Electron Tomography Reveals Rab6 Is Essential to the Trafficking of trans‐Golgi Clathrin and COPI‐Coated Vesicles and the Maintenance of Golgi Cisternal Number

Abstract

We have shown previously that Rab6, a small, trans-Golgi-localized GTPase, acts upstream of the COG and ZW10/RINT1 retrograde tether complexes to maintain Golgi homeostasis. Here, we present evidence from the unbiased and high-resolution approach of electron microscopy and electron tomography that Rab6 is essential to the trans-Golgi trafficking of two morphological classes of coated vesicles; the larger corresponds to clathrin-coated vesicles and the smaller to COPI-coated vesicles. Based on the site of coated vesicle accumulation, cisternal dilation, and the normal kinetics of cargo transport from the ER to Golgi followed by delayed Golgi to cell surface transport, we suggest that Golgi function in cargo transport is preferentially inhibited at the trans-Golgi/TGN. The >50% increase in Golgi cisternae number in Rab6-depleted HeLa cells that we observed may well be coupled to the trans-Golgi accumulation of COPI-coated vesicles; depletion of the individual Rab6 effector, myosin IIA, produced an accumulation of uncoated vesicles with if anything a decrease in cisternal number. These results are the first evidence for a Rab6-dependent protein machine affecting Golgi-proximal, coated vesicle accumulation and likely transport at the trans-Golgi and the first example of concomitant cisternal proliferation and increased Golgi stack organization under inhibited transport conditions.

Rab GAP cascade regulates dynamics of Ypt6 in the Golgi traffic

“…Mammalian Rab6 has been shown to localize and function in the TGN for the fusion of endosome-derived vesicles (13). However, Ypt6 does not appear to localize exclusively to TGN, because more than 50% of GFP-Ypt6 fluorescence overlaps with the cis-Golgi marker Sed5.…”

“…In mammalian cells, accumulating evidence suggests that Rab6 localizes at the trans-Golgi region and functions in retrograde protein transport and in Golgi structural organization (13,14). Ypt6 may very well function in the yeast Golgi maintenance.…”

Golgi enlargement in Arf-depleted yeast cells is due to altered dynamics of cisternal maturation

“…5). Interestingly, when mammalian cells are depleted of the Rab6 GTPase, secretion slows and the number of Golgi cisternae per stack increases (Storrie et al, 2012). It is possible that Rab6 depletion increases the persistence time of Golgi cisternae.…”

Retrograde Traffic from the Golgi to the Endoplasmic Reticulum

“…Through a slow transport pathway, Shiga toxin lacking a KDEL retrieval sequence and the Golgi-resident galactosyltransferase can reach the ER in a Rab6A-dependent manner (Girod et al 1999). The further analysis of the pathway is hampered by the fact that Rab6 is also involved in COPI-and clathrin-dependent trafficking steps at the Golgi (Storrie et al 2012), demanding careful dissection of the different roles of Rab6A and illustrating the need to identify Rab6A effectors specific for an individual pathway. To complicate the issue, Rab6-COPI-independent (Chen et al 2003) and Rab6-COPI-dependent pathways have also been reported (Smith et al 2009) that appear to function in parallel with the classical COPI retrograde Golgi-ER pathway.…”