An Elaborate Classification of SNARE Proteins Sheds Light on the Conservation of the Eukaryotic Endomembrane System

The Caenorhabditis elegans defecation motor program (DMP) is a highly coordinated rhythmic behavior that requires two GABAergic neurons that synapse onto the enteric muscles. One class of DMP mutants, called anterior body wall muscle contraction and expulsion defective (aex) mutants, exhibits similar defects to those caused by the loss of these two neurons. Here, we demonstrate that aex-2 encodes a G-protein-coupled receptor (GPCR) and aex-4 encodes an exocytic SNAP25 homologue. We found that aex-2 functions in the nervous system and activates a Gs␣ signaling pathway to regulate defecation. aex-4, on the other hand, functions in the intestinal epithelial cells. Furthermore, we show that aex-5, which encodes a pro-protein convertase, functions in the intestine to regulate the DMP and that its secretion from the intestine is impaired in aex-4 mutants. Activation of the Gs␣ GPCR pathway in GABAergic neurons can suppress the defecation defect of the intestinal mutants aex-4 and aex-5. Lastly, we demonstrate that activation of GABAergic neurons using the light-gated cation channel channelrhodopsin-2 is sufficient to suppress the behavioral defects of aex-2, aex-4, and aex-5. These results genetically place intestinal genes aex-4 and aex-5 upstream of GABAergic GPCR signaling. We propose a model whereby the intestinal genes aex-4 and aex-5 control the DMP by regulating the secretion of a signal, which activates the neuronal receptor aex-2. T he Caenorhabditis elegans defecation motor program (DMP)is a highly coordinated series of three muscle contractions that are executed every 45 sec [ Fig. 1A and supporting information (SI) Movie S1]. The cycle is initiated by a posterior body wall muscle contraction (pBoc), followed 2-3 sec later by an anterior body wall muscle contraction (aBoc). About 1 sec after the aBoc, enteric muscles contract, thus causing the expulsion (Exp) of intestinal contents. The process repeats itself Ϸ45 sec later with little variability in the timing of contractions (1). A genetic screen for mutants that displayed defects in the DMP isolated mutants defective in each of the three muscle contractions, known as pbo, abo, and exp (1). The screen also recovered mutants defective in the last two muscle contractions (aBoc and Exp [aex]) and mutants defective in the cycle periodicity (i.e., longer or shorter than normal DMP cycling times) (1). Molecular studies of these mutants have suggested that the behavior is orchestrated through the communication between the intestine, GABAergic neurons, and muscle.The periodicity of the DMP is regulated by the C. elegans intestine, a single-cell layer tube of polarized epithelial cells joined by gap junctions (2, 3). Intestinal Ca 2ϩ oscillations with Ϸ45-sec periodicity appear to play a central role in this timing. They consist of a posterior-to-anterior Ca 2ϩ wave whose levels peak in the posterior and anterior intestinal cells just before the pBoc and aBoc contractions, respectively (3-5). Mutations in genes involved in the maintenance of Ca 2ϩ oscillations or i...

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“…T14G12.2 encodes a homologue of the SNARE protein SNAP25 (Fig. S3) (15,16). These findings implicate aex-4 in exocytosis.…”

Section: Results Aex Mutants Are Primarily Defective In Exp and Onlymentioning

confidence: 86%

Intestinal signaling to GABAergic neurons regulates a rhythmic behavior in Caenorhabditis elegans

Mahoney

Luo

Round

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

110

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“…The SNAP-25 linker must be seen as an integral part of this machine and it is tempting to speculate that the fusion of the Qb and Qc-SNARE motifs might have evolved as an adaptation toward calcium triggering of exocytosis. (Kloepper et al, 2007). The 10 amino acids important for exocytosis triggering are marked by a shaded box.…”

