We have carried out biochemical and genetic characterization of yeast AP complexes. Of the 13 potential AP subunits identified in the yeast genome, four have been previously assigned to AP-3 (Cowles et al., 1997a
; Panek et al., 1997
). The results reported here indicate that eight of the remaining subunits make up two distinct AP complexes, AP-1 and AP-2R. The extra medium subunit can associate with β1 when overexpressed, raising the possibility of an alternative form of AP-1. These findings argue that yeast express three principal AP complexes. Only β1-containing complexes exhibit physical and genetic interactions with clathrin, yet elimination of all four subunits of the major AP-1 form does not affect growth, clathrin-dependent maturation of α-factor precursor, or assembly of clathrin coats. Cells lacking all three β subunits were subjected to a wide survey of protein trafficking pathways. Except for anticipated defects in AP-3-dependent transport to the vacuole, mutant cells sustained normal levels of pheromone receptor endocytosis, α-factor maturation, vacuolar protein sorting, and clathrin-coated vesicles. We conclude that AP complexes are not obligatory for clathrin-coated vesicle formation and clathrin-mediated protein sorting events in yeast.
Sequence comparisons between yeast and mammalian AP complex subunits indicate that these proteins have been conserved during evolution (Cowles et al., 1997a
; Panek et al., 1997
). In view of this conservation, as high as 50% amino acid identity, it is surprising that subunit deletions cause trafficking defects solely in the case of AP-3. In earlier studies, which involved single or double subunit deletions, the innocuous consequences of AP-1 and AP-2R mutations could theoretically be attributed to activity of incomplete AP complexes (Phan et al., 1994
; Rad et al., 1995
; Stepp et al., 1995
). Consistent with this possibility, comparisons of synthetic interactions between single AP-1 subunit deletions and chc1-ts
indicate that the extent of α-factor maturation defects varies depending on the subunit that is eliminated (Figure ). We therefore sought to inactivate AP-1 completely by generating a strain lacking the four AP-1 subunits (β1, γ, μ1, and ς1). No defects were detected in this strain. Because Apm2p can associate with β1, at least when overexpressed, a residual contribution of this subunit to clathrin-dependent processes in the ap1-null
strain might be envisioned. However, even when Apm2p is overexpressed, it cannot functionally replace μ1 in apm1Δ chc1-ts
cells (Stepp et al., 1995
). Nor does deletion of APM2
accentuate defects in apm1Δ chc1-ts
cells (Stepp et al., 1995
). These observations, combined with the absence of other AP-1 subunits in the ap1-null
strain, makes it improbable that Apm2p substitutes in any significant way for AP-1. Given the likelyhood that deletion of β1, γ, μ1, and ς1 abolishes AP-1 activity, the lack of phenotypes in the ap1-null
strain indicates that AP-1 is not necessary for normal clathrin function.
The relationship of Apm2p to AP-1 remains to be established. A requirement for overexpression to detect reproducible association of Apm2p with β1 suggests that either the Apm2p-containing AP-1-like complex is much less abundant than AP-1, or Apm2p does not normally associate with β1. Two-hybrid interactions between Apm2p and Apl4p(γ) have been observed (Huang and Lemmon, personal communication), favoring the idea that Apm2p is part of an AP-1-like complex. However, the absence of phenotypes associated with disruption of APM2
in either wild-type, chc1-ts
, or AP-1 subunit deletion strains (Stepp et al., 1995
) leaves the significance of these associations uncertain.
Functional redundancy between AP complexes could obscure a role for AP-1 in clathrin-dependent transport steps in cells expressing wild-type clathrin. However, our studies provide both biochemical and genetic evidence against this idea. In vitro binding assays with GST fusions to the three β subunits showed clathrin binding only to β1. Additionally clathrin was coimmunoprecipitated with AP-1 but not AP-2R or AP-3. These findings suggest that of the three AP complexes, only AP-1 is capable of associating with clathrin. As a genetic test for functional substitution of AP-1 by AP-2R and/or AP-3, genes encoding all three β subunits were deleted. We selected β subunits as targets to disrupt AP function based on our analysis of synthetic interactions between AP-1 subunit deletions and chc1-ts
, which demonstrate that deletion of β1 is equivalent to deletion of all four AP-1 subunits. In agreement with the importance of β subunits in AP function, mutation of the AP-3 β subunit is effective in blocking the AP-3 pathway (Cowles et al., 1997a
; Stepp et al., 1997
). However, despite disruption of all three β subunits, we were unable to detect defects in clathrin-dependent trafficking pathways. The concordance of results from both biochemical and genetic approaches prompts us to discount the idea of redundant function between the three AP complexes.
Could there be another, uncharacterized AP complex capable of substituting for AP-1? Analysis of the yeast genome sequence suggests that this possibility is remote. When mammalian or yeast AP subunits are used to search the yeast genome sequence, the most highly related sequences constitute the known set of 13 AP subunits (Cowles et al., 1997a
; Panek et al., 1997
). Beyond this group, sequence matches are of limited length and marginal statistical significance. Thus, the 13 AP-related proteins probably represent the complete contingent of AP subunits in yeast.
