In this study we report that Vps27 is mainly found in a phosphorylated form in vivo and that this phosphorylation occurs in the C-terminal domain of the protein. By screening kinases involved in endocytosis for a general defect in the VPS pathway, we identified Pkh1/2 as the kinases phosphorylating Vps27. In addition, we demonstrated that Pkh1/2 phosphorylates Vps27 in vivo and in vitro. We identified the serine 613 as critical for proper phosphorylation of Vps27 in vivo and as the target of Pkh2 phosphorylation in vitro. Moreover, Vps27 phosphorylation is required for MVB sorting of cargo like Cps1 and Ste2, a missorting most likely due to the impaired recruitment of ESCRT-I to endosomes. Indeed in a pkh2 pkh1-ts
mutant, the ESCRT-I subunit Vps28 is partly mislocalized to the cytoplasm. This defect is also observed in cells bearing vps27S613A
as the sole source of Vps27. Thus Vps27 phosphorylation by Pkh1/2 on serine 613 regulates the recruitment of ESCRT-I to endosomes.
In S. cerevisiae
endocytosis is dependent on sphingoid base (Zanolari et al., 2000
). It was shown that Pkh1/2 kinases are activated by sphingoid base and are required for the internalization step of endocytosis (Friant et al., 2001
). The overexpression of these kinases can restore endocytosis in the lcb1-100
mutant deficient in sphingolipid synthesis. Here we show that in addition to its role in the early step of endocytosis, Pkh2 also displays a general defect in the VPS pathway and can directly phosphorylate Vps27 to regulate ESCRT-I recruitment to endosomes. Thus Pkh1/2 kinases also play an important role at a later stage of endocytosis. Consistent with the observation of Marchal et al. (2001)
, we showed that the yck1 yck2-ts
mutant did not display a general VPS pathway defect (). Of interest, it was recently shown that Yck2 is palmitoylated, leading to its anchorage at the plasma membrane (Roth et al., 2011
). This plasma-membrane specific localization of Yck2 might explain why it cannot act directly at later stage of endocytosis. In contrast, we showed that the pkh2 pkh1-ts
mutant displays a strong secretion of vacuolar hydrolases, attesting to a general VPS pathway defect ( and Supplemental Figure S2) and that this defect was not restored by the phosphomimetic vps27S613D
mutant (Supplemental Figure S2 and ), suggesting that Vps27 was not the only Pkh1/2 effector. Furthermore, we showed that ypk1Δ
mutant displays a CPY secretion phenotype (), suggesting that the Pkh1/2 kinases could also act on its downstream effector Ypk1 to regulate the vacuolar sorting of hydrolases. The Pkh1/2 kinases might also act as a protein platform to regulate the trafficking function of some additional effector(s) via protein–protein interactions, as the pkh2K208R
mutant that is considered as kinase inactive did not recapitulate the strong VPS defect displayed by the pkh2 pkh1-ts
Vps27 binds directly to the UEV domain of the ESCRT-I subunit Vps23 and recruits the whole complex to endosomes. It was shown by pull-down experiments with recombinant GST fusion of a truncated version of Vps27 that residues 431–485, including the 447
-1 and 523
-2 motifs of Vps27, were required for interaction with the UEV domain of Vps23 (Bilodeau et al., 2003
). However, Katzmann et al. (2003
) showed that the residues 581–622 and the 581
motif of Vps27 control Vps23 endosomal localization in vivo. Of interest, recent interaction and structural studies confirmed the interaction between the PDSP motifs of Vps27 and an N-terminal motif on the UEV domain of Vps23 (Ren and Hurley, 2011
). However, this interaction motif was not essential for the MVB sorting of Cps1 (Ren and Hurley, 2011
). Here we show that the S613 residue in the C-terminal domain of Vps27 (581–622) is required to trigger the recruitment of the whole ESCRT-I complex to endosomes, as Vps28-GFP was mislocalized in its absence. Moreover, the single mutation of the phosphorylation site S613 is sufficient to alter the recruitment of the ESCRT-I complex by Vps27, and this without altering the capacity of Vps27 to bind to Vps23 (Supplemental Figure S5, B and C). We hypothesize that the function of this phosphorylation is to regulate the recruitment of ESCRT-I, perhaps by facilitating the accessibility to the PSDP motifs. In mammalian cells the Vps27 homologue Hrs is phosphorylated on residue Y334, which is just upstream of the 348
