Arabidopsis Sec21p and Sec23p homologs. Probable coat proteins of plant COP-coated vesicles

Intracellular protein transport between the endoplasmic reticulum (ER) and the Golgi apparatus and within the Golgi apparatus is facilitated by COP (c oat protein)-coated vesicles. Their existence in plant cells has not yet be en demonstrated, although the GTP-binding proteins required for coat format ion have been identified. We have generated antisera against glutathione-S- transferase-fusion proteins prepared with cDNAs encoding the Arabidopsis Se c21p and Sec23p homologs (AtSec21p and AtSec23p, respectively). The former is a constituent of the COPI vesicle coatomer, and the latter is part of th e Sec23/24p dimeric complex of the COPII vesicle coat. Cauliflower (Brassic a oleracea) inflorescence homogenates were probed with these antibodies and demonstrated the presence of AtSec21p and AtSec23p antigens in both the cy tosol and membrane fractions of the cell. The membrane-associated forms of both antigens can be solubilized by treatments typical for extrinsic protei ns. The amounts of the cytosolic antigens relative to the membrane-bound fo rms increase after cold treatment, and the two antigens belong to different protein complexes with molecular sizes comparable to the corresponding non plant coat proteins. Sucrose-density-gradient centrifugation of microsomal cell membranes from cauliflower suggests that, although AtSec23p seems to b e preferentially associated with ER membranes, AtSec21p appears to be bound to both the ER and the Golgi membranes. This could be in agreement with th e notion that COPII vesicles are formed at the ER, whereas COPI vesicles ca n be made by both Golgi and ER membranes. Both AtSec21p and AtSec23p antige ns were detected on membranes equilibrating at sucrose densities equivalent to those typical for in vitro-induced COP vesicles from animal and yeast s ystems. Therefore, a further purification of the putative plant COP vesicle s was undertaken.