DISCUSSIONS and PERSPECTIVES

The main purpose of this work was to describe the novel gene, lnp-1. For studying this topic, we have chosen C. elegans, a well-known model organism intensely utilized for numerous studies involving synaptic neurotransmission. The control of vesicle-mediated transport in the nerve cells is an important aspect of synapse function, development and maintenance.

Numerous cellular mechanisms were involved in the regulation of the neurotransmission, such as the intracellular levels of calcium and cyclic nucleotides, the protein phosphorylation and dephosphorylation, and the localization and translation of synaptic proteins. Hence, the activity, location, and levels of key synaptic proteins are central determinants of synaptic efficacy.

Ubiquitination regulates the amount and subcellular localisation of target proteins, qualifying as a candidate mechanism to regulate synaptic transmission (DiAntonio and Hicke, 2004; Hegde, 2004). Recent work demonstrated that ubiquitination regulates neurotransmission by mediating receptor internalization and degradation via the endocytic pathway, thus controlling the abundance of the postsynaptic receptors (Colledge et al., 2003; Schwartz, 2003). Furthermore, some RING finger-containing proteins, such as ZNRFs (Araki and Milbrandt, 2003), SCRAPPER (Yao et al., 2007) and RPM-1 (Nakata et al., 2005), bind presynaptic terminal proteins necessary for exocytosis, such as syntaxin 1 (Chin et al., 2002), RIM1 (Yao et al., 2007) and DLK-1 (Nakata et al., 2005), and thus participate in the establishment and maintenance of neuronal transmission and plasticity via their ubiquitin ligase activity. Numerous studies indicate that ubiquitin-mediated protein metabolism may regulate a variety of processes, including long-term memory formation (Lopez-Salon et al., 2001), synapse formation (DiAntonio et al., 2001), and the establishment of neuropathic pain (Moss et al., 2002). Coordinated regulation of pre- and postsynaptic protein levels via UPS may function not only in synaptogenesis and long-term responses, but also in activity-dependent short-term responses (Dobie and Craig, 2007).

In this work, we determined lnp-1 expression pattern, its subcellular distribution, and also analysed the behavioural phenotypes of lnp-1 mutants. We showed that LNP-1 is involved in the distribution of synaptic vesicle cargo (Ghila and Gomez, 2008). Furthermore, we provided data demonstrating the interaction between LNP-1 and SIAH-1, the ortholog of the Drosophila SINA E3/ubiquitin ligase. These proteins are co-distributed in the nervous system, further supporting the hypothesis that LNP-1–SIAH-1 couple might be involved in a common molecular pathway, most probably the ubiquitination/proteasome pathway.

LNP-1 was initially described in mammals, where it belongs to the lnp-evx2-hox cluster.

In the central nervous system, LNP is expressed in the ventral neural tube, extending to the upper part of the rombencephalon, in the presumptive cerebellum, in the dorsal neurons and in the columns of the ventral interneurons, as well as in some regions of the midbrain and forebrain (Spitz et al., 2003). Recently, it has been reported that the cognitive defects (e.g.

severe cerebellar hypoplasia) observed in a patient carrying break points in the vicinity of this

cluster might be caused by an alteration of the lnp expression in the central nervous system (Dlugaszewska et al., 2006). However, with the exception of these two reports, no evidence for a neuronal function of LNP has been described before this work.