Section: Discussionmentioning

confidence: 99%

The SNAP-25 Linker as an Adaptation Toward Fast Exocytosis

Nagy

Milošević

Mohrmann

et al. 2008

MBoC

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The assembly of four soluble N-ethylmaleimide-sensitive factor attachment protein receptor domains into a complex is essential for membrane fusion. In most cases, the four SNARE-domains are encoded by separate membrane-targeted proteins. However, in the exocytotic pathway, two SNARE-domains are present in one protein, connected by a flexible linker. The significance of this arrangement is unknown. We characterized the role of the linker in SNAP-25, a neuronal SNARE, by using overexpression techniques in synaptosomal-associated protein of 25 kDa (SNAP-25) null mouse chromaffin cells and fast electrophysiological techniques. We confirm that the palmitoylated linker-cysteines are important for membrane association. A SNAP-25 mutant without cysteines supported exocytosis, but the fusion rate was slowed down and the fusion pore duration prolonged. Using chimeric proteins between SNAP-25 and its ubiquitous homologue SNAP-23, we show that the cysteine-containing part of the linkers is interchangeable. However, a stretch of 10 hydrophobic and charged amino acids in the C-terminal half of the SNAP-25 linker is required for fast exocytosis and in its absence the calcium dependence of exocytosis is shifted toward higher concentrations. The SNAP-25 linker therefore might have evolved as an adaptation toward calcium triggering and a high rate of execution of the fusion process, those features that distinguish exocytosis from other membrane fusion pathways. INTRODUCTIONThe soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are central to vesicular trafficking (Fasshauer, 2003;Jahn and Scheller, 2006;Rizo et al., 2006) and characterized by a stretch of 60 -70 amino acids, known as the SNARE motif (Terrian and White, 1997;Weimbs et al., 1997). The assembly of four SNARE domains into a coiled-coil bundle is a general mechanism in fusion of intracellular membrane compartments. The bundle is held together by layers of hydrophobic interaction, with the exception of the middle layer (layer "0"), which is formed by an arginine and three glutamines (Sutton et al., 1998). The SNARE domains can be subdivided into four homologous groups, named after their contribution to the zero layer: R, Qa, Qb, and Qc (Fasshauer et al., 1998b). SNARE assembly requires the participation of one domain from each group, resulting in a certain degree of specificity Scales et al., 2000a).In most cases, the four SNARE-domains are encoded by separate membrane-targeted proteins, but the SNAREs driving the fusion of vesicles with the plasma membrane (exocytosis) are special in that three proteins provide the four domains (Weimbs et al., 1998;Fukuda et al., 2000). One of the SNAREs in this pathway, exemplified by the best-known isoform synaptosomal-associated protein of 25 kDa (SNAP-25), seems to have been created by fusion of the Qb and Qc SNAREs. This arrangement necessitates a flexible linker, which runs back along the complex from the C-terminal end of the first (Qb) SNARE-domain and connects to the Nterminal end of the second SN...

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“…Further refinement of the Q-SNARE proteins based on sequence conservation identifies each as a member of the Qa, Qb, or Qc subfamily (Kloepper et al 2007). SNARE-dependent membrane fusion is though to proceed through a conserved mechanism in which three Q-SNARES (Qa, Qb, and Qc) and one R-SNARE zipper together from the N-terminal side of the SNARE motif toward the membrane (Sudhof and Rothman 2009).…”

Section: Snare Protein-dependent Membrane Fusionmentioning

confidence: 99%

Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway

2013

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The secretory pathway is responsible for the synthesis, folding, and delivery of a diverse array of cellular proteins. Secretory protein synthesis begins in the endoplasmic reticulum (ER), which is charged with the tasks of correctly integrating nascent proteins and ensuring correct post-translational modification and folding. Once ready for forward traffic, proteins are captured into ER-derived transport vesicles that form through the action of the COPII coat. COPII-coated vesicles are delivered to the early Golgi via distinct tethering and fusion machineries. Escaped ER residents and other cycling transport machinery components are returned to the ER via COPI-coated vesicles, which undergo similar tethering and fusion reactions. Ultimately, organelle structure, function, and cell homeostasis are maintained by modulating protein and lipid flux through the early secretory pathway. In the last decade, structural and mechanistic studies have added greatly to the strong foundation of yeast genetics on which this field was built. Here we discuss the key players that mediate secretory protein biogenesis and trafficking, highlighting recent advances that have deepened our understanding of the complexity of this conserved and essential process. TABLE OF CONTENTS Abstract 383Introduction 384

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An Elaborate Classification of SNARE Proteins Sheds Light on the Conservation of the Eukaryotic Endomembrane System

Cited by 232 publications

References 73 publications

Intestinal signaling to GABAergic neurons regulates a rhythmic behavior in Caenorhabditis elegans

Intestinal signaling to GABAergic neurons regulates a rhythmic behavior in Caenorhabditis elegans

The SNAP-25 Linker as an Adaptation Toward Fast Exocytosis

Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway

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