In this study, we have characterized the role of endocytic trafficking in mTORC1 signaling and described how blocking a particular endocytic step, the early/late endosomal transition, affects the amino acid–sensing branch of mTORC1 signaling. When early/late endosome conversion is blocked by either overexpression of Rab5CA or knockdown of hVps39, hybrid endosomes are formed that contain markers for both early and late endosomes. mTOR localizes to these hybrid endosomes under amino acid–replete conditions, but does not signal to downstream effectors. Because overexpression of Rheb, but not hyperactivation of endogenous Rheb, can rescue the inhibition of mTORC1 by Rab5CA, we infer that the localization of mTOR to these hybrid endosomes decreases the interaction of mTOR with endogenous Rheb ().
Figure 5. Model of endosome conversion and mTORC1 signaling. The model shows a normal early-to-late endosome conversion (left) and a mixed early/late endocytic compartment (right). In order for mTOR to be properly activated in the late endocytic compartment in (more ...)
There is strong evidence implicating an evolutionarily conserved role for endocytic trafficking in TOR signaling. Work in D. melanogaster
has linked dTOR to the endocytic system, revealing both general and specific roles for dTOR in endocytosis (Hennig et al., 2006
). In mammalian cells, the Rag GTPases have been implicated in the amino acid input to mTOR signaling (Kim et al., 2008
; Sancak et al., 2008
). In yeast, the Rag homologues Gtr1p and Gtr2p are required for the nutrient-driven movement of amino acid permeases from endosomes to the plasma membrane (Gao and Kaiser, 2006
). Furthermore, a vacuole-associated complex containing Grt2p, Ego1p, and Ego3p are required for reinitiation of proliferation after rapamycin-induced senescence (Dubouloz et al., 2005
). A subsequent study linked the yeast HOPS complex to this same process (Zurita-Martinez et al., 2007
). Binda et al. (2009)
have recently published that in yeast, Vps39 acts as a Rag GEF whose activity contributes to TOR activation. We cannot rule out such a role for mammalian hVps39. However, if hVps39 knockdown led to a loss of Rag activation, then we would have expected to see a disruption of mTOR localization. Instead, we still observe mTOR localization to the hybrid early/late endosomes in hVps39 knockdown cells, but mTOR does not signal to S6K1. Given that Rag is required for mTOR endosomal localization (Sancak et al., 2008
), this would suggest that Rag GTP loading is preserved in hVps39 knockdown cells.
Rab5 is a pivotal player in early/late endosomal conversion. Rab5 cycles between the cytosol and early endosomes, with GDP-bound Rab5 found mostly in the cytosol and GTP-bound Rab5 found predominantly on early endosomal membranes (Stenmark et al., 1994
; Ullrich et al., 1994
). GTP-bound Rab5 recruits numerous effector proteins that serve multiple functions in endocytic trafficking, including EEA1, which is required for homotypic early endosome fusion, and the class C Vps/HOPS complex, which recruits and activates Rab7 (Wurmser et al., 2000
; Rink et al., 2005
). The distinct identity and function of early versus late endosomes is in part defined by whether they contain Rab5 or Rab7 on their membranes. Thus, a key step during early/late endosomal conversion is both the recruitment of Rab7 and the extraction of Rab5 from the early endosomal membrane. Rab5CA apparently blocks early/late endosome conversion through the failure of the constitutively GTP-bound Rab5 to be extracted from the early endosomal membrane (Rink et al., 2005
). Knockdown of hVps39, a member of the HOPS complex and a GEF for Rab7, also leads to a block in early/late endosomal conversion by inhibiting the recruitment and activation of Rab7. Zerial and coworkers (Rink et al., 2005
) speculate that during early to late endosomal conversion, a negative feedback loop takes place, wherein recruitment and GTP-loading of Rab7 by hVps39 leads to decreased Rab5 activation, most likely through a Rab7 effector protein with GAP activity toward Rab5. This deactivation of Rab5 allows the subsequent extraction of Rab5 from endosomal membranes.
Although the precise mechanism by which the early/late endosomal conversion facilitates amino acid–stimulated mTORC1 signaling remains unclear, we suggest that blocking endosomal conversion decreases the ability of Rheb to interact with mTOR. Endogenous Rheb may be localized to late endosomes, whereas overexpression of Rheb leads to its mislocalization to other compartments (Sancak et al., 2008
). This explains why overexpressed Rheb activates mTOR in the absence of amino acids; presumably the Rheb-mTOR interaction is driven by mass action in this case, and no longer requires the amino acid–stimulated movement of mTOR to late endosomes. Our Rheb overexpression data are consistent with this hypothesis, because mTOR signaling in Rab5CA-expressing cells is not rescued by constitutive hyperactivation of endogenous Rheb in TSC2−/− MEFs, but is rescued by overexpression of Rheb. These data suggest that overexpressed Rheb can interact with mTOR in the hybrid endosomes formed in Rab5CA-expressing cells, whereas endogenous Rheb cannot. Unfortunately, attempts to measure the binding of endogenous mTOR and Rheb has been unsuccessful even in control cells, and the localization of endogenous Rheb by fractionation or immunofluorescence has not yet been achieved, as has been described elsewhere (Buerger et al., 2006
; Sancak et al., 2008
). During the preparation of this article, a study describing the localization of GFP-Rheb to the endoplasmic reticulum (ER) and Golgi was published (Hanker et al., 2009
). However, overexpression of Rheb activates mTORC1 signaling even in the absence of amino acid–stimulated mTORC1 translocation (Sancak et al., 2008
), suggesting that the localization of GFP-Rheb may not mimic that of endogenous Rheb.
Our results indicate that some distinct characteristic of late endosomes is vital for mTORC1 signaling, presumably related to the targeting of Rheb. One key distinction between early and late endosomes is their luminal pH, with endosomes maintaining a lower intraluminal pH than early endosomes (Murphy et al., 1984
; Yamashiro and Maxfield, 1987a
). Changes in intraluminal pH can affect the recruitment and activation of membrane associated GTPases. For example, endosomal recruitment of the small GTPase Arf6 and its GEF, ARNO, is blocked by inhibition of the vacuolar ATPase and subsequent increase in intraluminal pH (Maranda et al., 2001
). Similar results have been observed for βCOP and Arf1 binding to early endosomes (Gu and Gruenberg, 2000
). Another distinction between early and late endosomes is their lipid compositions. Late but not early endosomes contain the lipid lysobisphosphatidic acid (LBPA; Matsuo et al., 2004
). LBPA helps regulate late endosomal cholesterol levels through an associated protein Alix (Chevallier et al., 2008
). Late endosomal cholesterol levels are lower than in the plasma membrane or recycling compartment but higher than in the ER (Mesmin and Maxfield, 2009
). GTPase association with distinct membranes depends in part on the lipid composition of the target membrane (ten Klooster and Hordijk, 2007
). Future work will address whether mTORC1 requires the distinct intraluminal pH and lipid composition of late endosomes for productive interactions with Rheb and for signaling to downstream effectors.