Rab2 purification and interaction with protein kinase C iota/lambda and glyceraldehyde-3-phosphate dehydrogenase.
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.
Ellen J
EJ
Tisdale
D000818:Animals; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D020693:rab2 GTP-Binding Protein / Q000302:isolation & purification* / Q000378:metabolism*; D000099293:Protein Kinase C-lambda
D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D007527:Isoenzymes; D011493:Protein Kinase C; D011994:Recombinant Proteins; D020693:rab2 GTP-Binding Protein; D000099293:Protein Kinase C-lambda