The term ERC refers to a set of endocytic membranes, essentially tubules, that usually concentrate in the perinuclear region of the cell and in which membrane components following the endocytic recycling pathway travel. Few proteins were shown to localize to the ERC (Prekeris et al., 1998
; Lin et al., 2001
; Lindsay et al., 2002
; Lindsay and McCaffrey, 2002
; Meyers and Prekeris, 2002
; Wallace et al., 2002a
). However, how these different proteins intervene in ERC function is largely unknown (Maxfield and McGraw, 2004
In the present study, we have identified a novel protein, Rffl, that, when overexpressed in HeLa cells, localized to and induced the condensation of endosomal structures displaying TfR, Rab5, and Rab11 markers into several perinuclear globular structures. In contrast, the localization of EEA1, another marker of the early endocytic endosomes was not altered by Rffl overexpression. Finally, lysosomal markers, like Lamp2, or markers of secretory organelles, including the TGN, were not affected by Rffl overexpression. Consistently with confocal microscopy data, ultrastructural analysis showed that Rffl was mainly located on densely packed tubulovesicular membrane profiles. Altogether, these data suggest that the compartment affected by Rffl overexpression is the ERC.
Furthermore, recycling of TfR was inhibited by Rffl overexpression. Indeed, the aggregated recycling endosomes were accessible to endocytosed TfR-Tf complexes. However, recycling from the Rffl-positive compartment was delayed, Tf being still detected in the perinuclear region of transfected cells after several hours of chase. Recycling of membrane proteins can occur through at least two different routes that appear to depend on different molecular machineries (Hao and Maxfield, 2000
; Sheff et al., 2002
). Rffl is involved in the recycling of only part of endocytosed TfR, suggesting that it affects the dynamics of a subdomain of recycling endosomes. The cumulative inhibitory effects of Rffl overexpression and of LY294002 treatment on Tf recycling suggests that Rffl acts mainly on a PtdIns(3)-kinase independent recycling pathway. This is consistent with our observations that Rffl affects the ERC and the proposal that, in HeLa cells, PtdIns(3)-kinase activity is required for recycling directly from early endosomes, a recycling route that bypasses the ERC (van Dam et al., 2002
We observed a major redistribution of Rab5 immunostaining upon Rffl overexpression. In normal cells, Rab5 overlaps extensively with EEA1 (Simonsen et al., 1998
; Lawe et al., 2000
), whereas it shows little colocalization with Rab11 (Sonnichsen et al., 2000
). In transfected cells, both Rab5 and Rab11 but not EEA1 concentrate in the aggregated recycling endosomes. This is a striking observation because EEA1 is a Rab5 effector (Simonsen et al., 1998
) and perturbations of the endocytic pathway that affect Rab5 without affecting EEA1 have not been reported so far. The way in which Rab5-positive endocytic membranes are disturbed by Rffl overexpression is unknown. However, it is unlikely to rely on direct physical interactions between Rab5 and Rffl, because the two proteins failed to interact in a yeast two-hybrid assay (F.Coumailleau and M.Cohen-Tannoudji, unpublished observations).
Concerning late endosomes, we noticed that some CD63-positive vesicles showed a tendency to be distributed around the aggregated Rffl-GFP–positive recycling endosomes. These surrounding endosomes appeared at the electron microscopy level as vacuoles with a limited amount of internal CD63-containing membranes and may correspond to early multivesicular bodies (MVB). We observed some Rffl staining on the limiting membrane of MVB-like endosomes, suggesting that Rffl overexpression might also perturb this compartment but to a much lesser extent than ERC.
