Identification and characterization of an 18-kilodalton, VAMP-like protein in suspension-cultured carrot cells

Polyclonal antibodies raised against rat vesicle associated membrane protein-2 (VAMP-2) recognized, in carrot (Daucus carota) microsomes, two major polypeptides of 18 and 30 kD, respectively. A biochemical separation of intracellular membranes by a sucrose density gradient co-localized the two polypeptides as resident in light, dense microsomes, corresponding to the endoplasmic reticulum-enriched fractions. Purification of coated vesicles allowed us to distinguish the subcellular location of the 18-kD polypeptide from that of 30 kD. The 18-kD polypeptide is present in the non-clathrin-coated vesicle peak. Like other VAMPs, the carrot 18-kD polypeptide is proteolyzed by tetanus toxin after separation of coatomers. Amino acid sequence analysis of peptides obtained by digestion of the 18-kD carrot polypeptide with the endoproteinase Asp-N confirms it to be a member of the VAMP family, as is suggested by its molecular weight, vesicular localization, and toxin-induced cleavage.