Rubicon controls endosome maturation as a Rab7 effector

Abstract

The activation and recruitment of the small GTPase Rab7 to early endosome is a critical step for early to late endosome maturation, a process that requires the class III phosphatidylinositol 3-kinase (PI3KC3) and GTPase regulators. However, the molecular mechanism underlying Rab7 activation and endosome maturation is still poorly defined. Here we report that Rubicon, a component of the PI3KC3 complex, prevents endosome maturation through differential interactions with Rab7 and UVRAG. UVRAG activates PI3KC3 and C-VPS/HOPS, a guanine nucleotide exchange factor that catalyzes the exchange of GDP for GTP on Rab7. We demonstrate that Rubicon sequesters UVRAG from C-VPS/HOPS. Active GTP-bound Rab7 competes for Rubicon binding and releases UVRAG to associate with C-VPS/HOPS, which in turn promotes further loading of Rab7 with GTP. This feed-forward loop ensures rapid amplification of GTP-bound Rab7 and consequent stimulation of endosome maturation. Hence, Rubicon serves as a previously unknown Rab7 effector to ensure the proper progression of the endocytic pathway.autophagy | endocytosis | epidermal growth factor | autophagosome | Barkor/Atg14(L)

Autophagy and human diseases

“…Both spastizin-mutated fibroblast cells derived from HSP patients and spastizin knockdown cells showed impaired autophagic flux and accumulation of autophagosomes due to reduced autophagosome-lysosome fusion [44]. However, as this complex also plays an important role in the endocytic pathway [46][47][48][49], and as spastizin is not present on the autophagic membranes [44], whether spastizin specifically regulates autophagosome-lysosome fusion needs to be clarified.…”

The role of membrane-trafficking small GTPases in the regulation of autophagy

“…Rubicon has been shown to inhibit the maturation and fusion steps during autophagy (Matsunaga et al, 2009;Zhong et al, 2009). Interestingly, rubicon appears to sequester UVRAG from the C-Vps complex and block Rab7 activation (Sun et al, 2010), suggesting that it terminates Rab7-dependent maturation of autophagosomes by modulating UVRAG in the C-Vps complex (Tabata et al, 2010) (Fig. 3A).…”

Mule determines the apoptotic response to HDAC inhibitors by targeted ubiquitination and destruction of HDAC2

“…The protocol for producing recombinant proteins from insect cells was the same as the tandem affinity purification (TAP) procedure described before (Sun et al 2010a). For recombinant proteins purified from mammalian cells, the method was adopted from the previously mentioned TAP protocol as follows: 293T cells were cultured in DMEM supplemented with 10% FBS.…”

“…pCDNA5/FRT/TO and pCDNA4/TO (Invitrogen) were modified as previously described (Sun et al 2008(Sun et al , 2010a. The Mule coding sequence was inserted between BamHI and NotI sites to generate the reading frame of Mule-Flag, which was used for establishing stable cell lines.…”