Rab6 coordinates a novel Golgi to ER retrograde transport pathway in live cells

We visualized a fluorescent-protein (FP) fusion to Rab6, a Golgi-associated GTPase, in conjunction with fluorescent secretory pathway markers. FP-Rab6 defined highly dynamic transport carriers (TCs) translocating from the Gol gi to the cell periphery. FP-Rab6 TCs specifically accumulated a retrograde cargo, the wild-type Shiga toxin B-fragment (STB), during STB transport fr om the Golgi to the endoplasmic reticulum (ER), FP-Rab6 TCs associated inti mately with the ER, and STB entered the ER via specialized peripheral regio ns that accumulated FP-Rab6. Microinjection of antibodies that block coatom er protein I (COPI) function inhibited trafficking of a KDEL-receptor FP-fu sion, but not FP-Rab6. Additionally, markers of COPI-dependent recycling we re excluded from FP-Rab6/STB TCs. Overexpression of Rab6:GDP (T27N mutant) using T7 vaccinia inhibited toxicity of Shiga holotoxin, but did not alter STB transport to the Golgi or Golgi morphology. Taken together, our results indicate Rab6 regulates a novel Golgi to ER transport pathway.