Rabphilin Potentiates Soluble<i>N</i>-Ethylmaleimide Sensitive Factor Attachment Protein Receptor Function Independently of rab3

Staunton, Jane; Ganetzky, Barry; Nonet, Michael L.

doi:10.1523/jneurosci.21-23-09255.2001

“…To determine whether RBF-1 is an effector of RAB-27 in vivo, we looked at RBF-1 localization in aex-3 and aex-6 mutant animals. We found that GFP-tagged RBF-1 (RBF-1::GFP) is mislocalized from synapse-rich regions in aex-3(js815) and aex-6(sa24) mutant animals, but RBF-1::GFP is normally localized in rab-3(js49) animals ( Figure 5A; Staunton et al, 2001). We also noted that RBF-1::GFP fluorescence is weaker in aex-6 mutant animals than in wild type.…”

mentioning

confidence: 78%

“…Single mutants rab-3(js49) and rbf-1(js232) Staunton et al, 2001). Double mutants were made by standard crossing procedures jsIs682; jsEx740, jsEx740, jsIs423; jsIs423,.…”

Section: Strains Usedmentioning

confidence: 99%

“…The jsIs682 (GFP::RAB-3) transgene rescues the aldicarb resistance of rab-3(js49) and is likely a functional GFP fusion (our unpublished data). jsIs423 (RBF-1::GFP) was a spontaneous integration of jsEx86 (Staunton et al, 2001). jsEx623 (NM1030-rab-27 promoter driving GFP) and jsEx740 (NM1112-GFP::RAB-27) were also used (see Supplemental Figure S1 for rab-27 clones used).…”

Section: Strains Usedmentioning

confidence: 99%

“…In vertebrates, rabphilin was originally identified as a Rab3 effector (Shirataki et al, 1993;Li et al, 1994;Ostermeier and Brunger, 1999); however, this does not seem to be the case in C. elegans (Staunton et al, 2001). Similarly, studies in mice have shown that rabphilin is not required for Rab3 function (Schluter et al, 1999).…”

Section: Rab-27 Regulates Rbf-1mentioning

confidence: 99%

“…However, C. elegans rabphilin interacts with RAB-27 but not RAB-3 via glutathione S-transferase (GST) pull-down, in vitro coimmunoprecipitation, and yeast two-hybrid assays (Fukuda, 2003a;Fukuda et al, 2004). Previous work demonstrated that rabphilin acts independently of RAB-3 in vertebrates and invertebrates and that rabphilin potentiates soluble N-ethylmaleimidesensitive factor attachment protein receptor (SNARE) function in invertebrates (Schluter et al, 1999;Staunton et al, 2001). Recent work by Tsuboi et al demonstrates that rabphilin regulates dense core vesicle docking by binding to the SNARE protein SNAP25 (Tsuboi and Fukuda, 2005).…”

Section: Introductionmentioning

confidence: 99%

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Regulation of Synaptic Transmission by RAB-3 and RAB-27 inCaenorhabditis elegans

Mahoney

¹

,

Liu

²

,

Itoh

³

et al. 2006

MBoC

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Rab small GTPases are involved in the transport of vesicles between different membranous organelles. RAB-3 is an exocytic Rab that plays a modulatory role in synaptic transmission. Unexpectedly, mutations in the Caenorhabditis elegans RAB-3 exchange factor homologue, aex-3, cause a more severe synaptic transmission defect as well as a defecation defect not seen in rab-3 mutants. We hypothesized that AEX-3 may regulate a second Rab that regulates these processes with RAB-3. We found that AEX-3 regulates another exocytic Rab, RAB-27. Here, we show that C. elegans RAB-27 is localized to synapse-rich regions pan-neuronally and is also expressed in intestinal cells. We identify aex-6 alleles as containing mutations in rab-27. Interestingly, aex-6 mutants exhibit the same defecation defect as aex-3 mutants. aex-6; rab-3 double mutants have behavioral and pharmacological defects similar to aex-3 mutants. In addition, we demonstrate that RBF-1 (rabphilin) is an effector of RAB-27. Therefore, our work demonstrates that AEX-3 regulates both RAB-3 and RAB-27, that both RAB-3 and RAB-27 regulate synaptic transmission, and that RAB-27 potentially acts through its effector RBF-1 to promote soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) function. INTRODUCTIONNeurotransmitter release is accomplished by the fusion of neurotransmitter-filled synaptic vesicles at the presynaptic nerve terminal. This process occurs through a series of highly regulated steps that include synaptic vesicle transport, docking/tethering, priming, fusion, endocytosis, recycling, and neurotransmitter refilling (Sudhof, 2004). Several genes have been assigned roles in the various steps of the synaptic vesicle cycle. Rab3 (termed RAB-3 in Caenorhabditis elegans), a member of the Rab family of small GTPases, regulates synaptic transmission, possibly through the docking, priming, or fusion steps Schluter et al., 2004).Rabs act in a variety of cell types and regulate vesicular transport between organelles. Rabs cycle on and off membranes via a GTP-dependent mechanism (Zerial and McBride, 2001). Rab activity is regulated by two proteins, which act in an antagonistic manner. The guanine nucleotide exchange factor (GEF) exchanges GTP for GDP, and the GTPase activating protein activates the intrinsic GTPase activity of a Rab (Bernards, 2003). GDP bound Rabs are held off of the membrane by a GDP dissociation inhibitor (Wu et al., 1996). The GTP/membrane-bound form of Rabs typically binds to a variety of effectors that regulate particular steps of membrane transport and cell signaling (Zerial and McBride, 2001;Spang, 2004).Rab3 was once thought to play a central role in regulating release. However, recent work has shown that a quadruple knockout of all four isoforms of Rab3 in mice only results in a 30% reduction in evoked synaptic response (Schluter et al., 2004). This is consistent with work in C. elegans showing that mutations in the single rab-3 gene cause only mild defects in synaptic transmission . Surprisingly, more dramatic phe...

