Tisdale Ellen J
Methods Enzymol. 2005;403:381-91. doi: 10.1016/S0076-6879(05)03033-8.
The small GTPase Rab2 is essential for membrane trafficking in the early secretory pathway. Rab2 associates with vesicular tubular clusters (VTCs) located between the endoplasmic reticulum (ER) and the Golgi complex. VTCs function as transport intermediates and sort anterograde-directed cargo from recycling proteins. Rab2 selectively recruits atypical protein kinase C iota/lambda (aPKCiota/lambda) and glyceraldehyde-3-phosphate (GAPDH) to VTCs where aPKCiota/lambda phosphorylates GAPDH. Both aPKCiota/lambda and GAPDH bind directly to Rab2 and this interaction ultimately results in COPI recruitment and the release of retrograde-directed vesicles. This chapter describes a protocol to purify recombinant Rab2 from Rab2 cDNA transformed bacteria and methods to assess recombinant Rab2 biological activity. Additionally, in vivo and in vitro assays are outlined that are employed to demonstrate Rab2 interaction with the downstream effectors aPKCiota/lambda and GAPDH.
小GTP酶Rab2对早期分泌途径中的膜运输至关重要。Rab2与位于内质网（ER）和高尔基体复合体之间的囊泡管状簇（VTCs）相关联。VTCs作为运输中间体，从循环利用的蛋白质中分拣顺行定向的货物。Rab2选择性地将非典型蛋白激酶Cι/λ（aPKCι/λ）和甘油醛-3-磷酸（GAPDH）招募到VTCs，在那里aPKCι/λ使GAPDH磷酸化。aPKCι/λ和GAPDH都直接与Rab2结合，这种相互作用最终导致COPI的招募和逆行定向囊泡的释放。本章描述了从Rab2 cDNA转化细菌中纯化重组Rab2的方案以及评估重组Rab2生物学活性的方法。此外，还概述了用于证明Rab2与下游效应器aPKCι/λ和GAPDH相互作用的体内和体外试验。
