Autophagy is a tightly regulated membrane rearrangement process that ensures lysosome-dependent bulk degradation of cytosolic proteins or organelles, and is highly conserved in eukaryotic cells, as seen with the endocytic pathway1
. In response to environmental stresses, portions of cytoplasmic constituents are engulfed by a unique membrane structure, the phagophore, as it elongates to form a double- or multiple-membrane-bound compartment called the autophagosome. Newly synthesized autophagosomes then undergo extensive remodelling to acquire degradative capabilities. The remodelling process, also known as autophagosomal maturation, involves sequential fusion of autophagosomes with endocytic vesicles (early and late endosomes) and lysosomes, producing degradative autolysosomes. The sequestered material is then degraded into building blocks for synthesis of macromolecules and energy production1-3
Although the autophagic pathway has been studied extensively, little is known about the molecular mechanism underlying the conversion of autophagosome to degradative autolysosome2
. It is generally thought that autophagosomal maturation probably has similar features as the progression of endosomes to lysosomes, a complex process that involves a number of vesicle-trafficking components4-9
. In fact, inhibition of lysosomal fusion by depletion of lysosomal membrane proteins or lysosomal enzymes inhibits both autophagic and endocytic degradation10-12
. Furthermore, morphologic convergence between autophagic and endocytic pathways has been frequently observed at the ‘early and late endosomes’ node, as well as lysosomes in the cell13-15
. Thus, autophagy, particularly the late stages of autophagic maturation, may interconnect with the endocytic pathway by sharing similar machinery for the concomitant lysosome fusion process.
A prerequisite for vesicle fusion is vesicle tethering. The tethering events at the yeast endosome/vacuole (equivalent to mammalian lysosomes) have been thoroughly studied and shown to require the class C vacuolar protein sorting (Vps) complex16-19
. The core class C Vps complex (hereafter referred to as C-Vps), including Vps11, Vps16, Vps18 and Vps33 exists into two configurations: the HOPS complex (for homotypic vacuole fusion and protein sorting), which contains two additional subunits (Vps39/Vam6 and Vps41), acts at the vacuole20,21
, whereas the class C core vacuole/endosome tethering (CORVET) complex has Vps3 (human Vps39 (hVps39) homologue) and Vps8 (Vps41 homologue) instead, and functions at the endosome17
. Both complexes interact with Ypt–Rab GTPase and are thought to couple Rab activation and SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment receptors) assembly during fusion17,22
. Although the specific function of each individual subunit remains to be established, Vps39 has been shown to confer GTPase exchange factor (GEF) activity to Ypt7p, the yeast Rab7 orthologue. In mammalian cells, hVps39 regulates the recruitment/activation of Rab7 onto the Rab5-labelled early endosomes, a process called Rab conversion23,24
. Although the role of C-Vps in mammals is unclear, mutations in the Drosophila melanogaster
homologues of C-Vps components induce defects in lysosomal protein transport as well as autophagosome maturation25,26
, suggesting that the role of C-Vps may be conserved in multicellular organisms.
Previously we reported the identification of a UV-irradiation-resistance-associated gene (UVRAG) as a Beclin1-interacting protein27
. UVRAG association with Beclin1 enhances phophatidylinositol-3-OH kinase class III (PI(3)KC3) activity and induces autophagosome formation27
. In our continuing effort to characterize the functions of UVRAG, we report here that UVRAG interacts with the C-Vps tethering complex. Independently of Beclin1, UVRAG interaction with C-Vps stimulates autophagosome maturation and endosomal fusion, thereby enhancing both autophagic and endocytic protein degradation. Thus, our study provides a mechanism for regulation of UVRAG-mediated autophagy, and it extends the function of UVRAG from an autophagy effector to an important factor in multiple membrane-trafficking events.