There are many membrane traffic pathways for which no coats have yet been identified, and this is what prompted us initially to look for adaptor-related proteins that might be components of novel types of coats. In a previous report we showed that two recently described proteins, p47 (μ3) and β-NAP (β3B), are associated with each other in an adaptor-like complex (Simpson et al., 1996
). We have now identified the remaining subunits of the complex and have further investigated its function. We have named the complex AP-3, by analogy with AP-1 and AP-2.
The identification of the missing subunits of the AP-3 complex relied upon the availability of random cDNA sequences in the EST database. Based on the degree of homology between μ3 and μ1/μ2 and between β3 and β1/β2 we predicted that the δ and σ3 subunits would be relatively distant homologues of α/γ and of σ1/σ2, respectively. Because the complex is fairly abundant, it was easy to find entries in the database for both types of subunits; indeed, a recent search revealed over 50 mammalian ESTs for the δ subunit alone. Interestingly, there are now additional homologues of the β, μ, and σ subunits in the database, suggesting that there is likely to be at least one more type of AP complex.
Both the μ and the β subunit have neuronal-specific as well as ubiquitously expressed isoforms; however, no neuronal-specific isoforms have been found for either of the other two subunits, δ and σ. Although we cannot rule out the possibility that such isoforms may exist, candidates for them do not appear to be present in the EST database, in spite of the fact that many of the entries are from brain and that both μ3B and β3B can be found as multiple “hits.” Why are there neuronal-specific isoforms of two of the subunits? Is μ3B always associated with β3B and μ3A with β3A, or can the subunits “mix and match”? We do not yet know the answers to either of these questions, but studies on the μ and β subunits of the AP-1 and AP-2 complexes may provide some clues. μ1 and μ2 have been shown to bind to tyrosine-based sorting signals (Ohno et al., 1995
), and recently μ3 has also been shown to bind such sequences (Dell Angelica et al., 1997). Possibly μ3A and μ3B recognize different repertoires of signals. β1 and β2 have been shown to bind to clathrin, an activity that involves the hinge domain of the protein (Gallusser and Kirchhausen, 1993
; Shih et al., 1995
). The AP-3 complex is not clathrin associated (Simpson et al., 1996
) but may interact with another type of scaffolding protein, and such an interaction would be likely to involve the β3 subunit, with the two different β3 isoforms possibly interacting with different types of scaffolds. Alternatively, it may be that only one of the two proteins has a neuronal-specific role. The μ and β subunits of the AP-1 and AP-2 complexes show a strong interactaction with each other (Page and Robinson, 1995
; Seaman et al., 1996
), and it is possible that neuronal-specific isoforms of μ3 and β3 may have coevolved because there is an obligatory coupling between them.
In our previous study we were able to localize the neuronal-specific complex using antibodies against β3B (Simpson et al., 1996
). The present study confirms and extends our earlier findings by localizing the endogenous AP-3 complex in nonneuronal cells. We have shown that the complex is brefeldin A–sensitive in vivo as well as in vitro and have confirmed that it is associated with both perinuclear and more peripheral membranes. What do these peripheral membranes correspond to? There is only very limited colocalization of either δ or β3B with endosomal markers, suggesting that the complex is more likely to be involved in a biosynthetic pathway than in an endocytic one. Our earlier study indicated that some of the complex is associated with the TGN (Simpson et al., 1996
), and the more peripheral labeling may represent a post-TGN compartment. This compartment may also be able to receive endocytosed proteins without being a conventional type of endosome. The presence of a μ subunit in the complex indicates that it plays a role in the sorting of proteins containing tyrosine-based signals (Ohno et al., 1995
; Dell'Angelica et al., 1997
). Such signals have been shown to function in the post-Golgi biosynthetic pathway as well as in the endocytic pathway and may be used to send proteins to any one of several destinations: the (basolateral) plasma membrane, endosomes, lysosomes, or back to the TGN (Humphrey et al., 1993
; Matter and Mellman, 1994
). Which of these pathways might the complex mediate?
