Our results are consistent with the idea that the FP-Rab6 membrane system defines a separate compartment that corresponds to a Golgi→ER transport pathway containing specific retrograde cargo and exhibiting distinct functional requirements. The FP-Rab6 compartment comprises all FP-Rab6 elements, the Golgi-associated pool, tubular and globular trafficking elements, and peripheral corner regions. It is separate from some of the traditionally defined morphological compartments (endosomes, lysosomes, and Golgi→plasma membrane transport carriers), and partly overlaps with others (Golgi, ER). FP-Rab6 dynamics in live cells emphasized elements that were neglected in static images, thus revealing morphological features of endogenous Rab6 that had previously been overlooked ( Goud et al. 1990
; Antony et al. 1992
). Since endogenous Rab6 and FP-Rab6 have common features ( ), FP-Rab6 dynamics are most likely not induced by overexpression of the FP-Rab6 fusion protein.
During the period when STB underwent transport from the Golgi to the ER, it accumulated in FP-Rab6 trafficking structures and together with FP-Rab6 in distinctive corner regions. At later times, when the STB has been shown to cycle between the ER and Golgi ( Johannes et al. 1997
), it continued to traffic in FP-Rab6 elements, although to a lesser extent. These observations indicate that FP-Rab6 trafficking elements are retrograde TCs, and are consistent with the idea that the initial vectorial pulse of STB transport concentrates it in FP-Rab6 retrograde carriers, resulting in strong coincidence at early times. At later times, as STB partitions between the Golgi and ER and begins cycling, only a proportion undergoes Golgi→ER retrograde transport during a given period, resulting in weaker coincidence. Since STB and FP-Rab6 trafficking behavior can be observed independently, it is unlikely that one or the other induces the transport carriers, and further support the ideas that FP-Rab6 does not induce trafficking elements, and that STB uses a preexisting cellular pathway.
The coincidence of FP-Rab6 and STB in distinctive peripheral corner regions prompted us to investigate these regions further. STB dissipated into the ER network directly from these regions, indicating that they are ER entrance sites ( b, and supplemental movies at http://www.jcb.org/cgi/content/full/147/4/743/DC1). We cannot exclude that STB entered the ER at other locations or along the entire ER network; we conclude only that ER entrance from the corner regions was particularly pronounced. Other transport steps are also concentrated at specialized sites: ER exit occurs in specialized regions that recruit COPII coat proteins to the membrane to concentrate and sort anterograde cargo from the ER ( Balch et al. 1994
; Bannykh et al. 1996
; Presley et al. 1997
; Scales et al. 1997
), and delivery of secretory cargo also appears to be directed to a specialized area of the plasma membrane, a targeting patch, in both yeast and polarized mammalian cells ( Finger and Novick 1998
; Grindstaff et al. 1998
STB traverses endosomes before it arrives in the Golgi ( Mallard et al. 1998
), so it remained possible that the STB which coincided with FP-Rab6 trafficking elements was in endosomes. This is, however, highly unlikely since we observed STB in FP-Rab6 trafficking structures long after the bulk of STB had exited endosomal structures (after 5 h at 37°C), and since STB blocked in the endosomes at 19.5°C did not coincide with FP-Rab6 (not shown). Consistent with this, FP-Rab6 peripheral elements never colocalized with a number of different endosomal markers, including transferrin receptor and FP-Rab5 in fixed cells, and labeled transferrin in live cells (continuous uptake; not shown). It is also highly unlikely that FP-Rab6 trafficking elements represent TCs containing post-Golgi anterograde cargo, despite superficial similarities ( Hirschberg et al. 1998
; Toomre et al. 1999
). When imaged in live cells, Golgi-to-plasma membrane TCs, revealed by four separate FP markers, exhibited different behavior, trafficked to different regions of the cell, and did not coincide in live cell double-labeling experiments (White, J., unpublished observations).
Since FP-Rab6 coincided with a retrograde cargo during Golgi→ER transport, we expected FP-Rab6 TCs to coincide with proteins involved in retrieval of ER residents. This was, however, not the case. FP-Rab6 was separate from KDELR-FP in live cells, and COPI components and endogenous KDELR in fixed cells. Consistent with this, STB segregated away from KDELR-FP in live cells; we observed no coincidence at early times of STB transport, and only partial coincidence from 2 to 5 h. Even when both proteins were in the ER network, they excluded one another, appearing as separate domains within the ER network. These results indicate that STB traffics predominantly in the FP-Rab6 pathway. The partial coincidence of STB and KDELR-FP-positive structures may be due to specific retrograde transport in these structures, or may be mis-sorting into this pathway at higher STB levels. Since KDELR-FP cycles between the ER and the Golgi, a subset of KDELR-FP trafficking may represent an anterograde transport step, leading to the additional possibility that STB cycles to the Golgi in anterograde structures that contain KDELR-FP, and to the ER in FP-Rab6 structures.
