Rab33b and Rab6 are functionally overlapping regulators of Golgi homeostasis and trafficking.

We used multiple approaches to investigate the coordination of trans and medial Rab proteins in the regulation of intra-Golgi retrograde trafficking. We reasoned that medially located Rab33b might act downstream of the trans Golgi Rab, Rab6, in regulating intra-Golgi retrograde trafficking. We found that knockdown of Rab33b, like Rab6, suppressed conserved oligomeric Golgi (COG) complex- or Zeste White 10 (ZW10)-depletion induced disruption of the Golgi ribbon in HeLa cells. Moreover, efficient GTP-restricted Rab6 induced relocation of Golgi enzymes to the endoplasmic reticulum (ER) was Rab33b-dependent, but not vice versa, suggesting that the two Rabs act sequentially in an intra-Golgi Rab cascade. In support of this hypothesis, we found that overexpression of GTP-Rab33b induced the dissociation of Rab6 from Golgi membranes in vivo. In addition, the transport of Shiga-like toxin B fragment (SLTB) from the trans to cis Golgi and ER required Rab33b. Surprisingly, depletion of Rab33b had little, if any, immediate effect on cell growth and multiplication. Furthermore, anterograde trafficking of tsO45G protein through the Golgi apparatus was normal. We suggest that the Rab33b/Rab6 regulated intra-Golgi retrograde trafficking pathway must coexist with other Golgi trafficking pathways. In conclusion, we provide the first evidence that Rab33b and Rab6 act to coordinate a major intra-Golgi retrograde trafficking pathway. This coordination may have parallels with Rab conversion/cascade events that regulate endosome, phagosome and exocytic processes.