In C. elegans, the expression pattern of the plnp-1::GFP transgenic animals as well as the immunofluorescence staining showed that LNP-1 is enriched in the nervous system. Similar to the distribution of pre-synaptic proteins in unc-104/KIF1A kinesin mutant, the subcellular localization of LNP-1 is affected in these animals; however, LNP-1 seems not to be a component of synaptic structures. The overall expression pattern of LNP-1 does not resemble that of synaptic proteins, which are enriched in synapse-rich regions, such as the nerve ring, the ventral and dorsal nerve cords, but missing from structures without synapses (commissures or cell bodies). Conversely, LNP-1 is broadly localized in neuronal cells, including cell bodies and commissures. This pattern is common for the molecules involved in vesicular trafficking, such as the motor proteins (e.g. UNC-104/KIF1A kinesin (Zhou et al., 2001) or UNC-116/Kinesin heavy chain (Sakamoto et al., 2005), or regulator proteins like UNC-16, a c-Jun N-terminal kinase (JNK) signalling scaffold protein (Byrd et al., 2001). In lnp-1 mutant backgrounds, the mislocalization of RAB-3::Venus and SNB-1::GFP cargo proteins, which are no longer enriched in the synaptic structures, strongly support the assumption that LNP-1 may function as regulator in UNC-104/kinesin-mediated distribution of synaptic vesicle components. RAB-3, a small GTPase that associates with synaptic vesicles, has an important role in the docking process (Nonet et al., 1997). SNB-1 is essential for the regulation of the fusion/docking events at the synapse (Nonet et al., 1998). Both proteins function to regulate synaptic transmission and their mislocalization results in neurotransmission defects (Iwasaki et al., 1997). Therefore, the behavioural deficiencies observed in lnp-1 mutants, such as the increased resistance to the aldicarb drug, slight locomotory and egg-laying defects, are consistent with the abnormal localization of these synaptic proteins. Surprisingly, lnp-1 mutants exhibit only mild behavioural alterations, suggesting that these mutants retain sufficient RAB-3 and SNB-1 associated with pre-synaptic structures, which allow the neutrotransmitter release. This may reflect either redundancy of LNP-1 function by alternative pathway(s) or that these lnp-1 mutants are hypomorphic lnp-1 alleles; however we do not favour this last hypothesis, as most of the gene is deleted in these alleles.

In order to better understand the molecular mechanisms of LNP-1 function, we searched for interaction partners. Using a yeast two-hybrid system, LNP-1 was found to interact with SIAH-1. These two proteins are conserved during evolution from invertebrates to human.

The vertebrate lnp and siah are expressed at low levels during embryogenesis in many tissues and share common domains of high expression in the eyes, heart, genitalia, and nervous system (Della et al., 1993; Spitz et al., 2003). The C. elegans lnp-1 is part of one group of coregulated genes highly enriched for neuronal and muscle genes. siah-1 is also part of this

specific group (Kim et al., 2001), suggesting that these proteins may play a role in a common biological pathway.

We provided evidence for physical interaction between LNP-1 and SIAH-1 in C.

elegans, using immunoprecipitation assays. Furthermore, worms expressing both lnp-1 and siah-1 transgenes display high level of colocalization within the nervous system, including sensory neurons and inter neurons of the head and the tail and motor neurons of the ventral neuronal cord. Endogenous colocalization of the two proteins is also confirmed by immunofluorescence staining. Importantly, siah-1(RNAi) and siah-1(tm1968) worms exhibited similar behavioural phenotypes similar to those of lnp-1(RNAi) and lnp-1 mutants suggesting that LNP-1 and SIAH-1 are acting together in vivo. The fact that the lnp-1(tm733);siah-1(tm1968) double mutant, as well as siah-1(tm1968) treated with lnp-1(dsRNA), are able to rescue the defects seen in single mutants, but lnp-1 mutants worms treated with siah-1(dsRNA) are displaying a phenotype similar to single mutants suggest that the truncated form of SIAH-1 (N-terminal part) might bypass LNP-1 requirement and suffice for SIAH-1 function. All these data indicated that the interaction between complete and operational LNP-1 and SIAH-1 proteins is compulsory to fulfil their tasks in wild-type worms. Although at the moment the physiological significance of this interaction is not well understood, previous studies in Drosophila and vertebrates open attractive possibilities. Drosophila and vertebrate sina/siah have been shown to regulate neuronal development and function by mediating the ubiquitin-dependent degradation of a number of neuronal target proteins. Several studies identified a relative large number of SIAH substrates, which are listed in table 1.