The prevailing paradigm for clathrin coat formation, established primarily through studies of mammalian clathrin, assigns key roles for AP complexes in assembly of the clathrin lattice at appropriate membranes and in cargo collection (Schmid, 1997
; Hirst and Robinson, 1998
). In contrast to our results, AP subunit mutations in filamentous fungi, nematodes, fruit flies, and mice have readily discernable phenotypes, supporting the central importance of AP complexes in clathrin-mediated protein transport in these organisms (Lee et al., 1994
; Keon et al., 1995
; González-Gaitán and Jäckle, 1997
; Zizioli et al., 1999
). If AP complexes are unnecessary for clathrin function in yeast, then it is likely that other factors subserve clathrin assembly and cargo selection functions. Among the expanding list of proteins associated with clathrin coats, there are a number of candidates that could provide appropriate activities. Mammalian neuronal AP180 binds clathrin and stimulates lattice assembly in vitro (McMahon, 1999
). Two recently identified yeast homologues of AP180 also interact with clathrin and could be assembly factors (Wendland and Emr, 1998
). However, deletion of both yeast AP180-encoding genes together with apl2
) has no deleterious effects on growth, pheromone receptor endocytosis, or α-factor maturation (Yeung, Payne, and Wendland, unpublished results). Similar results have been obtained in analyses of cells lacking the six AP large subunits and the two yeast AP180s (Huang et al., 1999
). Other newly discovered clathrin-interacting proteins such as Epsin (Chen et al., 1998
) and its yeast homologues Ent1p and Ent2p (Wendland et al., 1999
) interact with clathrin and may promote coat assembly. Further genetic analysis of these proteins will be needed to assess their role in clathrin coat assembly. Precedents for cargo collection by proteins other than AP complexes have been established through studies of mammalian cells. In the case of β-adrenergic receptor endocytosis, nonvisual arrestins bind both the receptor and clathrin heavy chain, thereby functioning as adaptors to direct receptors into clathrin-coated vesicles (Goodman et al., 1996
). Although a clear homologue of arrestin has not been identified in yeast, analogous adaptors could exist. Alternatively, there may not be a need for a unique adaptor protein. For example, a peptide containing the endocytosis targeting signal from the low-density lipoprotein receptor interacts with the N-terminal globular domain of clathrin heavy chain (Kibbey et al., 1998
), suggesting that clathrin might act directly to collect certain cargo. These examples suggest a diversification of clathrin assembly and cargo collection activities even in mammalian cells, where the importance of AP complexes is well established. Perhaps under the optimal growth conditions used in laboratory experiments, alternatives to AP-1 assume a more significant role in clathrin-mediated transport in yeast.
Our results clarify structural and functional distinctions between yeast AP complexes and offer additional insights into the relationship between yeast and mammalian APs. Previously, the synthetic growth and α-factor maturation defects caused by combination of chc1-ts
with AP-1 subunit deletions were interpreted as evidence for AP-1 association with clathrin (Phan et al., 1994
; Rad et al., 1995
; Stepp et al., 1995
). The specific physical interaction of clathrin with AP-1 and β1 in vitro now provides more direct evidence that AP-1 is a clathrin-associated complex. Thus, yeast AP-1 mimics mammalian AP-1 in both the primary sequence of subunits and physical interaction of the β subunit with clathrin. Although the consequences of subunit deletion appear to be substantially more severe in animal cells (Zizioli et al., 1999
), the genetic and physical interactions between yeast AP-1 and clathrin suggest that the similarity between mammalian and yeast AP-1 extends to a functional level. As proposed above, the more subtle functional contribution of yeast AP-1 may be attributable to the artificial nature of laboratory growth conditions. In contrast to AP-1, yeast AP-3 does not bind clathrin in our assays. This finding is consistent with genetic experiments indicating that AP-3 acts in a clathrin-independent pathway for membrane protein transport from the Golgi apparatus to vacuoles (Vowels and Payne, 1998a
). Although mammalian AP-3 resembles its yeast cognate by acting in membrane protein sorting to lysosomes, the relationship to clathrin is less clear. Mammalian β3 interacts with clathrin in vitro, and AP-3 can be colocalized with clathrin coats in vivo (Dell’Angelica et al., 1998
). However, AP-3 does not copurify with clathrin-coated vesicles (Simpson et al., 1996
). Resolution of these apparent discrepancies should establish the extent of similarity between yeast and mammalian AP-3 complexes. The third yeast AP complex that we defined, AP-2R, displays the highest primary sequence similarity to mammalian AP-2. However, in other ways AP-2R is clearly distinct from AP-2. Unlike mammalian AP-2, which shares a highly similar clathrin-binding β subunit with AP-1 (84% identity; Kirchhausen et al., 1989
), AP-2R contains a β subunit that is only 24% identical to yeast β1 and does not appear to bind to clathrin. Furthermore, mammalian AP-2 associates with endocytic clathrin-coated vesicles, whereas a role for AP-2R in endocytosis has not been detected, nor have we observed synthetic interactions between AP-2R subunit deletions and chc1-ts
. Identification of a role for yeast AP-2R awaits additional experiments.
In summary, the first comprehensive description of AP complexes in a single organism is now emerging from studies of S. cerevisiae. Three major, functionally distinct complexes have been described: AP-1, and perhaps an alternative form with a different medium subunit, acts in a clathrin-dependent protein sorting pathway from the TGN; AP-2R probably acts in a clathrin-independent pathway, but the identity of this pathway has not been uncovered; and AP-3 acts in clathrin-independent traffic of membrane proteins from the Golgi apparatus to vacuoles. Elimination of AP function results in AP-3 pathway defects but otherwise appears to be insignificant for clathrin-dependent events. Our results imply the existence of factors other than AP complexes, which play central roles in clathrin coat assembly and cargo selection.