Batoko H, Zheng H Q, Hawes C, Moore I
Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom.
Plant Cell. 2000 Nov;12(11):2201-18. doi: 10.1105/tpc.12.11.2201.
We describe a green fluorescent protein (GFP)-based assay for investigating membrane traffic on the secretory pathway in plants. Expression of AtRab1b(N121I), predicted to be a dominant inhibitory mutant of the Arabidopsis Rab GTPase AtRab1b, resulted in accumulation of a secreted GFP marker in an intracellular reticulate compartment reminiscent of the endoplasmic reticulum. This accumulation was alleviated by coexpressing wild-type AtRab1b but not AtRab8c. When a Golgi-targeted and N-glycosylated variant of GFP was coexpressed with AtRab1b(N121I), the variant also accumulated in a reticulate network and an endoglycosidase H-sensitive population appeared. Unexpectedly, expression of AtRab1b(N121I), but not of the wild-type AtRab1b, resulted in a reduction or cessation of vectorial Golgi movement, an effect that was reversed by coexpression of the wild type. We conclude that AtRab1b function is required for transport from the endoplasmic reticulum to the Golgi apparatus and suggest that this process may be coupled to the control of Golgi movement.
我们描述了一种基于绿色荧光蛋白（GFP）的检测方法，用于研究植物分泌途径中的膜转运。预测拟南芥Rab GTP酶AtRab1b的显性抑制突变体AtRab1b（N121I）的表达，导致一种分泌型GFP标记物在细胞内网状区室中积累，该区域让人联想到内质网。通过共表达野生型AtRab1b可缓解这种积累，但共表达AtRab8c则不能。当将一种靶向高尔基体且经N-糖基化修饰的GFP变体与AtRab1b（N121I）共表达时，该变体也在网状网络中积累，并出现了对内切糖苷酶H敏感的群体。出乎意料的是，AtRab1b（N121I）的表达而非野生型AtRab1b的表达，导致高尔基体的定向移动减少或停止，而共表达野生型可逆转这种效应。我们得出结论，从内质网到高尔基体的转运需要AtRab1b的功能，并表明该过程可能与高尔基体移动的控制相关。