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C 2‐Domain Proteins Involved in Membrane Traffic

Rizo

¹

2004

Handbook of Metalloproteins

0

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C 2 ‐domains are the second‐most abundant Ca 2+ ‐binding modules in nature. Their most common activity is Ca 2+ ‐dependent phospholipid binding, but other types of interactions have been observed for these versatile protein modules. A variety of proteins involved in membrane traffic contain multiple C 2 ‐domains, which often appear as two tandem C 2 ‐domains as in the case of synaptotagmins and rabphilin. The structures of the C 2 ‐domains of these proteins consist of a conserved β‐sandwich, formed by two four‐stranded β‐sheets, with variable loops at the top and the bottom that sometimes contain α‐helices. Multiple Ca 2+ ‐ions bind in a tight cluster at the top loops, usually without inducing substantial conformational changes. Instead, electrostatic changes caused by Ca 2+ binding induce the Ca 2+ ‐dependent interactions of C 2 ‐domains, which can also be aided by hydrophobic interactions and partial coordination of Ca 2+ by the target molecules. Extensive analysis of the structure and Ca 2+ ‐binding properties of the two synaptotagmin 1 C 2 ‐domains has helped design genetic experiments that have strongly supported the notion that this protein acts as the major Ca 2+ sensor in neurotransmitter release. These studies have also suggested that Ca 2+ ‐dependent phospholipid binding underlies the function of synaptotagmin 1 in triggering release.

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C 2‐Domain Proteins Involved in Membrane Traffic

Rizo¹

2004

Encyclopedia of Inorganic and Bioinorganic Chemistry

0

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C 2 ‐domains are the second‐most abundant Ca 2+ ‐binding modules in nature. Their most common activity is Ca 2+ ‐dependent phospholipid binding, but other types of interactions have been observed for these versatile protein modules. A variety of proteins involved in membrane traffic contain multiple C 2 ‐domains, which often appear as two tandem C 2 ‐domains as in the case of synaptotagmins and rabphilin. The structures of the C 2 ‐domains of these proteins consist of a conserved β‐sandwich, formed by two four‐stranded β‐sheets, with variable loops at the top and the bottom that sometimes contain α‐helices. Multiple Ca 2+ ‐ions bind in a tight cluster at the top loops, usually without inducing substantial conformational changes. Instead, electrostatic changes caused by Ca 2+ binding induce the Ca 2+ ‐dependent interactions of C 2 ‐domains, which can also be aided by hydrophobic interactions and partial coordination of Ca 2+ by the target molecules. Extensive analysis of the structure and Ca 2+ ‐binding properties of the two synaptotagmin 1 C 2 ‐domains has helped design genetic experiments that have strongly supported the notion that this protein acts as the major Ca 2+ sensor in neurotransmitter release. These studies have also suggested that Ca 2+ ‐dependent phospholipid binding underlies the function of synaptotagmin 1 in triggering release.

show abstract

Rabphilin Potentiates SolubleN-Ethylmaleimide Sensitive Factor Attachment Protein Receptor Function Independently of rab3

Cited by 57 publications

References 56 publications

Regulation of Synaptic Transmission by RAB-3 and RAB-27 inCaenorhabditis elegans

Regulation of Synaptic Transmission by RAB-3 and RAB-27 inCaenorhabditis elegans

C 2‐Domain Proteins Involved in Membrane Traffic

C 2‐Domain Proteins Involved in Membrane Traffic

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