Biological Function Ligase Cellular Target(s) Associated

Proteins Reference Cell cycle (mitosis) SIAH-1 Kinesin like DNA binding

protein (Kid) Table 1. E3 ubiquitin-protein ligase SIAH family has more than one target protein. Similarly, substrates may be degraded in vivo by more than one ligase or pathway (PO4 – phosphorylated substrate).

SIAH promotes the ubiquitination and the degradation of synphilin-1 and synaptophysin, two proteins associated with synaptic vesicles that are implicated in neurotransmitter release (Nagano et al., 2003; Wheeler et al., 2002). These findings suggest that SIAH could be involved in the regulation of neurotransmitter release by promoting ubiquitination of pre-synaptic vesicle proteins. Recently, new mechanisms involving protein degradation through the ubiquitin-proteasome system have been shown to play an important role in synaptic regulation, including synapse development and size, vesicle cycling, neurotransmitter release, receptor trafficking and synaptic plasticity (DiAntonio and Hicke, 2004). The ubiquitin proteasome complex consists of three major complexes: E1, an ubiquitin activating enzyme, E2, an ubiquitin conjugating enzyme, and E3, an ubiquitin ligase. Monoubiquitination and multiple monoubiquitinations will usually lead to changes in protein function or to its targeting to the endocytic pathway. Addition of more than four ubiquitin molecules at a single site of the protein target (polyubiquitination) leads to its degradation by the proteasome complex (Amerik and Hochstrasser, 2004; Smalle and Vierstra, 2004). Ubiquitination not only regulates protein degradation but also plays an important role in membrane trafficking (d'Azzo et al., 2005).

In this work, we showed that the E3/ubiquitin ligase activity of SIAH-1 is conserved in C.

elegans. Operational LNP-1 and SIAH-1 proteins are required for normal distribution of pre-synaptic vesicle proteins like SNB-1 and RAB-3 and seem to be implicated in the regulation of postsynaptic proteins. Moreover, LNP-1–SIAH-1 couple is involved in the regulation of synaptic pools of GLR-1, most probably through ubiquitination. Two independent research groups reported that SIAH-1A mediates ubiquitination and degradation of group 1 metabotropic glutamate receptors in vertebrates (Kammermeier and Ikeda, 2001; Moriyoshi et al., 2004).

Ishikawa et al. showed not only that Siah-1A directly interacts with group 1 mGluRs, but they also suggested a novel modulatory mechanism mediated by a competitive interaction between Ca2+/CaM and Siah-1A with the cytoplasmic tail of these glutamate receptors (Ishikawa et al., 1999). In C. elegans, synaptic pools of glutamate receptors GLR-1 are regulated through complex mechanisms involving numerous proteins, including CaMKII, ubiquitin, APC and LIN-23 (subunit of Skp1/Cullin/F-box) E3-ubiquitin ligases (Burbea et al., 2002; Dreier et al., 2005;

Juo and Kaplan, 2004; Rongo and Kaplan, 1999). APC and LIN-23 E3-ligases are also involved in the degradation of !-catenin, which modulates the !-catenin/Wnt pathway to alter postsynaptic GLR-1 levels (Dreier et al., 2005). In vertebrates, SIAH-1 regulates the !-catenin levels via p53-inducible phosphorylation-independent pathway that involves a complex set of SIAH-1 interactions with SIP, Skp, Ebi and APC (Figure 1, reviewed by (Polakis, 2001). Our results suggest that this pathway might be conserved in C. elegans, SIAH-1 might regulate !-catenin and GLR-1 levels through a similar mechanism, which in addition involves LNP-1.

