A role for Rab30 in retrograde trafficking and maintenance of endosome-TGN organization.

Rab30 is a poorly characterized small GTPase. Here we show that Rab30 is localised primarily to the TGN and recycling endosomes in a range of cell types, including primary neurons; minor levels of Rab30 were also detected throughout the Golgi stack and early endosomes. Silencing of Rab30 resulted in the dispersal of both early and recycling endosomes and TGN compartments in HeLa cells. By analyzing cargo trafficking in Rab30-silenced and Rab30-overexpressing HeLa cells, we demonstrate that Rab30 plays a role in retrograde trafficking of TGN38 from endosomes to the Golgi, but has no apparent role in the endocytic recycling of the transferrin receptor to the plasma membrane. Five interactive partners with Rab30 were identified by pull-down and MS analysis using GFP-tagged Rab30 mutant, Rab30(Q68L). Two of the interactive partners identified were Arf1 and Arf4, known regulators of endosome to TGN retrograde transport. Knockdown of Arf1 and Arf4 results in GFP-Rab30 decorated tubules arising from the recycling endosomes, suggesting association of Rab30 with tubular carriers. Overall our data demonstrates a role for Rab30 in regulating retrograde transport to the TGN and maintenance of endosomal-TGN organization.

A role for Rab11 in the homeostasis of the endosome-lysosomal pathway.

The small GTPases Rab11a and 11b are key regulators of membrane transport, localised to the recycling endosomes and also early endosomes. The function of Rab11 within the recycling pathway has been well defined, however, the role of Rab11 at the early endosomes remains poorly characterised. Here, we have generated HeLa cell lines devoid of either Rab11a or Rab11b using CRISPR/Cas9 to functionally dissect the roles of these two Rab11 family members in recycling and in the endosomal-lysosomal system. Both Rab11a and Rab11b contribute to the dynamics of tubulation arising from recycling endosomes whereas Rab11a has the major role in recycling of transferrin receptor. Deletion of either Rab11a or Rab11b resulted in the formation of enlarged early endosomes and perturbation of the endosomal-lysosomal pathway. Strikingly, Rab11a knock-out cells showed an increased density of functional late endosomes/lysosomes as well as lysotracker-positive organelles which were primarily concentrated in a perinuclear location, indicating that the homeostasis of the endosome/lysosome pathway had been perturbed. Moreover, in Rab11a knockout cells there was a functional defect in the intracellular recycling of the cation-independent mannose 6-phosphate receptor (CI-M6PR) between the late endosomes and the TGN, a defect associated with enhanced degradation of CI-M6PR. Expression of wild-type Rab11a in Rab11a knockout cells rescued the late endosome/lysosome phenotype. Overall, these results indicate that Rab11a and Rab11b have overlapping and distinct functions and that Rab11a, unexpectedly, plays a central role in the homeostasis of endosomal-lysosomal biogenesis.

Amyloid precursor protein traffics from the Golgi directly to early endosomes in an Arl5b- and AP4-dependent pathway.

The intracellular trafficking and proteolytic processing of the membrane-bound amyloid precursor protein (APP) are coordinated events leading to the generation of pathogenic amyloid-beta (Aß) peptides. The membrane transport of newly synthesized APP from the Golgi to the endolysosomal system is not well defined, yet it is likely to be critical for regulating its processing by ß-secretase (BACE1) and γ-secretase. Here, we show that the majority of newly synthesized APP is transported from the trans-Golgi network (TGN) directly to early endosomes and then subsequently to the late endosomes/lysosomes with very little transported to the cell surface. We show that Arl5b, a small G protein localized to the TGN, and AP4 are essential for the post-Golgi transport of APP to early endosomes. Arl5b is physically associated with AP4 and is required for the recruitment of AP4, but not AP1, to the TGN. Depletion of either Arl5b or AP4 results in the accumulation of APP, but not BACE1, in the Golgi, and an increase in APP processing and Aß secretion. These findings demonstrate that APP is diverted from BACE1 at the TGN for direct transport to early endosomes and that the TGN represents a site for APP processing with the subsequent secretion of Aß.