Indication for differential sorting of the rat v-SNARE splice isoforms VAMP-1a and -1b.

Abstract

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are essential constituents of the intracellular trafficking machinery. The variable C-terminus in the 2 rat VAMP-1 splice isoforms VAMP-1a and -1b potentially acts as a sorting signal, because similar changes at the C-terminal end of a human VAMP-1 splice isoform resulted in its sorting to mitochondria. To evaluate the differences in the subcellular localization of these two v-SNARE proteins, VAMP-1a and -1b proteins tagged with green fluorescent protein (GFP) and red fluorescent protein (RFP) were expressed in HeLa, COS-7, and MDCK cells and evaluated by conventional confocal as well as total internal reflection fluorescence microscopy. Regions consistent with the endoplasmic reticulum and Golgi apparatus demonstrated a major overlap of both signals. In the periphery, vesicular structures were observed that mainly expressed one of the 2 isoforms. Within our experimental settings, we could not observe sorting of any of the 2 isoforms to mitochondria or peroxisomes, whereas both isoforms were found expressed in a minor subset of singular vesicles, which sporadically appeared to co-localize with the exocyst marker EXOC3/Sec6. Because vesicular structures were seen that expressed only one of the two splice variants, it is possible that VAMP-1a and VAMP-1b are sorted to distinct cellular compartments that require further characterization.