motif required for TSG101 interaction, upon EGF stimulation (Urbe et al., 2000
; Steen et al., 2002
). These observations allow us to speculate that Hrs–TSG101 interaction might also be modulated by phosphorylation. The role of Hrs phosphorylation remains unclear; it was suggested that it triggers relocation of Hrs from cytosol to endosome (Urbe et al., 2000
), but was also described as responsible for Hrs cytosolic relocation and degradation (Stern et al., 2007
). Using subcellular fractionation, we observed no major differences in Vps27 distribution upon mutation of the S613 residue, suggesting that the phosphorylation of this residue does not alter Vps27 endosomal localization (Supplemental Figure S5A). An explanation for the importance of Hrs phosphorylation in ESCRT-I recruitment to endosomes is that phosphorylation of a residue lying next to the P(T/S)AP motifs might trigger a conformational change resulting in better accessibility of this motif to ESCRT-I (TSG101/Vps23) binding.
Several other residues of Vps27 can be phosphorylated (Gruhler et al., 2005
; Smolka et al., 2007
; Albuquerque et al., 2008
). We analyzed the steady-state phosphorylation status of point mutants of these residues (S155,157A; S274A; S279,280A; S495A; and T497A) and found none required for steady-state phosphorylation of Vps27 (Supplemental Figure S1). However, this does not exclude a role in regulating Vps27 cellular functions and thus the MVB pathway in different environmental or stress conditions. Indeed, two of these residues, S157 and S495, in the FYVE and C-terminal region, respectively, are hyperphosphorylated upon α-factor treatment (Gruhler et al., 2005
). It will be interesting to further investigate the role of the phosphorylation of these residues and to identify the kinases involved in these modifications.
In the ubiquitin-binding domain containing protein, ubiquitination is involved in intramolecular inhibition of the ubiquitin binding (Hoeller et al., 2006
). Phosphorylation can regulate ubiquitination of proteins; for example, in the case of the uracil permease Fur4 phosphorylation of the PEST sequence is required for proper ubiquitination and subsequent internalization of the protein (Marchal et al., 2000
). Thus phosphorylation could also regulate the ubiquitination of Vps27 and thus its ability to bind ubiquitinated cargoes. It has been shown that the ESCRT-0 protein STAM (Hse1 in yeast) ubiquitination is increased upon down-regulation of the scaffold leucine-rich repeat kinase LRRK1, and this regulates endosomal trafficking of the EGF receptor (Hanafusa et al., 2011
). Thus a similar mechanism could exist for the regulation of Vps27 ubiquitin binding via its UIM motif. It will be important to further characterize the role of the phosphorylation of the different residues of Vps27.
Phosphorylation of Vps27 at residue S613 is important for the regulation of ESCRT function, and thus the dephosphorylation step must also play an important role in this process. One protein phosphatase candidate is the phosphatase 2A (PP2A). Indeed, Friant et al. (2000)
showed that its loss of activity, as well as Pkc1 overexpression, can suppress sphingoid base requirement for endocytosis. This suggests that PP2A acts in the same pathway as Pkh1/2 to regulate endocytosis. Further investigations should be carried out to decipher the role of this phosphatase in the regulation of MVB function.
In conclusion, we showed that phosphorylation of Vps27 is crucial for endosomal recruitment of the ESCRT machinery and thus required for proper MVB sorting, which gives new insight into the regulation of the ESCRT machinery assembly. Considering the high degree of conservation of the MVB pathway throughout evolution, we propose that this might be a conserved regulatory mechanism of MVB function.