TY  - GEN
N2  - The Endoplasmic Reticulum (ER) to Golgi transport is mediated by COPII vesicle in yeast and mammalian cell. COPII coats consist of the small GTPase Sar1p and the heterodimeric protein complexes Sec23/24 and Sec13/31. COPII mediated sorting occur when protein cargoes exit the ER. Although the principles of ER-to-GA transport organization in plant cells are supposed to be similar to those in yeast and mammalian systems, evidence in support of such an assertion is largely circumstantial. Moreover, there is a substantial body of evidence that emphasizes the differences, such as the apparent absence of the intermediate compartment in plants, large numbers of GA stacks moving along the ER and the differences in the organization of the cytoskeleton involved in interrelationships between the ER and GA. In this study, in vitro and in vivo approaches were employed to understand the function of the COPII vesicles in plant cells. In vitro, we have set up a budding assay which we could monitor the in vitro formation of COPII vesicle using ER-rich microsome, 30% (NH4)2SO4 cytosol GMP-PNP and ATP regenerating system. The vesicle budding was enhanced when ER-rich microsome from a Sec12 overproducer and more Sar1p are available in the budding mixture. Putative COPII vesicles were isolated from a flotation gradient at 41% sucrose fraction and observed as 50 nm in diameter vesicle under the electric microscope. The ability of the cytosolic tail of a plant p24 protein to bind COPI and COPII subunits from plant and animal sources in vitro has been examined. We have found that a dihydrophobic motif in the -7,-8 position (relative to the cytosolic carboxy-terminus) is responsible for binding of COPII subunits from both Arabidopsis and rat liver cytosol. However, unlike rat liver cytosol, COPI from plant sources has a stronger affinity for p24 cytosolic tails than COPII. Only in the absence of the dilysine motif in the -3,-4 position (which strongly cooperates with the dihydrophobic motif in the -7,-8 position in binding COPI) or after COPI depletion could we observe COPII binding to the p24 tail with plant cytosol. In order to visualize ERESs in tobacco BY-2 cells we have employed two different approaches: a) direct visualization of endogenous COPII proteins (Sar1, Sec13, Sec23) in cell lines stably expressing ER- and Golgi-localized GFP-markers by immunoflourescence microscopy, and b) visualization of ER-bound Sec13 by expression of a Sec13-GFP construct in cells transiently expressing ER- and Golgi-localized RFP markers. In both cases ERESs considerably outnumber Golgi stacks, and some ERESs colocalize with Golgi stacks. Dual wavelength live cell imaging of ERESs (Sec13-GFP) and Golgi stacks (Man1-RFP) demonstrates that, as they move, Golgi stacks collect ERESs at their periphery. ERESs do not disappear as a result of BFA treatment, despite considerable morphological changes in the Golgi apparatus. Prevention of ER-export through expression of a Sar1 mutant locked in the GDP state leads to the partial loss of visible ERESs.
ID  - heidok4840
AV  - public
A1  - Yang, Yaodong
Y1  - 2004///
TI  - From the Endoplasmic Reticulum to the Golgi Apparatus : In Vitro and In Vivo Approaches to Understanding COPII Vesicle Function in Plant Cells
KW  - COPII 
KW  -  COPI 
KW  -  p24 
KW  -  Arabidopsis 
KW  -  BY2vesicle budding 
KW  -  ERES
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/4840/
ER  -