Clathrin-mediated (Grunwald et al., 2004) and/or cholesterol-dependent (Glodowski et al., 2007) endocytosis is also required for the regulation of GLR-1 glutamate receptors. Following internalization from the plasma membrane, GLR-1::GFP is sorted into either of these two

post-endocytic pathways (clathrin-mediated or cholesterol-dependent endocytosis). Degradation of GLR-1::GFP requires ubiquitin-mediated sorting into the multivesicular bodies (MVBs)/lysosome pathway, whereas UNC-108/Rab-2 mediates recycling trafficking of GLR-1::GFP from Syntaxin-13-positive endosomes (Chun et al., 2008). A large-scale mapping of human protein-protein interactions by mass spectrometry predicted that LNP-1 interacts with CHMP1A (Ewing et al., 2007), component of the ESCRT-III (endosomal sorting required for transport complex III), which is required for MVBs formation and sorting of endosomal cargo proteins into MVBs. If this interaction will be confirmed, then LNP-1 – SIAH-1 couple might represent the missing link between the ubiquitination of GLR-1s and their targeting to the MVBs.

In the last four years, tremendous work on the regulation of GLR-1 receptors in C.

elegans enabled the identification of an increasing number of proteins involved in this process, like UNC-11/AP180 (Grunwald et al., 2004), APC (Juo and Kaplan, 2004), XBR-1 (Shim et al., 2004), SOL-1 (Walker et al., 2006b; Zheng et al., 2006; Zheng et al., 2004), STG-1 (Walker et al., 2006a), LIN-12 (Chao et al., 2005), LIN-23 (Dreier et al., 2005), KEL-8 (Schaefer and Rongo, 2006), RAB-10 (Glodowski et al., 2007), CDK-5 (Juo et al., 2007), UNC-108/Rab2 (Chun et al., 2008). To this collection of proteins, we add two more proteins: LNP-1 and SIAH-1.

Figure 1. Protein-Protein Interactions in the Ebi (upper) and !-TrCP (lower) Complexes“N” indicates N terminus, “F” indicates F box, “PPP” represents phosphorylation of !-catenin, “E2” indicates a ubiquitin conjugating enzyme, and “MCR” is the mutation cluster region (from Polakis, 2001)

Elucidation of the molecular mechanisms employed by LNP/ubiquitin proteasome system to regulate synapse function might represent an essential aspect of understanding the pathological processes that underlie some central nervous system disorders in humans. Several studies had shown that disruption of motor-proteins/microtubule-based transport within the axon could be involved in neurodegenerative disorders, including Alzheimer disease (AD), Huntington disease (HD), amyotrophic lateral sclerosis (ALS), Parkinson disease (PD), and Charcot-Marie-Tooth disease type 2A, (reviewed in (Duncan and Goldstein, 2006; Gunawardena and Goldstein, 2005). Moreover, the phenotypes associated with these diseases become more severe with the age, leading to death. lnp-1 mutants have reduced life span and show signs of early neurodegeneration, suggesting once more an involvement of LNP-1 in synaptic trafficking.

Parkin, a gene mutated in Parkinson’s disease, encodes an ubiquitin E3 ligase, which was involved in the stabilization of the microtubule network through direct and strong binding mediated by three independent domains (Yang et al., 2005). It has been reported that parkin is associated with fast axonal transport of pre-synaptic proteins (Mizuno et al., 2001). SIAH interacts with both "-tubulin and the kinesin-like DNA binding protein (KiD) and it has been hypothesized that SIAH may regulate chromosome movement through the microtubule network by regulating the KiD levels (Germani et al., 2000). Our immunofluorescence data in NGF-treated PC12 cells revealed that both LNP and SIAH, the mammalian orthologs of LNP-1 and SIAH-1 respectively, were colocalized with kinesin. Their distribution was depending of an intact microtubule network in nocodazole-treated fibroblast cells. Furthermore, the colocalization between LNP, SIAH and synaptic vesicle proteins, like synaptophysin, in neuronal processes suggested that the interaction between LNP/LNP-1 and SIAH/SIAH-1 might be conserved during evolution. In the light of these data is tempting to propose that LNP/LNP-1 and SIAH/SIAH-1 proteins may regulate microtubule-kinesin-based vesicle transport through the ubiquitination pathway, and consequently regulate synaptic function. However, several questions remain open; for example, does LNP-1/SIAH-1 function as an E3/ubiquitin ligase in vivo? How do these two proteins regulate synaptic function in vivo? Is SNB-1 ubiquitinated in vivo? Are pre-synaptic proteins, like SNB-1 and RAB-3, regulated through a mechanism similar to that of postsynaptic proteins, like GLR-1?