An important clue is provided by the finding that the δ subunit is the mammalian homologue of the Drosophila garnet
gene product. The garnet
mutant alleles that have been described only appear to affect pigmentation, yet the gene is expressed ubiquitously, not just in pigment cells. In addition, although a number of alleles have been identified, so far none of those tested by Northern blotting have been found to be nulls; thus, the protein may be essential (Lloyd, V., personal communication). Of the two alleles shown in Fig. , g53d
is tissue specific, suggesting that the mutation is in the 5′ upstream regulatory region, while g3
contains an insertion within the coding portion of the gene (Lloyd, V., personal communication). Thus, the g3
mutation is likely to affect all the cells in the flies' bodies, yet the animals are viable and in particular do not appear to have any neurological problems. This is in contrast to flies with conditional mutations in two other genes encoding membrane traffic proteins, dynamin and NSF, where incubation at a nonpermissive temperature causes a block in neurotransmission (Chen et al., 1991
; Van der Bliek and Meyerowitz, 1991; Pallanck et al., 1995
). Although more information is needed about the activity of the mutant protein, the g3
phenotype appears to be inconsistent with a role previously proposed for both the β3 and μ3 subunits in synaptic vesicle biogenesis (Pevsner et al., 1994
; Newman et al., 1995
The most likely explanation for the garnet
phenotype is a defect in the delivery of proteins to pigment granules. The pigments themselves are small molecules, but the granules must also contain a specific set of proteins for synthesizing, transporting, and/or storing the pigments, and it is possible that the sorting of these proteins may be impaired in the garnet
flies. But what role might the complex play in nonpigment cells? In mammalian cells, pigment granules have been shown to be like modified lysosomes. Thus, patients with Chediak Higashi syndrome, or mice with the beige
mutation, have not only giant lysosomes but also giant melanosomes (Burkhardt et al., 1993
). Less is known about the formation of pigment granules in flies, but it seems likely that they too are lysosome-like in origin. This possibility is supported by the discovery that VPS18
, a yeast gene involved in the sorting of proteins to the lysosome-like vacuole (Robinson et al., 1991
), is homologous to another fly eye color gene, deep orange
(Reider, S., and S. Emr, personal communication).
Taken together, these observations suggest that the AP-3 complex may play a role in trafficking from the TGN to the lysosome. However, a coat already exists for this pathway: the AP-1 and clathrin-containing coat. One possibility is that both coats are involved in this pathway but that they act at different stages or sort different types of cargo molecules. Alternatively, the role of the AP-3 coat in lysosomal biogenesis may be less direct. For instance, it may be involved in a different pathway, such as trafficking to the plasma membrane or recycling back to the TGN, but defective sorting may result in a certain amount of “scrambling” of proteins and interfere with other pathways as well. Specialized organelles such as pigment granules may be particularly susceptible to such missorting and may also be especially sensitive to problems at earlier stages of the secretory pathway (e.g., ER to Golgi), as has been shown for the specialized secretory granule-like trichocysts of Paramecium
(Gautier et al., 1994
). There are numerous Drosophila
eye color genes, many of which have not yet been cloned, and it seems likely that some of these other genes will also be found to encode proteins involved in membrane traffic.
Although the garnet
phenotype provides important information about the role of the AP-3 complex, further studies will be required to establish this role definitively. One promising lead comes from the observation that another genetically tractable organism, Sarcharomyces cerevisiae
, contains genes encoding homologues of all four types of AP subunits, including some (e.g., SCYPL195W [δ] and SCYJL024C [σ3]) whose protein products are much more closely related to components of the AP-3 complex than to components of either the AP-1 or the AP-2 complexes. Targeted gene disruptions may indicate whether the complex is involved in trafficking to the vacuole, to the plasma membrane, or in some other pathway. Microinjection and/or immunodepletion experiments, using the antibodies we have generated that recognize the nonneuronal complex in its native form, may also help to define the role of the complex. AP-3 is not the only novel coat component that has recently been identified. Database searches indicate that there is at least one other AP-type complex, and there may be more. Monoclonal antibodies have identified p200 as yet another possible coat component (Narula and Stow, 1995
), and electron microscopy has revealed the existence of lace-like coats associated with the TGN (Ladinsky et al., 1994
). Thus, there are pathways in the cell that require coats and coats that require pathways, and the next step will be to fit the two together.