Our results suggest that the sorting nexin Grd19/Snx3p functions as a cargo-specific accessory component of the retromer complex, which is required for endocytic recycling of the Fet3p–Ftr1p iron transporter. In support of this model, we report that in null mutants of grd19 and each of the five retromer subunits, Fet3p–Ftr1p is missorted to the vacuole when cells are grown under conditions that favor recycling, that Grd19p directly binds a sequence in Ftr1p required for endocytic recycling, that Grd19p-GFP and Vps17p-RFP colocalize substantially on tubular endosomes, and that Grd19p can be chemically cross-linked to the retromer complex in vivo. Although the full range of functions and the specific mechanisms by which retromer operates in membrane trafficking have not been elucidated, it is clear that it is a general endosomal sorting factor required for the proper sorting and export of a diverse set of cargo molecules from endosomes. The work presented in this study is relevant for understanding how cargo is identified by retromer, and the results suggest that that the repertoire of retromer-dependent cargos is extended by its interaction with Grd19p. We speculate that Grd19p functions with retromer in a manner analogous to vesicle coat protein adapters that link cargo selection to coat protein recruitment.
Recycling of Fet3p–Ftr1p is likely to be initiated through recognition of Ftr1p by Grd19p because the PX domain of Grd19p binds PtdIns(3)P with the highest affinity of all the PX domains encoded in the yeast genome (Yu and Lemmon, 2001
). In contrast, the PX domains of the retromer subunits Vps5p and Vps17p bind PtdIns(3)P with at least 100-fold lower affinity than Grd19p (Yu and Lemmon, 2001
). Preliminary analysis of the binding interaction between Grd19p and the Ftr1p recycling signal by surface plasmon resonance indicates that the affinity is relatively weak (Kd
between 10 and 100 μM; unpublished data), which is consistent with PtdIns(3)P binding providing the driving force for recruitment of Grd19p to endosomes. Because the abundances of Grd19p and Vps17p appear to be similar (), Grd19p is expected to preferentially accumulate on early endosomes containing relatively low amounts of PtdIns(3)P. Retromer will subsequently load onto endosomes as they accrue higher levels of PtdIns(3)P during maturation, facilitated by interactions with other factors, such as Grd19p and cargo molecules. In this manner, Grd19p could serve as both a coincidence sensor that detects the presence of Fet3p–Ftr1p on PtdIns(3)P-containing endosomes and as an adaptor that recruits retromer to cargo to initiate export from the endosome. Grd19p and retromer appear to be sufficient for export from the endosome because we have not observed any recycling defects in other known cargo-sorting factors, including the clathrin adaptor AP-1 complex, other sorting nexins, or the recently identified GSE–EGO complex involved in endosome-to-plasma membrane sorting of the general amino acid permease Gap1p (Gao and Kaiser, 2006
; Table S1). Once exported from the endosome, Fet3p–Ftr1p is probably delivered to the Golgi for resecretion because deletion of the Golgi Rab GTPase Ypt6p and its regulators also results in defective recycling, and because the Ftr1p C-terminal tail can direct Vps10p from the endosome back to the Golgi.
In yeast, five proteins have been identified that are sorted via the Grd19p–retromer pathway. These include native proteins that cycle between the Golgi and endosomes, Ste13p, Kex2p, and Pep12p (Nothwehr et al., 2000
; Hettema et al., 2003
), and Fet3p–Ftr1p, which uses the Grd19p–retromer pathway to be sorted back to the plasma membrane. The Golgi retrieval signals in Ste13p and Kex2p contain key aromatic residues, although their relevance to Grd19p-mediated sorting is not clear because Grd19p (supplied in a cell extract) still bound well to the cytoplasmic tail of Ste13p, even when the aromatic residue-based retrieval signal had been deleted (Voos and Stevens, 1998
). Consistent with this, mutation of the single aromatic residue within the Ftr1p recycling signal (Phenylalanine323 to Alanine) did not affect recycling (unpublished data), so a more systematic analysis of this signal is required to identify its key features. It is also interesting that vacuolar targeting of the Ftr1p mutant lacking the Grd19p binding site (Ftr1pΔ319-328) was not so robust, perhaps implying that other sorting determinants are present within the C-terminal tail of Ftr1p. Inasmuch as the available data suggest that the sequences of the signals which confer Grd19p- and retromer-dependent trafficking are diverse, Grd19p probably recognizes structural features of cargo proteins rather than a strict linear amino acid sequence.
Importantly, our results showing that Grd19p directly recognizes Ftr1p and Ste13p and links them to retromer establish for the first time how a sorting nexin (other than Snx1) and retromer cooperate to recognize cargo. Although direct interactions between cargo and any subunit of retromer have not yet been confirmed using purified proteins, the current view posits that cargo is recognized by Vps35p, although other sorting nexins, including human Snx1, also have the capacity to bind cytoplasmic regions of some endocytic cargo proteins (Wang et al., 2002
). Moreover, the recent discovery that Vps26 has an arrestin-like structure raises the possibility that Vps26p, like arrestins, may also interact with cargo (Shi et al., 2006
). Another possible function of retromer is suggested by the crystal structure of Vps29, which has a protein fold resembling that of phosphoesterases (Collins et al., 2005
; Wang et al., 2005
), and recent studies have shown that Vps29p exhibits protein phosphatase activity (Damen et al., 2006
). Regardless of the specific functions of the individual retromer subunits, it is clear that multiple cargo recognition mechanisms must contribute to the general function of retromer in endosomal sorting because Grd19p is not required for all retromer-dependent trafficking.
In mammalian cells, the early endosomal system is comprised of vacuolar domains connected to an extensive network of tubules, which are enriched in integral membrane cargo proteins that are subsequently sorted to a variety of different organelles (Bonifacino and Rojas, 2006
). On the basis of the large surface area/volume ratio of tubes compared with spherical structures, it has been proposed that the packaging of integral membrane proteins into tubes is a highly efficient geometry-based mechanism for segregating membrane and lumenal components (Helenius et al., 1983
; Marsh et al., 1986
; Geuze et al., 1987
). Human retromer appears to be involved in this sorting mechanism through its cargo-binding activities and the ability of the BAR domain–containing Snx1 subunit to sense regions of high membrane curvature (Carlton et al., 2004
). The role of this geometric sorting mechanism in yeast is not known due, in large part, to the difficulty in visualizing cargo within domains of yeast endosomes by light microscopy. However, visualization of yeast endosomes by electron microscopy has provided clear evidence that a subset of them is indeed tubular in shape (Prescianotto-Baschong and Riezman, 1998
; Quenneville et al., 2006
). These results support the notion that retromer-mediated sorting in yeast and mammalian cells involves the same mechanisms, and they open the door to evaluating the roles of various retromer proteins and auxiliary factors, such as Grd19p in the biogenesis of these organelles. We expect that the interaction between Grd19p and retromer in yeast holds true for the human orthologues, as this interaction could explain the observation that overexpression of human Snx3 in cultured cells leads to a huge expansion of tubular early endosomal compartments through enhanced recruitment of the BAR domain–containing Snx1 component of retromer (Xu et al., 2001
The results presented in this work demonstrate that recognition of recycling protein cargo by retromer can be initiated by a sorting nexin (Grd19p) that functions as an adaptor to link cargo to the cellular recycling machinery. With the identification of the endocytic recycling machinery and insight into how it mediates recycling of Fet3p–Ftr1p, the opportunity now exists to explore how, in response to changes in extracellular iron concentration, the iron transporter is channeled into either recycling or degradative pathways.