Plant Physiol, January 2000, Vol. 122, pp. 25-34
Identification and Characterization of an 18-Kilodalton,
VAMP-Like Protein in Suspension-Cultured Carrot Cells1
Marine
Gasparian,
Michele
Pusterla,
Barbara
Baldan,
Patrick M.
Downey,
Ornella
Rossetto,
Patrizia Polverino
de
Laureto,
Francesco
Filippini,
Mario
Terzi, and
Fiorella Lo
Schiavo*
Dipartimento di Biologia (M.G., M.P., B.B., F.F., M.T., F.L.S.),
Centro Ricerche Interdipartimentale Biotechnologie Innovative
(P.M.D., P.P.d.L.), and Dipartimento di Scienze Biomediche (O.R.),
Università di Padova, Viale Giuseppe Colombo 3, 35131 Padova, Italy.
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.
1
This research was supported by the Biotechnology
Program of the European Community (grant no. BIO 4 CT 960689 to M.T.).
*
Corresponding author; e-mail loschiav{at}civ.bio.unipd.it; fax
39-049-8276280.
© 2000 American Society of Plant Physiologists
