Neuronal nitric oxide synthase (nNOS) is concentrated at synaptic junctions in brain and motor endplates in skeletal muscle. Here, we show that the N-terminus of nNOS, which contains a PDZ protein motif, interacts with similar motifs in postsynaptic density-95 protein (PSD-95) and a related novel protein, PSD-93.nNOS and PSD-95 are coexpressed in numerous neuronal populations, and a PSD-95/nNOS complex occurs in cerebellum. PDZ domain interactions also mediate binding of nNOS to skeletal muscle syntrophin, a dystrophin-associated protein. nNOS isoforms lacking a PDZ domain, identified in nNOSdelta/delta mutant mice, do not associate with PSD-95 in brain or with skeletal muscle sarcolemma. Interaction of PDZ-containing domains therefore mediates synaptic association of nNOS and may play a more general role in formation of macromolecular signaling complexes.

Nitric oxide (NO) is synthesized in skeletal muscle by neuronal-type NO synthase (nNOS), which is localized to sarcolemma of fast-twitch fibers. Synthesis of NO in active muscle opposes contractile force. We show that nNOS partitions with skeletal muscle membranes owing to association of nNOS with dystrophin, the protein mutated in Duchenne muscular dystrophy (DMD). The dystrophin complex interacts with an N-terminal domain of nNOS that contains a GLGF motif. mdx mice and humans with DMD evince a selective loss of nNOS protein and catalytic activity from muscle membranes, demonstrating a novel role for dystrophin in localizing a signaling enzyme to the myocyte sarcolemma. Aberrant regulation of nNOS may contribute to preferential degeneration of fast-twitch muscle fibers in DMD.

Soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) proteins of the vesicle-associated membrane protein (VAMP) and syntaxin families play a central role in vesicular trafficking through the formation of complexes between proteins present on vesicle and target membranes. Formation of these complexes is proposed to mediate aspects of the specificity of vesicle trafficking and to promote fusion of the lipid bilayers. In order to further understand the molecular mechanisms that organize membrane compartments, we have characterized seven new mammalian proteins of the VAMP and syntaxin families. The proteins are broadly expressed; however, syntaxin 13 is enriched in brain and VAMP 8 in kidney. The seven novel SNAREs localize in distinct patterns overlapping with Golgi, endosomal, or lysosomal markers. Our studies support the hypothesis that evolutionary radiation of these two gene families gave rise to sets of proteins whose differential expression and combinatorial associations define and organize the membrane compartments of cells.The distribution and restriction of molecules to membrane compartments is an essential process of eukaryotic cells. Distinct organelles of the secretory pathway are synthesized and maintained by budding of transport vesicles from a donor compartment followed by fusion of these vesicles with an acceptor membrane (1). The molecular mechanisms responsible for vesicle biogenesis, protein sorting, and membrane fusion are not yet fully understood. While yeast genetics, in vitro biochemistry, and studies of synaptic vesicles have identified many of the components essential for these processes (2-4), the full repertoire of important proteins and their mechanisms of action are yet to be determined. One particularly interesting issue is how a vesicle loaded with specific cargo recognizes the appropriate target. It is becoming clear that several independent mechanisms contribute to the specificity of vesicle trafficking, and it is the sum of these multiple layers of specificity that results in a process with high fidelity (5).A vesicle-target membrane recognition event mediated by interaction of integral membrane proteins of the vesicle (vSNAREs) 1 and target (t-SNAREs) membranes represents one layer of targeting specificity, acting at the final step of membrane fusion (6, 7). This process has been extensively studied in the mammalian presynaptic nerve terminal, where formation of a heterotrimeric complex between the v-SNARE, VAMPs 1 or 2, and the t-SNAREs syntaxin 1 and SNAP-25 is thought to serve as a membrane recognition mechanism and may drive fusion of the lipid bilayers (8, 9). These proteins have subsequently been found to be prototypic members of gene families that span species as well as membrane compartments (6). For example, syntaxin homologs in yeast have been localized to the Golgi (Sed5p) (10), endosomes (Pep12p) (11), lysosomes (Vam3p) (12), and the plasma membrane (Sso1p and Sso2p) (13). In particular, the SNAREs present on yeast vacuoles have been extensivel...

The proposed cis-Golgi vesicle receptor syntaxin 5 was found in a complex with Golgi-associated SNARE of 28 kDa (GOS-28), rbet1, rsly1, and two novel proteins characterized herein: rat sec22b and membrin, both cytoplasmically oriented integral membrane proteins. The complex appears to recapitulate vesicle docking interactions of proteins originating from distinct compartments, since syntaxin 5, rbet1, and GOS-28 localize to Golgi membranes, whereas mouse sec22b and membrin accumulate in the endoplasmic reticulum. Protein interactions in the complex are dramatically rearranged by N-ethylmaleimide-sensitive factor. The complex consists of two or more subcomplexes with some members (rat sec22b and syntaxin 5) in common and others (rbet1 and GOS-28) mutually exclusively associated. We propose that these protein interactions determine vesicle docking/fusion fidelity between the endoplasmic reticulum and Golgi.