The regulation of postsynaptic proteins via ubiquitination has been described in many studies (Ehlers, 2003), but little is known about the regulation of presynaptic proteins by ubiquitination (Dobie and Craig, 2007; Yao et al., 2007). Our work might represent the foundation of a unified view of the mechanisms involved in synaptic proteins trafficking, regardless of their pre- or postsynaptic membranes localisation.

6. BIBLIOGRAPHY

Aboobaker, A., and Blaxter, M. (2003a). Hox gene evolution in nematodes: novelty conserved. Curr Opin Genet Dev 13, 593-598.

Aboobaker, A.A., and Blaxter, M.L. (2003b). Hox Gene Loss during Dynamic Evolution of the Nematode Cluster. Curr Biol 13, 37-40.

Ackley, B.D., Harrington, R.J., Hudson, M.L., Williams, L., Kenyon, C.J., Chisholm, A.D., and Jin, Y.

(2005). The two isoforms of the Caenorhabditis elegans leukocyte-common antigen related receptor tyrosine phosphatase PTP-3 function independently in axon guidance and synapse formation. J Neurosci 25, 7517-7528.

Ahringer, J. (1996). Posterior patterning by the Caenorhabditis elegans even-skipped homolog vab-7.

Genes Dev 10, 1120-1130.

Ahringer, J. (2006). Reverse genetics. WormBook.

Akert, K. (1972). [The ultrastructure of synapses]. Arkh Anat Gistol Embriol 62, 5-8.

Albertson, D.G., and Thomson, J.N. (1976). The pharynx of Caenorhabditis elegans. Philos Trans R Soc Lond B Biol Sci 275, 299-325.

Alfonso, A., Grundahl, K., Duerr, J.S., Han, H.P., and Rand, J.B. (1993). The Caenorhabditis elegans unc-17 gene: a putative vesicular acetylcholine transporter. Science 261, 617-619.

Alfonso, A., Grundahl, K., McManus, J.R., and Rand, J.B. (1994). Cloning and characterization of the choline acetyltransferase structural gene (cha-1) from C. elegans. J Neurosci 14, 2290-2300.

Alkema, M.J., Hunter-Ensor, M., Ringstad, N., and Horvitz, H.R. (2005). Tyramine Functions independently of octopamine in the Caenorhabditis elegans nervous system. Neuron 46, 247-260.

Amerik, A.Y., and Hochstrasser, M. (2004). Mechanism and function of deubiquitinating enzymes.

Biochim Biophys Acta 1695, 189-207.

Andrews, N.W., and Chakrabarti, S. (2005). There's more to life than neurotransmission: the regulation of exocytosis by synaptotagmin VII. Trends Cell Biol 15, 626-631.

Avery, L., and Horvitz, H.R. (1989). Pharyngeal pumping continues after laser killing of the pharyngeal nervous system of C. elegans. Neuron 3, 473-485.

Ballif, B.A., Villen, J., Beausoleil, S.A., Schwartz, D., and Gygi, S.P. (2004). Phosphoproteomic analysis of the developing mouse brain. Mol Cell Proteomics 3, 1093-1101.

Bamber, B.A., Beg, A.A., Twyman, R.E., and Jorgensen, E.M. (1999). The Caenorhabditis elegans unc-49 locus encodes multiple subunits of a heteromultimeric GABA receptor. J Neurosci 19, 5348-5359.

