The findings presented in this paper provide evidence that the three members of the mammalian GGA family act together to mediate the sorting of MPRs into transport vesicles at the TGN. The experiments also reveal a role for the GGAs in maintaining the architecture of the trans-Golgi and TGN. Using confocal microscopy, the three GGAs were found to colocalize in the TGN, and cryo-immunogold EM showed that all three GGAs were present in the same clathrin-coated buds and vesicles. These results are in agreement with prior reports on the colocalization of the GGAs at the TGN (Boman et al., 2000
; Dell'Angelica et al., 2000
; Hirst et al., 2000
Although these morphologic findings are consistent with the three GGAs acting together to package cargo into transport vesicles, they do not exclude the possibility that the various GGAs function independently. To address this issue, we performed binding experiments demonstrating that the various GGAs bind to each other, consistent with the GGAs forming a complex on the membrane. Further evidence for complex formation was obtained by cross-linking experiments after recruitment of the GGAs onto Golgi-enriched membranes.
These experiments showed that GGA1 and GGA2 could be cross-linked on the membrane, but not in the cytosol. Due to the inability to detect GGA3 in bovine adrenal cytosol, we were unable to determine whether GGA3 was also present in the Golgi-associated complex. However, this is likely in view of the direct interaction of GGA3 with the other GGAs in the pull-down assays.
Together, these findings indicate that the individual GGAs are recruited as monomers from the cytosol onto the Golgi in an ARF–GTP-dependent manner, and then form a complex that subsequently interacts with AP-1 at regions of clathrin-coated bud formation (Doray et al., 2002c
). The multivalency of the assembled complex could serve to enhance the recruitment of cargo molecules and accessory proteins involved in vesicle formation.
The precise nature of the interactions of the GGAs with each other remains to be explored. Our preliminary findings indicate that multiple domains are involved, as both the VHS and the ear domains of GGA2 bind GGA3. Potentially, the VHS domain of GGA2 could bind to the internal acidic cluster/dileucine motif within the hinge segment of GGA3 (Doray et al., 2002b
). Likewise, the ear domain of GGA2, which is homologous to the γ-appendage of AP-1, could bind to the hinge domain of GGA3 (Doray et al., 2002c
). Furthermore, crystal structures of the GAT domains of the GGAs have revealed a conserved binding site that is predicted to interact with coiled-coil domain-containing proteins (Suer et al., 2003
). Thus, the GAT domains that have a predominantly coiled-coil structure might contribute to inter-GGA binding by interacting with each other. Because the various domains of the GGAs can be separately expressed, it should be possible to analyze the nature of these interactions in vitro. While this manuscript was in preparation, Wakasugi et al. (2003)
reported that the GGA3 short form is the predominant form of GGA3 expressed in cell lines and all tissues except brain. This form has a unique VHS domain that lacks a region around helix 6 implicated in binding acidic cluster/dileucine motifs (Misra et al., 2002
; Shiba et al., 2002
). Even though this form of GGA3 is unlikely to be directly involved in the cargo protein recognition, it was found on similar TGN membranes as GGA1 by immunofluorescence. Our analyses indicate that the function of the GGA3 short form could be to stabilize the complex formed by the three GGAs on the TGN membranes.
Further evidence that the GGAs act together was obtained with the post-transcriptional gene silencing experiments using RNAi to knockdown the individual GGAs. At the morphological level, the loss of any one GGA resulted in the others being redistributed from the TGN to the cytosol (). This is consistent with the three GGAs needing to form a complex on the TGN membrane to maintain a stable association with this organelle. A key finding was that the knockdown of any one GGA was associated with maximal missorting of cathepsin D, a process that is dependent on the function of the MPRs. In contrast to yeast, where the two GGAs compensate for each other's absence (Dell'Angelica et al., 2000
; Hirst et al., 2000
), the requirement for all three GGAs to maintain the MPR sorting function supports the notion that the GGAs act together in mammalian cells. This is not the only difference between the yeast and mammalian GGA proteins. Although the mammalian GGAs cooperate with AP-1 in TGN-to-endosome transport (Doray et al., 2002c
), yeast GGAs and AP-1 appear to mediate independent trafficking pathways (Black and Pelham, 2000
It is curious that the knockdown of any one GGA was associated with a partial decrease in the levels of the other GGAs. Levels of the nontargeted GGAs could be restored by transfection of the silenced GGA or by treatment of the cells with the proteasome inhibitor MG132. The mechanism whereby depletion of one GGA results in enhanced proteasomal degradation of the other GGAs is not clear at this point. Perhaps an increase in the cytosolic pool of GGAs in the absence of membrane complex formation triggers this degradative pathway.
