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.
Gasparian
M
M
Dipartimento di Biologia, Universit¿a di Padova, Viale Giuseppe Colombo 3, 35131 Padova, Italy.
Pusterla
M
M
Baldan
B
B
Downey
P M
PM
Rossetto
O
O
de Laureto
P P
PP
Filippini
F
F
Terzi
M
M
Lo Schiavo
F
F
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D018699:Coated Vesicles / Q000378:metabolism; D018552:Daucus carota / Q000378:metabolism* / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D008565:Membrane Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D050683:R-SNARE Proteins; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013744:Tetanus Toxin / Q000494:pharmacology
D008565:Membrane Proteins; D010940:Plant Proteins; D050683:R-SNARE Proteins; D013744:Tetanus Toxin