Rffl is a previously uncharacterized protein that contains a zinc finger domain at both ends. The amino-terminal domain is similar to the well-characterized PtdIns(3)P-binding FYVE finger but presents noticeable departures from the consensus sequence in particular at the level of the core R(R/K)HHCR sequence, which is the principal site of interaction with PtdIns(3)P (Dumas et al., 2001
; Stenmark et al., 2002
). For this reason, we named it FYVE-like domain. Besides sequence similarities, the FYVE-like domain of Rffl shares other similarities with bona fide FYVE domains. Indeed, we have shown that it was necessary for the recruitment of Rffl to endocytic membranes. In addition, it is sufficient, if duplicated, to target GFP to endosomes. This is reminiscent of what has been described for the FYVE domains of EEA1 and Hrs (Stenmark et al., 1996
; Gillooly et al., 2000
; Lawe et al., 2000
). In these cases, the fact that isolated FYVE domains were not targeted to endosomes has been interpreted as due to a weak affinity for PtdIns(3)P that could be compensated by duplication. Similarly, the FYVE-like domain of Rffl may mediate weak interactions with components of recycling endocytic membranes, the nature and the identity of which remain to be identified. Importantly, major differences exist that distinguish the Rffl FYVE-like domain from bona fide FYVE domain. First, Rffl was found on membranes with characteristic of recycling endosomes and did not overlap with EEA1-positive early endosomes membranes. On the contrary, bona fide FYVE domain-containing proteins localize to PtdIns(3)P-containing membranes, i.e., early endosomes and internal vesicles of the MVB (Gillooly et al., 2000
). These proteins play important roles in endocytic membrane trafficking such as early endosome fusion (EEA1; Simonsen et al., 1998
), traffic from early endosome to recycling endosomes (Rabip4 and Rabenosyn-5; Nielsen et al., 2000
; Cormont et al., 2001
; de Renzis et al., 2002
), or from early endosome to late endosomes (Hrs and PikFYVE; Komada and Soriano, 1999
; Ikonomov et al., 2001
; Raiborg et al., 2002
) but are not involved in traffic from ERC to plasma membrane. A second important difference is that endosomal localization of full-length Rffl or DFL-GFP is independent of PtdIns(3)-kinase activity, whereas that of bona fide FYVE domain-containing proteins is not. This indicates that, as already suggested by the amino-acid sequence, Rffl FYVE-like domain does not bind to PtdIns(3)P. It is therefore not surprising that Rffl does not localize to PtdIns(3)P-enriched membranes. Of special interest will be the characterization of the natural ligand of the FYVE-like domain.
The second zinc finger domain of Rffl is a carboxy-terminal RING finger motif. Within the past few years, evidence has accumulated arguing in favor of a general role in ubiquitination for RING finger-containing proteins (Freemont, 2000
; Weissman, 2001
). Recently, it has been shown that SAKURA, the rat ortholog of Rffl, exhibited E3 ubiquitin ligase activity in vitro and ex vivo (Araki et al., 2003
). However, the RING domain of Rffl does not appear to participate in the recycling inhibition mediated by Rffl overexpression because similar perturbations of recycling endosomes were obtained upon transfection of full-length and RING finger-deleted version of Rffl (Rffl-ΔCter).
Exactly how Rffl could affect the morphology and the function of the ERC is unclear. However, it should be noted that inhibition of Tf recycling was obtained by overexpressing the full-length protein. This is in contrast with other proposed ERC regulatory proteins for which recycling was affected upon overexpression of constitutively active or inactive forms (Rab4, Rab11) or dominant negative mutated or truncated proteins (RCP, Rme1, Rab11BP/Rabphillin11), whereas the overexpression of full-length proteins had little or no effect on the Tf recycling pathway (van der Sluijs et al., 1992
; Ullrich et al., 1996
; Zeng et al., 1999
; Wilcke et al., 2000
; Lin et al., 2001
; McCaffrey et al., 2001
; Lindsay et al., 2002
). Therefore, the phenotype observed in cells overexpressing Rffl may be due to enhancement of the normal functions of Rffl, resulting in excess Rffl activity. Underlying this hypothesis is the assumption that in untransfected cells Rffl is a limiting component. When the FYVE-like domain of Rffl was removed, the protein remained cytosolic and inhibition of Tf recycling was no longer observed. This suggests that association of Rffl with ERC membranes is necessary for its interference with recycling. Interestingly, we found that DFL-GFP has an inhibitory effect on recycling. This could be the consequence of the occupancy of FYVE-like domain binding sites on ERC membranes by DFL-GFP and the consecutive displacement of endogenous Rffl or other proteins targeted to ERC membranes through similar mechanism.
In conclusion, we have characterized a new protein that, when overexpressed, alters both the morphology and the function of the ERC. Further studies on how Rffl overexpression inhibits recycling from ERC to the plasma membrane will certainly help to understand the molecular mechanisms underlying membranes proteins trafficking along this poorly characterized compartment.