Bamber, B.A., Richmond, J.E., Otto, J.F., and Jorgensen, E.M. (2005). The composition of the GABA receptor at the Caenorhabditis elegans neuromuscular junction. Br J Pharmacol 144, 502-509.

Bargmann, C.I., and Avery, L. (1995). Laser killing of cells in Caenorhabditis elegans. Methods Cell Biol 48, 225-250.

Bargmann, C.I., Hartwieg, E., and Horvitz, H.R. (1993). Odorant-selective genes and neurons mediate olfaction in C. elegans. Cell 74, 515-527.

Bargmann, C.I., and Horvitz, H.R. (1991). Chemosensory neurons with overlapping functions direct chemotaxis to multiple chemicals in C. elegans. Neuron 7, 729-742.

Bargmann, C.I., and Kaplan, J.M. (1998). Signal transduction in the Caenorhabditis elegans nervous system. Annu Rev Neurosci 21, 279-308.

Bedford, F.K., Kittler, J.T., Muller, E., Thomas, P., Uren, J.M., Merlo, D., Wisden, W., Triller, A., Smart, T.G., and Moss, S.J. (2001). GABA(A) receptor cell surface number and subunit stability are regulated by the ubiquitin-like protein Plic-1. Nat Neurosci 4, 908-916.

Beg, A.A., and Jorgensen, E.M. (2003). EXP-1 is an excitatory GABA-gated cation channel. Nat Neurosci 6, 1145-1152.

Berezikov, E., Bargmann, C.I., and Plasterk, R.H. (2004). Homologous gene targeting in Caenorhabditis elegans by biolistic transformation. Nucleic Acids Res 32, e40.

Bessereau, J.L. (2006). Insertional mutagenesis in C. elegans using the Drosophila transposon Mos1: a method for the rapid identification of mutated genes. Methods Mol Biol 351, 59-73.

Bessereau, J.L., Wright, A., Williams, D.C., Schuske, K., Davis, M.W., and Jorgensen, E.M. (2001).

Mobilization of a Drosophila transposon in the Caenorhabditis elegans germ line. Nature 413, 70-74.

Betz, A., Thakur, P., Junge, H.J., Ashery, U., Rhee, J.S., Scheuss, V., Rosenmund, C., Rettig, J., and Brose, N. (2001). Functional interaction of the active zone proteins Munc13-1 and RIM1 in synaptic vesicle priming. Neuron 30, 183-196.

Bies, J., Markus, J., and Wolff, L. (2002). Covalent attachment of the SUMO-1 protein to the negative regulatory domain of the c-Myb transcription factor modifies its stability and transactivation capacity. J Biol Chem 277, 8999-9009.

Bramham, C.R., and Wells, D.G. (2007). Dendritic mRNA: transport, translation and function. Nat Rev Neurosci 8, 776-789.

Bredt, D.S., and Nicoll, R.A. (2003). AMPA receptor trafficking at excitatory synapses. Neuron 40, 361-379.

Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77, 71-94.

Broadie, K.S., and Richmond, J.E. (2002). Establishing and sculpting the synapse in Drosophila and C.

elegans. Curr Opin Neurobiol 12, 491-498.

Brockie, P.J., Madsen, D.M., Zheng, Y., Mellem, J., and Maricq, A.V. (2001). Differential expression of glutamate receptor subunits in the nervous system of Caenorhabditis elegans and their regulation by the homeodomain protein UNC-42. J Neurosci 21, 1510-1522.

Brockie, P.J., and Maricq, A.V. (2003). Ionotropic glutamate receptors in Caenorhabditis elegans.

Neurosignals 12, 108-125.

Brockie, P.J., and Maricq, A.V. (2006). Building a synapse: genetic analysis of glutamatergic neurotransmission. Biochem Soc Trans 34, 64-67.

Brown, D.A. (2006). Acetylcholine. Br J Pharmacol 147 Suppl 1, S120-126.

