The results presented herein demonstrate that clathrin, GGA1, and AP-1 are associated with a population of vesicular-tubular carriers budding from the TGN. These carriers look larger and more pleiomorphic than conventional CCVs. After detaching from the TGN, the carriers move along microtubules for distances of up to ~10 μm toward the peripheral cytoplasm. The signal of the fluorescently labeled coat proteins associated with the carriers disappears at different times after reaching the cell periphery, probably due to uncoating. Some coated carriers, however, persist and can be seen engaging in fusion or kiss-and-run interactions with peripheral endosomes before they lose their coat. Thus, it is likely that a function of these coated carriers is to move cargo between the TGN and peripheral endosomes.
A cargo molecule transported by these intermediates is the CD-MPR, which interacts via acidic-cluster–dileucine signals with the VHS domain of the GGAs (Puertollano et al., 2001a
; Takatsu et al., 2001
; Doray et al., 2002a
). Indeed, we observed that the CD-MPR leaves the TGN on vesicular-tubular structures decorated with GGA1. Other transmembrane proteins such as the CI-MPR (Puertollano et al., 2001a
; Takatsu et al., 2001
; Zhu et al., 2001
), sortilin (Nielsen et al., 2001
), the low-density lipoprotein-related protein 3 (Takatsu et al., 2001
), and β-secretase (He et al., 2002
), also have acidic-cluster–dileucine signals that interact with the GGAs and might therefore be exported from the TGN on the same carriers. In contrast, recycling transferrin receptors and the VSV-G protein traffic in distinct sets of tubules and vesicles devoid of GGA1.
The presence of both clathrin and GGA1 on the same carriers adds to the evidence that the GGAs function in association with clathrin (Puertollano et al., 2001b
; Costaguta et al., 2001
; Mullins and Bonifacino, 2001
). The occurrence of AP-1 on these TGN-derived carriers, however, is intriguing because the exact role of AP-1 in sorting is currently a matter of debate. Our observations with GFP-γ1-adaptin agree with those of Huang et al. (2001)
obtained using YFP-μ1 to label the AP-1 complex. These authors reported that most YFP-μ1A–labeled vesicles also move from the TGN to the periphery of the cells. This sense of transport contrasts with the proposed role of AP-1 in recycling MPRs from endosomes to the TGN, inferred from the accumulation of MPRs in peripheral endosomes of μ1A-deficient fibroblasts (Meyer et al., 2000
). A possible explanation for these seemingly contradictory observations could be that AP-1 actually mediates removal of cargo from the intermediates as they move toward the cell periphery or upon their merge with peripheral endosomes. AP-1 has in fact been shown to function in the removal of membrane proteins from immature secretory granules after their budding from the TGN (Klumperman et al., 1998
). This would be analogous to the behavior of COPI, which is present on v
(VTCs) moving from endoplasmic reticulum (ER) exit sites to the Golgi complex (Presley et al., 1997
), even though it plays a role in protein recycling from the Golgi complex to the ER (Letourneur et al., 1994
). Another possibility is that AP-1 plays a role in sorting at the TGN, as previously assumed. Recent work suggests that the GGAs and AP-1 do cooperate to package MPRs into TGN-derived intermediates (Doray et al., 2002b
The TGN-derived coated intermediates described herein seem to belong to a growing family of large intracellular transport carriers, including VTCs, that mediate transport from the ER to the Golgi complex (Aridor et al., 1995
; Presley et al., 1997
) and PGCs involved in transport of VSV-G protein from the TGN to the plasma membrane (Hirschberg et al., 1998
; Polishchuk et al., 2000
). The large carriers described herein are the first ones shown to contain associated clathrin and GGA1. The decoration of tubules carrying the CD-MPR with GGA1 seem to occur in discontinuous manner, as if defining specific domains on the tubules. The CD-MPR was often more concentrated in the tubule domains containing associated GGA1, suggesting that the segregation of CD-MPR from other cargo molecules may persist after budding from the TGN.
The vesicular-tubular carriers containing CD-MPR and even the individual foci labeled for clathrin, GGA1, and AP-1 were apparently larger than plasma membrane-coated pits and conventional CCVs. This difference could be easily appreciated in microscopic fields where both types of clathrin-coated structures were visible (Figure D). Although the intensity of the fluorescence signal can impinge upon estimations of size by optical microscopy, in the case of clathrin it is reasonable to assume that the probability of GFP-labeled clathrin to be incorporated into clathrin lattices is the same in different parts of the cell. Thus, brighter clathrin-coated structures are also larger. Corroboration of the larger size of the TGN-derived intermediates was obtained by comparison with fluorescent beads of known size and similar brightness (Figure ). The larger size of the TGN-intermediates was not due to “streaking” because the speed of scanning (typically 20–30 μm/s) was much higher than the speed of the intermediates (~1 μm/s).
The fine structure of the TGN-derived carriers could not be resolved by fluorescence microscopy because of limits on the resolution by this technique (~200 nm under the conditions of our experiments; Inoue, 1989
). We envision that they consist of tubular or irregularly shaped membrane-bound organelles (akin to VTCs [Aridor et al., 1995
] or PGCs [Polishchuk et al., 2000
]) with 60- to 130-nm coated buds that define specific domains within these organelles. The apparently larger size of the coated foci relative to CCVs could be due to the presence of several 60- to 130-nm coated buds on the carriers. It is also possible that the larger foci represent clusters of CCVs that are somehow tethered together.
An important property of the TGN-associated coats studied herein is that they are constantly cycling between membranes and the cytosol. Even when vesicle budding from the TGN is inhibited by incubation at 20°C, the coats continue to exchange. Therefore, dissociation of the coats does not require formation of vesicular intermediates. In this regard, the clathrin GGA– and AP-1–containing TGN coats behave like plasma membrane clathrin coats (Wu et al., 2001
) and COPI (Presley et al., 2002
), which also cycle on and off membranes when vesicle budding is inhibited. We were unable to examine the dynamics of coats on the TGN-derived intermediates themselves because of their mobility. In any event, whether dynamically or statically, the intermediates do retain their coats until they reach the periphery. The coats could thus be directly responsible for the recruitment of motor molecules that effect tracking along microtubules. The kinesin superfamily protein KIF13A, for example, is a plus-end microtubule motor that interacts with the β1-adaptin subunit of AP-1 (Nakagawa et al., 2000
). The coats could also bind tethering proteins that allow interactions or fusion of the intermediates with endosomes. For instance, rabaptin-5 is an effector of the early endosomal rab4 and rab5 GTP-binding proteins (Stenmark et al., 1995
; Vitale et al., 1998
) that also interacts with the γ1-adaptin subunit of AP-1 (Shiba et al., 2002
). The persistence of the coats on the intermediates may thus enable their involvement in organelle targeting events, in addition to their roles in vesicle formation and cargo selection.
Of course, our observations do not rule out the existence of small CCVs that could mediate short-range transfer of cargo to the larger intermediates or to endosomes located in the vicinity of the TGN. In fact, the endosomal recycling compartment (Yamashiro et al., 1984
) and late endosomes (Rabinowitz et al., 1992
) are mostly concentrated in the juxtanuclear area of the cell. It would be virtually impossible to observe direct transfer of materials from the TGN to these compartments. Small CCVs could also pinch off from the large intermediates as they move toward the peripheral cytoplasm. Finally, small CCVs could be too weakly labeled or transient for detection. Nevertheless, our findings do reveal the existence of a previously unrecognized type of intermediate containing associated clathrin coats, which could be involved in long-range delivery of biosynthetic cargo to peripheral cellular locations.