ARHGEF10 directs the localization of Rab8 to Rab6-positive executive vesicles

Abstract

The function of ARHGEF10, a known guanine nucleotide exchange factor (GEF) for RhoA with proposed roles in various diseases, is poorly understood. To understand the precise function of this protein, we raised a monoclonal antibody against ARHGEF10 and determined its localization in HeLa cells. ARHGEF10 was found to localize to vesicles containing Rab6 (of which there are three isoforms, Rab6a, Rab6b and Rab6c), Rab8 (of which there are two isoforms, Rab8a and Rab8b), and/or the secretion marker neuropeptide Y (NPY)-Venus in a Rab6-dependent manner. These vesicles were known to originate from the Golgi and contain secreted or membrane proteins. Ectopic expression of an N-terminal-truncated ARHGEF10 mutant led to the generation of large vesicle-like structures containing both Rab6 and Rab8. Additionally, small interfering (si)RNA-mediated knockdown of ARHGEF10 impaired the localization of Rab8 to these exocytotic vesicles. Furthermore, the invasiveness of MDA-MB231 cells was markedly decreased by knockdown of ARHGEF10, as well as of Rab8. From these results, we propose that ARHGEF10 acts in exocytosis and tumor invasion in a Rab8-dependent manner.

In vivo identification of GTPase interactors by mitochondrial relocalization and proximity biotinylation

“…Rab6A: Rab6 is conserved in most, if not all, eukaryotes and acts on the trans Golgi network, particularly in recycling from endosomes (Pfeffer, 2011). Almost half of the top thirty hits with Rab6A-GTP are known Rab6 effectors, including p150/glued subunit of dynactin (DCNT1), the Rab39 GEFs DENDD5A and DENND5B, the dynein adaptor bicaudal-1/2, the Rho GEF ARHGEF1 and the coiled-coil proteins ERC1/2 and TMF1 (Short et al, 2002;Matanis et al, 2002;Fukuda et al, 2011;Shibata et al, 2016;Fridmann-Sirkis et al, 2004) (Grigoriev et al, 2007) ( Figure 4B). In addition, RABGAP1 and its paralog RABGAP1L have been reported to have GAP activity on Rab6 (Cuif et al, 1999).…”

“…Rab8A and Rab10: Rab8 and Rab10 arose from a gene duplication early in metazoan evolution and have related roles in exocytosis including sharing some effectors (Sato et al, 2014;Elias et al, 2012;Kloepper et al, 2012). The high scoring hits with the GTP-bound form of Rab8A include known effectors such as synaptotagmin-like 4 (SYTL4), the myosin MYO5A, the lipid phosphatase OCRL, the Rho GEF ARHGEF10, and the flavoprotein monooxygenases MICALL1 and MICAL3, with the latter four also being hits for Rab10 (Fukuda et al, 2002;Shibata et al, 2016;Sharma et al, 2009;Grigoriev et al, 2011;Sun et al, 2014) (Figure 4E,F). The Ste20 kinase MAP4K3 and its paralog MAP4K5 were hits with both Rab8A and Rab10.…”

CRISPR-based bioengineering of the Transferrin Receptor revealed a role for Rab7 in the biosynthetic secretory pathway

“…Some Rabs can act together in the exocytosis process, such as Rab6 and Rab8 (44) or Rab3 and Rab27 (45). We detected > 35% of TfR-eRUSH-containing vesicles harboring a Rab6-positive staining (Figure 3D), but our data suggest however that Rab6 and Rab7 do not intervene at the same stage of the secretory pathway and/or in the same type of vesicular transport, as shown by the absence of overlap between the Rab6 and Rab7 staining (Figure S3B).…”

Direct trafficking pathways from the Golgi apparatus to the plasma membrane

“…MICAL3 and Rab8 interact with each other and MICAL3 with ELKS, hinting at a functional complex for exocytosis. ARHGEF10, an exchange factor for RhoA has been shown to localise to Rab6/Rab8 positive carriers and is required for Rab8 recruitment suggesting that there is a small G protein cascade leading to carrier fusion [155,156]. The soluble biosynthetic cargo neuropeptide Y accumulates in pre-fusion ELKS dependent carriers, suggesting that this is the biosynthetic transport route [120].…”