The results presented here indicate that the mammalian GARP complex is required for the trafficking of CatD to lysosomes by enabling the recycling of the CI-MPR from endosomes to the TGN. In the absence of GARP, the CI-MPR accumulates in a population of small vesicles downstream of endosomes. The ensuing depletion of MPRs from the TGN causes newly synthesized CatD to be released into the extracellular medium as an uncleaved precursor instead of being sorted to endosomes and then to lysosomes. Loss of CatD, and likely of other mannose 6-phosphate–modified hydrolases, results in swelling of the lysosomes, a phenotype similar to that of lysosomal storage disorders.
Where does GARP act in this process? A previous study had suggested a mostly endosomal localization for mammalian GARP (Liewen et al., 2005
). However, our analyses of H4 and HeLa cells expressing Vps54-GFP indicate that GARP is mainly associated with the TGN ( and , B–D). It is then at this location that GARP must participate in CI-MPR trafficking. On the basis of the proposed function of yeast GARP (Conibear and Stevens, 2000
; Siniossoglou and Pelham, 2001
; Whyte and Munro, 2002
; Conibear et al., 2003
; Panic et al., 2003
), we think that mammalian GARP is involved in the tethering or docking of endosome-derived, retrograde transport carriers to the TGN. This would be followed by SNARE-mediated fusion and delivery of the CI-MPR into the TGN. Indeed, depletion of GARP causes a shift in the steady-state distribution of the CI-MPRs from a set of relatively large endosomal structures to myriad small vesicles scattered throughout the cytoplasm (, E, I, and L). A similar accumulation in small vesicles is observed for CI-MPR internalized from the plasma membrane (, D and J), indicating that these vesicles lie in the retrograde transport pathway. The small vesicles are lighter than endosomes or the Golgi complex () and lack markers of either of these organelles (). Importantly, they do not contain SNX2 (E), a component of the retromer complex that initiates the retrieval of the CI-MPR from endosomes by diverting the receptor into recycling tubules. Moreover, internalized CI-MPR passes through SNX2-positive endosomes before accumulating in the small vesicles (, M–O). This places the small vesicles past the tubular endosomal network (TEN; Bonifacino and Rojas, 2006
) through which the CI-MPR transits en route to the TGN (Arighi et al., 2004
). Thus, the small vesicles are likely intermediates in the transport between the TEN and the TGN.
Interestingly, the levels of CI-MPR are not changed by depletion of GARP despite its altered distribution (P). This is in contrast to the depletion of retromer, which results in lower levels of CI-MPR due to its diversion to lysosomes (Arighi et al., 2004
). This indicates that the small vesicles where the CI-MPR accumulates in the absence of GARP are past the point where default transport to lysosomes is possible.
The role of GARP in retrograde transport is not limited to CI-MPR trafficking because the recycling TGN protein, TGN46, and the bacterial toxin, STxB, are also prevented from reaching the TGN in GARP-depleted cells. In these cells, both TGN46 (F) and internalized STxB (, C and D) accumulate in small vesicles similar to those that contain the CI-MPR. However, some differences with the behavior of the CI-MPR are apparent. The total levels of TGN46 are decreased (P). In addition, STxB also accumulates in larger structures that colocalize with endosomal markers ( and data not shown). This suggests that some cargo proteins back up into endosomal compartments in the absence of GARP. Despite these differences, it is clear that GARP is required for the retrograde transport of different types of protein: recycling transmembrane proteins like the CI-MPR and TGN46, and a glycosphingolipid-binding luminal protein like STxB. GARP thus appears to function as a general mediator of retrograde transport to the TGN.
Previous studies have identified other proteins that function to tether retrograde transport intermediates to the TGN. Among these are the golgins, golgin-97 (Lu et al., 2004
), golgin-245 (Yoshino et al., 2005
), GCC88 (Lieu et al., 2007
), and GCC185 (Reddy et al., 2006
; Derby et al., 2007
). It is currently unclear why so many tethering factors would be involved in retrograde transport. One possibility is that they all cooperate to dock the same set of retrograde transport carriers to the TGN. An alternative possibility is that each participates in the docking of a different type of carrier, as defined by its origin or cargo. For example, GCC185 participates, together with Rab9 and TIP47, in retrieval of CI-MPR specifically from late endosomes (Reddy et al., 2006
). Another variation is exemplified by GCC88, which participates in retrograde transport of CI-MPR and TGN38 (the rat ortholog of human TGN46), but not STxB (Lieu et al., 2007
). This is consistent with the existence of multiple routes and carriers for retrograde transport. To the extent that we have analyzed it, the role of GARP appears to be general to various cargo proteins.
Although most of the CI-MPR accumulates in small vesicles in GARP-depleted cells, a fraction appears to concentrate in a juxtanuclear structure that colocalizes with Golgi markers (, I–K). This may indicate that some CI-MPR molecules are still delivered to the TGN in the absence of GARP, perhaps due to the action of the other tethering factors mentioned above. Alternatively, the juxtanuclear remnant may reflect a certain degree of inhibition of exit from the Golgi complex. Indeed, after prolonged (≥5 d) depletion of GARP, we observed that even the plasma-membrane–targeted VSV-G protein accumulates to some extent in the Golgi complex. This could point to an additional role of GARP in export from the Golgi complex. In this regard, interference with another tethering factor, golgin-97, has been shown to inhibit transport of the adhesion molecule, E-cadherin, from the Golgi complex to the basolateral surface of polarized epithelial cells (Lock et al., 2005
). However, accumulation in the Golgi complex could also be secondary to impaired retrieval of factors that are required for exit from the TGN, as has been previously proposed for yeast GARP (Conibear and Stevens, 2000
As would be expected for a complex that plays a general role in retrograde transport, GARP is essential for embryonic development and viability in the mouse (Schmitt-John et al., 2005
). Mouse embryos with homozygous disruption of the Vps54
gene fail to thrive and die at about day 12.5 postcoitum (Schmitt-John et al., 2005
). However, the mutant Wobbler mouse, which carries the missense mutation L967Q in Vps54 (Schmitt-John et al., 2005
), is viable though it exhibits motor neuron degeneration similar to that of amyotrophic lateral sclerosis (Boillee et al., 2003
). The different phenotypes of the Vps54 disruption and Wobbler mutants are likely explained by the ability of the Vps54(L967Q) mutant protein to assemble with the other subunits of GARP and to support sorting of CI-MPR, CatD, and TGN46 (). The expression levels of Vps54(L967Q), however, are lower than those of its wild-type counterpart (A), perhaps explaining the Wobbler motor neuron defect.
The demonstration of a role of the human GARP complex in retrograde transport further supports the notion that the core machinery for acid hydrolase sorting has been faithfully conserved from yeast to humans, to the point of utilizing similar proteins or complexes at virtually every step of the sorting pathways. This conservation undoubtedly stems from the essential nature of endosomal transport pathways and, in particular, retrograde transport from endosomes to the TGN for the maintenance of cellular homeostasis.