Burbea, M., Dreier, L., Dittman, J.S., Grunwald, M.E., and Kaplan, J.M. (2002). Ubiquitin and AP180 regulate the abundance of GLR-1 glutamate receptors at postsynaptic elements in C. elegans. Neuron 35, 107-120.

Buttner, C., Sadtler, S., Leyendecker, A., Laube, B., Griffon, N., Betz, H., and Schmalzing, G. (2001).

Ubiquitination precedes internalization and proteolytic cleavage of plasma membrane-bound glycine receptors. J Biol Chem 276, 42978-42985.

Byrd, D.T., Kawasaki, M., Walcoff, M., Hisamoto, N., Matsumoto, K., and Jin, Y. (2001). UNC-16, a JNK-signaling scaffold protein, regulates vesicle transport in C. elegans. Neuron 32, 787-800.

Cameron, P.L., Sudhof, T.C., Jahn, R., and De Camilli, P. (1991). Colocalization of synaptophysin with transferrin receptors: implications for synaptic vesicle biogenesis. J Cell Biol 115, 151-164.

Carnell, L., Illi, J., Hong, S.W., and McIntire, S.L. (2005). The G-protein-coupled serotonin receptor SER-1 regulates egg laying and male mating behaviors in Caenorhabditis elegans. J Neurosci 25, 10671-10681.

Chalfie, M., Sulston, J.E., White, J.G., Southgate, E., Thomson, J.N., and Brenner, S. (1985). The neural circuit for touch sensitivity in Caenorhabditis elegans. J Neurosci 5, 956-964.

Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W., and Prasher, D.C. (1994). Green fluorescent protein as a marker for gene expression. Science 263, 802-805.

Chao, M.Y., Larkins-Ford, J., Tucey, T.M., and Hart, A.C. (2005). lin-12 Notch functions in the adult nervous system of C. elegans. BMC Neurosci 6, 45.

Chapman, E.R. (2002). Synaptotagmin: a Ca(2+) sensor that triggers exocytosis? Nat Rev Mol Cell Biol 3, 498-508.

Chase, D.L., and Koelle, M.R. (2007). Biogenic amine neurotransmitters in C. elegans. WormBook, 1-15.

Chase, D.L., Pepper, J.S., and Koelle, M.R. (2004). Mechanism of extrasynaptic dopamine signaling in Caenorhabditis elegans. Nat Neurosci 7, 1096-1103.

Chen, Y.A., and Scheller, R.H. (2001). SNARE-mediated membrane fusion. Nat Rev Mol Cell Biol 2, 98-106.

Cheng, T.S., Chang, L.K., Howng, S.L., Lu, P.J., Lee, C.I., and Hong, Y.R. (2006). SUMO-1 modification of centrosomal protein hNinein promotes hNinein nuclear localization. Life Sci 78, 1114-1120.

Chevaillier, P. (1993). Pest sequences in nuclear proteins. Int J Biochem 25, 479-482.

Chilcote, T.J., Galli, T., Mundigl, O., Edelmann, L., McPherson, P.S., Takei, K., and De Camilli, P.

(1995). Cellubrevin and synaptobrevins: similar subcellular localization and biochemical properties in PC12 cells. J Cell Biol 129, 219-231.

Chin, L.S., Vavalle, J.P., and Li, L. (2002). Staring, a novel E3 ubiquitin-protein ligase that targets syntaxin 1 for degradation. J Biol Chem 277, 35071-35079.

Chun, D.K., McEwen, J.M., Burbea, M., and Kaplan, J.M. (2008). UNC-108/Rab2 Regulates Post-endocytic Trafficking in C. elegans. Mol Biol Cell.

Ciechanover, A., Heller, H., Elias, S., Haas, A.L., and Hershko, A. (1980). ATP-dependent conjugation of

Ciechanover, A., Heller, H., Elias, S., Haas, A.L., and Hershko, A. (1980). ATP-dependent conjugation of