A dominant negative mutant of sar1 GTPase inhibits protein transport from the endoplasmic reticulum to the Golgi apparatus in tobacco and Arabidopsis cultured cells.
Protein secretion plays an important role in plant cells as it does in animal and yeast cells, but the tools to study molecular events of plant secretion are very limited. We have focused on the Sar1 GTPase, which is essential for the vesicle formation from the endoplasmic reticulum (ER) in yeast, and have previously shown that tobacco and Arabidopsis SAR1 complement yeast sar1 mutants. In this study, we have established a transient expression system of GFP-fusion proteins in tobacco and Arabidopsis cultured cells. By utilizing confocal laser scanning microscopy, we demonstrate that a dominant negative mutant of Arabidopsis Sar1 inhibits the ER-to-Golgi transport of Golgi membrane proteins, AtErd2 and AtRer1B, and locates them to the ER. The same mutant Sar1 also blocks the exit from the ER of a vacuolar storage protein, sporamin. These results not only provide the first evidence that the Sar1 GTPase functions in the ER-to-Golgi transport in plant cells, but also prove that conditional expression of dominant mutants of secretory machinery can be a useful tool in manipulating vesicular trafficking.
Takeuchi
M
M
Molecular Membrane Biology Laboratory, RIKEN, Wako, Saitama 351-0198, Japan.
Ueda
T
T
Sato
K
K
Abe
H
H
Nagata
T
T
Nakano
A
A
D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D001693:Biological Transport, Active; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000235:genetics* / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D010947:Plants, Toxic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C086264:SAR protein, Lycopersicon esculentum; D049452:Green Fluorescent Proteins; D020558:GTP Phosphohydrolases; D020559:Monomeric GTP-Binding Proteins