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

Polyclonal antibodies raised against vat vesicle associated membrane protei n-2 (VAMP-2) recognized, in carrot (Daucus carota) microsomes, two major po lypeptides of 18 and 30 kD, respectively. A biochemical separation of intra cellular membranes by a sucrose density gradient co-localized the two polyp eptides as resident in light, dense microsomes, corresponding to the endopl asmic reticulum-enriched fractions. Purification of coated vesicles allowed us to distinguish the subcellular location of the 18-kD polypeptide from t hat of 30 kD. The 18-kD polypeptide is present in the non-clathrin-coated v esicle peak. Like other VAMPs, the carrot 18-kD polypeptide is proteolyzed by tetanus toxin after separation of coatomers. Amino acid sequence analysi s of peptides obtained by digestion of the 18-kD carrot polypeptide with th e endoproteinase Asp-N confirms it to be a member of the VAMP family, as is suggested by its molecular weight, vesicular localization, and toxin-induc ed cleavage.