As mentioned earlier in this section, the hypersecretion of cathepsin D by the GGA knockdown cells is most likely due to disordered MPR trafficking. In these cells, the CI-MPR was partially redistributed from the TGN to EEA1-positive early endosomal compartments, similar to what has been observed in AP-1 knockout cells (Meyer et al., 2000
). Even more striking was the significant exclusion of the CI-MPR from the CCVs of the TGN. This alteration in the steady-state distribution of the CI-MPR could be accounted for by several mechanisms. We have proposed that GGAs may bind MPRs in the trans-Golgi and bring them to AP-1–containing clathrin-coated membranes at the TGN, where the MPRs are then transferred to AP-1 (Doray et al., 2002c
; Ghosh and Kornfeld, 2003
). This was based on the finding that mutant MPRs that are incapable of binding to GGAs, but not impaired in binding to AP-1, are poorly incorporated into AP-1-containing clathrin-coated buds and vesicles at the TGN. In the absence of binding to membrane-associated GGAs, the MPRs may exit the Golgi via secretory pathways to the cell surface where they would be rapidly internalized into early endosomes. Not only would this decrease the packaging of the MPRs into AP-1 vesicular carriers at the TGN, it might shift the steady-state distribution of the MPRs toward the early endosome compartment. This would occur if the GGAs normally retain the MPRs in the terminal Golgi compartments and prevent premature exit via the plasma membrane–targeting pathway. In addition, GGAs, like AP-1, might be involved in early endosome-to-TGN retrieval of the MPRs, as well as in anterograde transport from the TGN. GGAs have been localized to early endosome-like punctate peripheral structures (Boman et al., 2000
; Dell'Angelica et al., 2000
; Hirst et al., 2000
), although in our immuno-EM analyses, GGAs have only occasionally been found on endosomes. It has also been reported that the COOH-terminal dileucine motif of the cation-dependent MPR is essential for retrograde trafficking (Tikkanen et al., 2000
) and the GGAs, unlike AP-1, have an absolute requirement for this dileucine motif to bind their cargo. This is consistent with the possibility that the GGAs play a role in the retrograde trafficking of the MPRs. However, because the CI-MPR and AP-1 colocalize in the early endosomes in the absence of GGAs, it is not clear that the GGAs serve to usher the receptors into AP-1 CCVs at this location. Another cytosolic protein, phosphofurin acidic cluster sorting protein 1, has been proposed to perform this function at the endosome (Crump et al., 2001
The finding that GGA knockdown results in morphologic alterations of the trans-Golgi and TGN extends the reports that overexpression of the various GGAs causes structural changes in the Golgi (Poussu et al., 2000
; Takatsu et al., 2000
). In addition, it has been reported that expression of a dominant-negative mutant of BIG2, an ARF-guanine nucleotide exchange factor that acts at the trans-Golgi/TGN, results in redistribution of GGA1 and AP-1 to the cytosol and membrane tubulation of the TGN (Shinotsuka et al., 2002
). Golgi localization of COPI remained unchanged, and the rest of the Golgi architecture was preserved. Although this phenotype exhibits similarities to the GGA knockdown cells, it differs in that the CI-MPR was found on the tubules emanating from the Golgi region rather than on EEA1-positive structures, as observed in our experiments. The distribution of β-GalT was not analyzed in that experiment, so it is uncertain whether the trans-Golgi was affected. However, TGN46 distribution was unaltered, indicating that the BIG2 dominant-negative mutant induced a selective alteration in TGN morphology. Further analyses are needed to decipher the exact role of the GGAs in maintaining Golgi morphology.
Finally, it should be noted that the GGAs, in addition to interacting with each other, also bind to AP-1 (Doray et al., 2002c
). In this case, the hinge regions of the GGAs bind to the γ-appendage of AP-1. Dissociation of GGA1 and GGA3 from AP-1 is mediated by phosphorylation of the GGA hinge domains by a casein kinase 2 that is associated with AP-1. It will be important to determine if the various phosphorylation sites on GGA1 and GGA3 regulate the formation and dissolution of the complexes that form on the Golgi membrane.