Unlike the transport of proteins from the ER to the Golgi or from the Golgi to the plasma membrane, the transport of MPRs from endosomes to the trans-Golgi is a relatively low volume process. For example, the ER-to-Golgi Rab1B GTPase is 40 times more abundant in mammalian cells than Rab9 (Soldati et al., 1995
), and that pathway may involve 40 times as many transport vesicles. The low volume of endosome to trans-Golgi transport together with the low abundance of the requisite molecular machinery have hindered detection of transport intermediates in previous studies. Although MPRs have been documented as cargo of clathrin-coated vesicles that leave the TGN en route to prelysosomal compartments (Klumperman et al., 1993
), little is known about the pathway that carries MPRs back to the Golgi.
We have shown for the first time that Rab9 is present on vesicles that bud off of Rab9-positive structures. In addition, small Rab9-positive vesicles can fuse with the Golgi complex. We do not yet know what proteins coat these vesicles, but a prediction of our model is that they bear TIP47 on their surfaces. In addition, Rab9 remains vesicle associated at least through the docking reaction and possibly until after the fusion process is complete.
We have shown that like early endosomal Rabs Rab9 occupies a discrete domain on late endosome membranes that include MPRs and is likely to also include other proteins needed for vesicle formation, docking, and fusion. Rab7 was generally adjacent to Rab9, although some vesicles were positive for one of the two Rabs and not the other. Because the two domains were so distinct, it remains possible that we are visualizing two discrete vesicles that are tightly attached to each other. However, their remarkably close association seen by video microscopy appeared more consistent with the Rabs occupying distinct domains in a single membrane-bound compartment. In either case, the Rabs define distinct membrane domains.
TIP47 is likely to enrich MPRs within the Rab9 domain. In support of this conclusion is our finding that a mutant TIP47 that binds poorly to Rab9 alters dramatically the appearance of Rab9-positive compartments. Our working model is that the mutant protein coassembles with native TIP47 to form an aberrant structure. In other work, we have shown that a myc-tagged TIP47 coassembles with endogenous wild-type TIP47. Such coassembled mutant complexes may sequester MPRs within late endosomes; indeed, cells expressing the TIP47 mutant endocytosed significantly less anti–MPR-IgG than control cells and showed altered morphology of Rab9-positive compartments. Altogether, these data suggest that TIP47, Rab9, and MPRs are present altogether in an endosomal subdomain.
Do the transport vesicles we have visualized contain MPRs? MPR transport from endosomes to the Golgi complex is completely dependent on Rab9 function (Lombardi et al., 1993
; Riederer et al., 1994
). In addition, we have shown here that Rab9 domains from which the vesicles bud are enriched in CI-MPRs. Unfortunately, we could not get enough label onto MPRs to detect their presence in nascent vesicles containing Rab9 protein. In addition, little of the CFP-CD-MPR is ever seen in late endosomes at steady-state, making it unusable as a cargo marker for the retrograde transport route. It seems very possible that the CFP on the cytoplasmic domain of the CD-MPR may slow the export of this receptor from the TGN.
Although some vesicles might be formed that do not contain MPRs, the simplest model is that MPRs are indeed contained within the Rab9-positive vesicles. Because Rab9 enhances the affinity of TIP47 for MPR cytoplasmic domains, it seems reasonable to propose that these proteins are packaged altogether into transport carriers. In any event, we never saw tubules emerge from the Rab9 domain nor did we detect CI-MPRs in tubules emerging from Rab9-positive structures.
Rab7 is believed to function in the homotypic fusion of late endosomes (Feng et al., 1995
; Bucci et al., 2000
). This is distinct from the function of Rab9, which is needed for MPR recycling to the Golgi complex (Lombardi et al., 1993
; Riederer et al., 1994
). It will be of interest to determine if organelles positive for Rab9 or Rab7 (but not both) are capable of fusing with each other. If, like Rab5 in early endosomes, Rab7 organizes the homotypic late endosome fusion machinery, it is possible that the Rab9+
organelles will have lost the capacity to fuse at high efficiency with the Rab7+
organelles. Our experiments cannot rule out the presence of nonfluorescent Rab7 in a Rab9-positive compartment. Yet the observations suggest an unappreciated possibility that formation of a Rab9-positive Rab7-negative organelle would yield a terminal compartment that can no longer fuse with other late endosomes; it would only retain the capacity to bud off Rab9 vesicles or be a target for autophagic consumption. Therefore, late endosomes may have a mechanism to retain Rab7 and Rab9 in distinct domains within a single membrane-bound organelle to avoid such an outcome.
We have tried to construct functional fluorescent TIP47 chimeras using GFP, YFP, and CFP attached to either the NH2 or COOH terminus of the protein without success. We made second generation constructs that contained a polyglycine linker between the fluorescent tag and the TIP47 protein at either end. In all cases, the resulting fusion proteins failed to associate with membranes unlike native TIP47. We will not give up our attempts to generate a functional fluorescent TIP47 protein; we hope to use it to establish whether TIP47 is indeed present on transport vesicles and unlike Rab9 falls off of the vesicles before docking and fusion events. Our working model is that TIP47 collects Rab9 and MPRs into nascent transport vesicles; TIP47 release would then permit Rab9-GTP recruitment of yet to be discovered docking factors that mediate the subsequent events. We hope that future experiments will enable us to test this working model.