The differences between ER retrieval and FP-Rab6/STB retrograde transport extend to the functional level. Previous studies indicate toxin proteins define two functionally different retrograde pathways to the ER. Jackson and coworkers have shown some toxin proteins require the KDEL-retrieval system for efficient transport to the ER, while others do not ( Jackson et al. 1999
). Girod and coworkers have recently shown that microinjection of anti-EAGE antibodies, potent inhibitors of COPI function in live cells ( Pepperkok et al. 1993
, Pepperkok et al. 1998
) inhibits Golgi→ER transport of toxin proteins which contain KDEL-like motifs. In contrast, transport of STB, and likewise the holotoxin, is unaffected (Girod, A., B. Storrie, J.C. Simpson, J.M. Lord, T. Nilsson, and R. Pepperkok, manuscript submitted for publication). Here, we show that FP-Rab6 trafficking is not inhibited by anti-EAGE microinjection, but KDELR-FP trafficking is reduced to 60% of control ( ). These results are consistent with the observation that modified STB reach the ER equally well with or without a KDEL retrieval signal. They also imply that trafficking is linked to transport of KDELR (and presumably other components of the ER retrieval system), a relationship that is often assumed but which has not been shown. It was previously unclear whether the toxin protein pathway was preexisting or a consequence of the toxin itself, but the coincidence of FP-Rab6 and STB in trafficking structures with the same functional characteristic strongly argues that STB uses a COPI-independent cellular pathway defined by FP-Rab6.
Our results suggest native Rab6 regulates this alternate Golgi→ER transport pathway. In previous studies, high overexpression of active forms of Rab6 (Q72L mutant or wild-type) stimulated retrograde transport from the Golgi to the ER, progressively relocating Golgi residents to the ER and indirectly inhibiting intra-Golgi anterograde transport ( Martinez et al. 1994
, Martinez et al. 1997
). This interpretation fits well with the general idea that Rab proteins are active in their GTP-bound form ( Novick and Zerial 1997
). Consistent with these observations, overexpression of Rab6:GDP (T27N) reduces toxicity of the holotoxin by inhibiting a Golgi→ER transport step ( ), without altering earlier transport steps or overall Golgi morphology. We note that overexpression of active forms of Rab6 does not in fact stimulate ER arrival of STB, because STB must be transported to the ER via an intact Golgi structure ( Johannes et al. 1997
), and upon overexpression of active Rab6 the Golgi redistributes to the ER and is no longer intact ( Martinez et al. 1997
). Taken together, these results imply that this pathway is activated by GTP-Rab6 and inhibited by GDP-Rab6.
Why would an alternate Golgi→ER pathway exist? Presumably it would be used by a subset of cellular components, not only by toxin proteins. Supporting this, verotoxin and Shiga-like toxins have homology to cellular proteins that are transported from the cell surface to the nuclear envelope, a subdomain of the ER ( Lingwood and Yiu 1992
; Maloney and Lingwood 1994
; Khine et al. 1998
). These components do not have a recognizable ER retrieval motif, yet appear to reach the ER. Other cellular components that return to the ER also lack a retrieval motif. Resident Golgi proteins slowly but continuously cycle through the ER (Cole, 1998), apparently via COPI-independent mechanisms ( Girod et al. 1999
). A transport pathway to the ER could be used for degradation of transmembrane proteins: certain transmembrane ER proteins may be extracted from the membrane by retrotranslocation to be targeted for proteasome-mediated degradation in the cytosol ( Ward et al. 1995
; Kopito 1997
; Plemper et al. 1997
). Transmembrane proteins from other intracellular compartments could also undergo ER-based degradation. Another rationale for an alternate pathway would be a mechanism to recycle lipid components to the ER. Intracellular lipid trafficking is not yet well characterized, but it is notable that STB binds a glycolipid receptor, globotriaosyl ceramide, at the cell surface ( Cohen et al. 1987
). It remains to be shown whether STB follows its glycolipid receptor as far as the ER.
The idea that Rab proteins define trafficking routes within the cell was proposed based primarily on the apparent specificity of their localization ( Simons and Zerial 1993
), but until this study has never been directly tested. Observation of Rab protein dynamics provides even further insight than simple localization: Previous studies localized native Rab6 to the Golgi ( Goud et al. 1990
; Antony et al. 1992
), but its cellular function there was unclear ( Martinez et al. 1994
, Martinez et al. 1997
). Observation of FP-Rab6 TCs and the accumulation of a specific retrograde cargo within them during Golgi→ER transport directly indicate a role for Rab6 in transport to the ER. Furthermore, microtubule-dependent translocation of FP-Rab6 TCs rationalizes the direct association of Rab6:GTP with Rabkinesin-6 ( Echard et al. 1998
). Our studies of FP-Rab6 dynamics in live cells provide the cellular context in which to interpret the function of Rab6 at the molecular level.