D53 is a novel endosomal SNARE-binding protein that enhances interaction of syntaxin 1 with the synaptobrevin 2 complex in vitro

Abstract

Synaptobrevin 2 (Sb2), syntaxin1 (Stx1), and synaptosomal-associated protein of 25 kDa (SNAP-25) are the main components of the soluble N -ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) complex involved in fusion of synaptic vesicles with the presynaptic plasma membrane. We report the characterization of D53, a novel SNARE-binding protein preferentially expressed in neural and neuro-endocrine cells. Its two-dimensional organization, established by the hydrophobic cluster analysis, is reminiscent of SNARE proteins. D53 contains two putative helical regions, one of which includes a large coiled-coil domain involved in the interaction with Sb2 in vitro. Following subcellular fractionation, endogenous D53 was specifically detected in the membrane-containing fraction of PC12 cells, where it co-immunoprecipitated with Sb2. Analysis by confocal microscopy showed that, in these cells, endogenous D53 co-localized partially with the transferrin receptor in early endosomes. In vitro assays revealed that binding properties of D53 to Stx1 and Sb2 are comparable with those of SNAP-25. Furthermore, D53 forms Sb2/Stx1/D53 complexes in vitro in a manner similar to SNAP-25. We propose that D53 could be involved in the assembly or disassembly of endosomal SNARE complexes by regulating Sb2/Stx interaction.

Evidence for structural and functional diversity among SDS-resistant SNARE complexes in neuroendocrine cells

“…Transmitter release from PC12 cells requires syntaxin 1, synaptobrevin 2 and SNAP-25 (Gerona et al, 2000;Lang et al, 2002;Proux-Gillardeaux et al, 2003;Quetglas et al, 2002). To identify SDS-resistant complexes of these proteins in the PC12 cell line, membrane proteins were extracted in SDS-sample buffer without boiling, separated by SDS-PAGE, blotted and analysed by immunostaining with antibodies directed against syntaxin, synaptobrevin and SNAP-25.…”

TPD52 expression increases neutral lipid storage within cultured cells

“…TPD52 is the founding member of the TPD52-like protein family, whose members share ∼50% sequence identity. At the molecular level, TPD52-like proteins exhibit functional redundancy, in that heterologous partners identified through yeast two-hybrid screens using a single TPD52-like bait also interact with related TPD52-like proteins (Wilson et al, 2001;Proux-Gillardeaux et al, 2003;Shahheydari et al, 2014). However, stable expression of TPD52 or its paralogue TPD52L1 in BALB/c 3T3 cells produced shared but also isoformspecific cellular effects (Lewis et al, 2007;Shehata et al, 2008a).…”

“…1A) directly interacts with ADRP. We first employed the yeast two-hybrid system which has previously been used to identify TPD52-binding partners (Boutros et al, 2003;Byrne et al, 1998;Proux-Gillardeaux et al, 2003;Shahheydari et al, 2014). Co-transfection of amino acids 1-415 ADRP or fulllength TIP47 bait and TPD52 prey constructs produced detectable Hf7c yeast growth on triple drop-out medium after 5 days and 3 days, respectively (Fig.…”

PrLZ, a Novel Prostate-Specific and Androgen-Responsive Gene of the TPD52 Family, Amplified in Chromosome 8q21.1 and Overexpressed in Human Prostate Cancer

“…Members of the TPD52 family are involved in protein-protein interaction (11)(12)(13). Annexin VI (14), MAL2 (13), syntaxin I, and VAMP2 (15) have been identified as interacting partners of TPD52 proteins. 14 -3-3, a crucial player in Ras signaling, vesicular transport, and cytoskeletal organization, was revealed recently as another interacting partner (11).…”