Report ESCRTs and Fab1 Regulate Distinct Steps of Autophagy

Medical Research Council National Institutefor Medical ResearchThe RidgewayMill HillLondon NW7 1AAUnited KingdomSummaryEukaryotes use autophagy to turn over organelles,proteinaggregates,andcytoplasmicconstituents.Theimpairment of autophagy causes developmental de-fects, starvation sensitivity, the accumulation of pro-tein aggregates, neuronal degradation, and cell death[1,2].Double-membranedautophagosomessequestercytoplasm and fuse with endosomes or lysosomes inhigher eukaryotes [3], but the importance of the endo-cytic pathway for autophagy and associated diseaseisnotknown.Here,weshowthatregulatorsofendoso-mal biogenesis and functions play a critical role inautophagy in Drosophila melanogaster. Genetic andultrastructuralanalysisshowedthatsubunitsofendo-somalsortingcomplexrequiredfortransport(ESCRT)-I,-IIand-III,aswellastheirregulatoryATPaseVps4andthe endosomal PtdIns(3)P 5-kinase Fab1, all are re-quired for autophagy. Although the loss of ESCRT orVps4functioncausedtheaccumulationofautophago-somes, probably because of inhibited fusion with theendolysosomal system, Fab1 activity was necessaryfor the maturation of autolysosomes. Importantly, re-duced ESCRT functions aggravated polyglutamine-induced neurotoxicity in a model for Huntington’sdisease. Thus, this study links ESCRT function withautophagy and aggregate-induced neurodegenera-tion, thereby providing a plausible explanation for thefact that ESCRT mutations are involved in inheritedneurodegenerative disease in humans [4].Results and DiscussionAnincreasedunderstandingofthemechanismsthatreg-ulate macroautophagy (hereafter referred to as autoph-agy) is critical because the dysregulation of autophagyis seen in many human pathologies, including cancerand neurodegeneration [2, 5]. Insight into the molecularmechanisms of autophagy stems largely from geneticscreens in yeast, which have identified two ubiquitin-like conjugation machineries necessary for autophago-some formation [1, 6, 7]. Although the requirement forthesemachinerieshasbeenconservedtohighereukary-otes,themolecularmechanismsofautophagictransportto the lysosomes in metazoans are less clear. In yeast,the autophagosome fuses directly with the vacuole(lysosome) in a step requiring the small GTPase, Ypt7/Rab7, the HOPS-C complex, and SNAREs [6, 8, 9].Incontrast, it is well established that fusion structures be-tweenautophagosomesandendosomalcompartments,called amphisomes, are formed in higher eukaryotes [3,10, 11]. This raises the question of whether autophagyin metazoans is controlled by components that regulateendosomebiogenesisandsortingfunctions.Endosomalsorting complexes required for transport (ESCRTs) areinteresting in this context because these complexesmediate multivesicular body (MVB) biogenesis and deg-radativeproteinsortingandbecausetheirdysfunctionisassociated with neurodegenerative disease in humans[4]. Another candidate regulator of autophagy is thePtdIns(3)P 5-kinase Fab1/PIKfyve, which controls late-endosomal membrane homeostasis, and whose in-activation is associated with an inherited form of fleckcorneal dystrophy [12].Autophagy in Drosophila is regulated by global nutri-tional and developmental cues such as amino acidavailability and hormonal signaling [13, 14]. In order toovercome confounding effects from such systemic in-fluences, we analyzed trafficking mutants in clones ofcells by using surrounding wild-type cells as a control.Mutant clones were induced by FLP-FRT technology inanimals expressing Gal4. The clones were recognizedby the loss of a upstream activating sequence (UAS)-myristylated red fluorescent protein (mRFP) transgenepresent on the homologous chromosome. In order tofollow autophagy, we used a transgene located on an-other chromosome that carries UAS-GFP-Atg8a (GFP:green fluorescent protein), which labels autophago-somes and autolysosomes (Figure 1A) [13, 15–17].To address the role of the endosomal sorting machin-eryin autophagy, we selected flies containing null muta-tions in members of ESCRT-I (vps28), -II (vps25), and -III(vps32) obtained in a screen for tumor suppressors [18,19] (T.V. and D.B., unpublished data). We first investi-gated clones of the ovarian follicular epithelium in stage8 or 9 egg chambers, in which no autophagy is normallydetectable.Interestingly,mutantfollicularepithelialcellsof vps28, vps25, and vps32 all accumulated intracellular