metal ions, EM reveals regularly spaced, cone-shaped structures of a size of approximately 50 nm attached to the presynaptic PM, which are known as dense projections (DPs) or, collectively, the "presynaptic particle web" or "presynaptic grid." It has been possible to isolate DP-like particles by subcellular fractionation, and even to reconstitute similar aggregates after previous solubilization, and many proteins of the presynaptic molecular machinery have been detected in them (3). These findings suggest that the presynaptic machinery may be organized into modular units of homogeneous size and shape, and possibly also of defined stoichiometry and spatial organization of its constituent proteins. An organized positioning of individual proteins and protein domains could allow these supramolecular assemblies to function as "molecular machines" and contribute to the high speed, precision, and fine-tuning that is characteristic for synaptic exocytosis. Morphological organization of active z...

Cholesterol in the diet can readily autoxidize and be absorbed and transported in plasma lipoproteins. Cholesterol oxides can also be endogenously produced in tissues via free-radical-induced reactions. Some cholesterol oxides, notably cholestane-3 beta, 5 alpha, 6 beta-triol and 25-hydroxycholesterol, have been shown to cause injury to vascular endothelial and smooth muscle cells, to alter LDL receptor function, to enhance cholesteryl ester accumulation, to inhibit prostacyclin production, and to induce experimental atherosclerosis alone or in combination with cholesterol. An epidemiological study examining relationships between atherosclerosis and plasma levels of cholesterol oxides as independent risk factors may provide additional insights regarding the roles of cholesterol oxides in atherogenesis.

Allelic differences in expression are important genetic factors contributing to quantitative trait variation in various organisms. However, the extent of genome-wide allele-specific expression by different modes of gene regulation has not been well characterized in plants. In this study we developed a new methodology for allele-specific expression analysis by applying Massively Parallel Signature Sequencing (MPSS), an open ended and sequencing based mRNA profiling technology. This methodology enabled a genome-wide evaluation of cis- and trans-effects on allelic expression in six meristem stages of the maize hybrid. Summarization of data from nearly 400 pairs of MPSS allelic signature tags showed that 60% of the genes in the hybrid meristems exhibited differential allelic expression. Because both alleles are subjected to the same trans-acting factors in the hybrid, the data suggest the abundance of cis-regulatory differences in the genome. Comparing the same allele expressed in the hybrid versus its inbred parents showed that 40% of the genes were differentially expressed, suggesting different trans-acting effects present in different genotypes. Such trans-acting effects may result in gene expression in the hybrid different from allelic additive expression. With this approach we quantified gene expression in the hybrid relative to its inbred parents at the allele-specific level. As compared to measuring total transcript levels, this study provides a new level of understanding of different modes of gene regulation in the hybrid and the molecular basis of heterosis.

Rim1 is a protein of the presynaptic active zone, the area of the plasma membrane specialized for neurotransmitter exocytosis, and interacts with Rab3, a small GTPase implicated in neurotransmitter vesicle dynamics. Here, we have studied the molecular determinants of Rim1 that are responsible for Rab3 binding, employing surface plasmon resonance and recombinant, bacterially expressed Rab3 and Rim1 proteins. A site that binds GTP-but not GDP-saturated Rab3 was localized to a short ␣-helical sequence near the Rim1 N terminus (amino acids 19 -55). Rab3 isoforms A, C, and D were bound with similar affinities (K d ‫؍‬ 1-2 M). Low affinity binding of Rab6A-GTP was also observed (K d ‫؍‬ 16 M), whereas Rab1B, -5, -7, -8, or -11A did not bind. Adjacent sequences up to amino acid 387, encompassing differentially spliced sequences, the zinc finger module, and the SGAWFF motif of Rim1, did not significantly contribute to the strength or the specificity of Rab3 binding, whereas a point mutation within the helix (R33G) abolished binding. This Rab3 binding site of Rim1 is reminiscent of the N-terminal ␣-helix that is part of the Rab3-binding region of rabphilin-3, and indeed we observed low affinity, specific binding of Rab3A (K d on the order of magnitude of 10 -100 M) to this region of rabphilin-3 alone (amino acids 40 -88), whereas additional sequences up to amino acid 178 are needed for high affinity Rab3A binding to rabphilin-3 (K d ‫؍‬ 10 -20 nM). In contrast, an N-terminal ␣-helix motif in aczonin, with sequence similarity to the Rab3-binding site of Rim1, did not bind Rab3A, -C, or -D or several other Rab proteins. These results were qualitatively confirmed in pull-down experiments with native, prenylated Rab3 from brain lysate in Triton X-100. Munc13 bound to the zinc finger domain of Rim1 but not to the rabphilin-3 or aczonin zinc fingers. Pull-down experiments from brain lysate in the presence of cholate as detergent detected binding to downstream Rim1 sequences, between amino acids 56 and 387, of syntaxin and of Rab3. The latter, however, was inhibited rather than stimulated by GTP.

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