A Conserved Mechanism of Synaptogyrin Localization

Abstract

We have studied the localization of synaptogyrin family members in vivo. Both native and green fluorescent protein (GFP)-tagged Caenorhabditis elegans synaptogyrin (SNG-1) are expressed in neurons and synaptically localized. Deletion and mutational analysis with the use of GFP-tagged SNG-1 has defined a 38 amino acid sequence within the C terminus of SNG-1 and a single arginine in the cytoplasmic loop between transmembrane domain 2 and 3 that are required for SNG-1 localization. These domains may represent components of signals that target synaptogyrin for endocytosis from the plasma membrane and direct synaptogyrin to synaptic vesicles, respectively. In chimeric studies, these regions were sufficient to relocalize cellugyrin, a nonneuronal form of synaptogyrin, from nonsynaptic regions such as the sensory dendrites and the cell body to synaptic vesicles. Furthermore, GFP-tagged rat synaptogyrin is synaptically localized in neurons of C. elegans and in cultured hippocampal neurons. Similarly, the C-terminal domain of rat synaptogyrin is necessary for localization in hippocampal neurons. Our study suggests that the mechanisms for synaptogyrin localization are likely to be conserved from C. elegans to vertebrates. INTRODUCTIONSynaptic vesicles (SVs) contain a restricted set of membrane proteins important for neuronal function (Sü dhof, 1995;Calakos and Scheller, 1996;Fernandez-Chacon and Sü dhof, 1999). The mechanisms responsible for targeting these proteins specifically to the SV membrane are poorly understood. Integral membrane proteins are synthesized on the rough endoplasmic reticulum and traffic through the Golgi network. Proteins exiting the trans-Golgi network (TGN) are sorted to different types of transport vesicles. SV proteins are sorted to synaptic vesicle precursors (preSVs), which differ from mature SVs both in morphology and protein composition (Tsukita and Ishikawa, 1980; Okada et al., 1995). Evidence supports both direct routing of SV proteins from TGN to preSVs and indirect routing via the plasma membrane (reviewed by Hannah et al., 1999). preSVs are subsequently transported along axonal processes to the nerve terminal by motor proteins of the kinesin superfamily (reviewed by Hirokawa, 1998). How preSVs mature after delivery to the nerve terminal is unknown. preSVs might fuse with an endosomal compartment and SVs generated by budding from the endosome. A second, but not mutually exclusive possibility, is that preSVs are delivered to the presynaptic plasma membrane at the nerve terminal and then retrieved by the same mechanism(s) used to recycle SVs after regulated exocytosis (Hannah et al., 1999; reviewed by Buckley et al., 2000). Regardless of the exact route of trafficking to the mature organelle, SV proteins must be sorted away from other membrane proteins at several stages during their life cycle.Signal sequences resident in proteins are thought to mediate the sorting of many proteins to cellular compartments. Several studies have identified domains and motifs necessary for correct localizati...

SYNGR1 is associated with schizophrenia and bipolar disorder in southern India

“…This mutation lies in the cytoplasmic loop between transmembrane domains 2 and 3 of the SYNGR1 protein. Experimental evidence from Caenorhabditis elegans synaptogyrin 1, and comparative sequence analysis of rat-synaptogyrin (rat-syngr) vs ratcellugyrin (rat-cgyr) protein, suggests the importance of this cytoplasmic domain in the synaptic localization of this protein (Zhao and Nonet 2001). Hence, the Lys99-Glu mutation might affect the selective localization of SYNGR1 protein to synaptic vesicles.…”

“…We have also identified two common exonic variations including an ins/del polymorphism located in exon 6. According to experimental analysis on C. elegans synaptogyrin 1, this ins/del polymorphism lies within the signaling sequence for synaptic localization of synaptogyrin 1 protein (Zhao and Nonet 2001), thereby suggesting a functional consequence of this polymorphism. This ins/del polymorphism also showed association with BPAD in our case-control analysis.…”

Mutations inCaenorhabditis elegansCytoplasmic Dynein Components Reveal Specificity of Neuronal Retrograde Cargo

“…The following antibodies were used: mouse anti-RAB-3 (Nonet et al, 1997), rabbit anti-synaptotagmin (SNT-1; Nonet et al, 1993), mouse anti-SNT-1, rabbit anti-synaptobrevin (SNB-1; Nonet et al, 1998), chicken anti-synaptogyrin (SNG-1; Zhao and Nonet, 2001), rabbit anti-MEC-7 (Savage et al, 1994), mouse anti-acetylated tubulin (Sigma, St. Louis, MO;Fukushige et al, 1999), mouse anti-UNC-104 (S. P. Koushika and M. L. Nonet, unpublished data), anti-UNC-70 (Moorthy et al, 2000), rabbit anti-DYN-1 (Labrousse et al, 1998), rabbit anti-Rim (Koushika et al, 2001), mouse anti-green fluorescent protein (GFP; Clonetech, Cambridge, UK; or Roche Molecular Biochemicals, Indianapolis, IN), rabbit anti-GFP (a gift from M. Linder, Washington University), rabbit anti-UNC-64 , and rabbit anti-DHC-1 (Gonczy et al, 1999). Appropriate secondary Alexa 488 and Alexa 568 antibodies were used (Molecular Probes, Eugene, OR).…”

Cellugyrin Induces Biogenesis of Synaptic-like Microvesicles in PC12 Cells

“…One report on the C. elegans synaptogyrin homolog suggested that an arginine in the cytoplasmic loop was critical for targeting to nerve endings (24). An arginine is present in cellugyrin's cytoplasmic loop as well, but targeting of the protein to SLMVs was maintained despite scrambling the position of that amino acid (pHis-SCR104 -112).…”

“…Previous studies performed with rat synaptogyrin 1 and the C. elegans synaptogyrin homolog showed that the carboxyl termini of those proteins were required for reaching nerve termini in cultured hippocampal neurons and intact worms (24). Therefore, we decided to focus our initial efforts on the C terminus of cellugyrin.…”