Rallying the Exocyst As an Autophagy Scaffold

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Rallying the Exocyst as an Autophagy Scaffold

Jean-Claude Farre´ 1 and Suresh Subramani1,* 1Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093-0322, USA *Correspondence: [email protected] DOI 10.1016/j.cell.2011.01.005

Protein scaffolds coordinate the assembly of many multicomponent signaling complexes. Bode- mann et al. (2011) now show that the exocyst, a protein complex involved in tethering transport vesicles to the plasma membrane, provides an assembly and activation platform for components of the autophagy machinery via a process requiring the GTPase RalB.

Macroautophagy (henceforth referred to Autophagosome formation consists of ates with Atg5 and Atg12, autophagy as autophagy) is a cellular degradation three steps: nucleation, expansion, and proteins involved in the ubiquitin-like pathway for the clearance of damaged fusion of the isolation membrane. Each system, cementing the association or superfluous proteins and organelles step involves a specific set of protein between the autophagy machinery and (Yang and Klionsky, 2010). Despite its complexes. The ULK (Unc-51-like kinase) the exocyst. importance in cellular homeostasis and and PI3K (phosphatidylinositol-3 kinase) Given the association between auto- in immunity against pathogens and complexes are most important for nucle- phagy and exocyst components and the despite accumulating knowledge re- ation, whereas the ubiquitin-like (Ubl) fact that the small GTPases RalA and garding the autophagy machinery itself, conjugation system and the mAtg9 (mam- RalB mobilize exocyst assembly (Moska- the early regulatory signals that activate malian autophagy-related gene 9) cycling lenko et al., 2002, 2003), the authors next autophagy are unknown. Furthermore, complex, which is involved in transit of inquire whether RalA and RalB also play several gaps remain in our understanding mAtg9 to and from the isolation mem- a role in autophagy. Indeed, Bodemann of how distinct autophagy complexes brane, facilitate expansion and closure et al. show convincingly that activation of assemble and collaborate (Mehrpour of the isolation membrane (Mehrpour RalB, but not RalA, in cervical cancer and et al., 2010). Bodemann et al. (2011) now et al., 2010). epithelial cell lines is necessary for auto- identify some of the early regulatory and The exocyst, a hetero-octameric phagy. They find that competitive in- assembly steps, revealing a role for the complex containing the proteins Sec3, hibitors of RalB inhibit the induction of exocyst, a complex that regulates post- Sec5, Sec6, Sec8, Sec10, Sec15, autophagy during starvation, whereas Golgi protein traffic. The authors demon- Exo70, and Exo84 (recently renamed constitutively activated forms of RalB stim- strate that nutrient deprivation, a condition EXOC1-EXOC8) is involved in the post- ulate autophagy even under nutrient-rich that promotes autophagy, activates the Golgi trafficking and tethering of vesicles conditions. Thus, RalB is both necessary Ras-like small GTPase RalB, which then to the plasma membrane (He and Guo, and sufficient for activation of autophagy. engages the effector protein and exocyst 2009; Munson and Novick, 2006). New RalB and its related partner RalA coop- component Exo84. This interaction pro- evidence implicating a role for the exocyst erate in mitogen-induced signaling during motes the assembly and activation of complex in signaling during pathogen oncogenic transformation by Ras. RalA is the autophagy complex using the exocyst infection (Chien et al., 2006) led the required to bypass normal restraints on as an assembly scaffold. authors to screen for proteins interacting cell proliferation, whereas RalB bypasses During metabolic stress, including star- with the exocyst subunit, Sec3. Using normal restraints on cell survival (Chien vation, autophagy promotes the degrada- a high-throughput yeast two-hybrid et al., 2006). Tumor cells have higher tion of cytoplasmic components by the screen, the authors found that both nega- levels of RalB, and cells depleted of lysosome, and the recycling of their tive and positive regulators of autophagy RalB exhibit survival defects (Bodemann constituents promotes cell survival (Mehr- interact with Sec3. The interactors include and White, 2008). These observations pour et al., 2010; Yang and Klionsky, Rubicon (RUN domain and cysteine-rich may be explained, in part, by the finding 2010). Autophagy involves formation of domain containing), an inhibitor of au- that RalB promotes cell survival during an isolation membrane, which elongates tophagy, as well as Atg14L, a component starvation by inducing autophagy. and fuses to form a double-membrane of the PI3K complex, and FIP200, part of Interestingly, the authors characterize vesicle called an autophagosome. The the ULK complex. Indeed, the authors two complexes containing both exocyst autophagosome encloses cytoplasmic find that several exocyst subunits (Sec3, and autophagy components: an auto- cargoes for delivery by fusion to the endo- Sec5, and Exo84) coimmunoprecipitate phagy-active and autophagy-inactive some or lysosome, eventually forming an with Rubicon and Atg14L. Additionally, complex. The RalB-Exo84 complex con- autolysosome. the core exocyst subunit, Sec8, associ- tains functional ULK and PI3K complexes,

172 Cell 144, January 21, 2011 ª2011 Elsevier Inc. suggesting that this complex both general autophagy and is active during starvation- the selective autophagic induced autophagy. In degradation of bacteria. contrast, upon inhibition of Together, the data are consis- RalB signaling, Rubicon, an tent with the idea that the exo- inhibitor of autophagy, asso- cyst functions as a scaffold for ciates with Exo84. The Sec5- the core autophagy machinery ULK-PI3K complex correlates in mammalian cells. with the inactive autophagy Earlier work showed that state and is more abundant RalB competes with phos- under nutrient-rich condi- phatidylinositol 3,4,5-tri- tions. These observations led sphosphate (PIP3) for binding the authors to propose a to the pleckstrin homology model for the activation of (PH) domain of Exo84 (Mos- autophagy (Figure 1). They kalenko et al., 2003). It is suggest that assembly of the therefore exciting to hypothe- ULK and PI3K complexes on size that the binding of RalB Exo84 triggers autophagy by to Exo84 may trigger the generating an autophagy- movement of the complex active complex. In contrast, from a PIP3-enriched environ- interaction between these ment, such as the plasma complexes and Sec5 creates membrane or recycling endo- an autophagy-inactive com- somes, to the autophago- plex that is either a preinitiation some assembly site. complex unable but poised to The precise mechanism trigger autophagy or is a signal linking the activities of the termination complex for the ULK and PI3K complexes to process. Consistent with this the elongation and completion model, endogenous mamma- Figure 1. A Model for Exocyst Function in Autophagy of the isolation membrane by Bodemann et al. (2011) provide evidence that the exocyst, a protein complex lian target of rapamycin involved in post-Golgi protein trafﬁc, may function as a scaffold for the the Ubl and mAtg9 cycling complex 1 (mTORC1), which assembly of autophagy complexes. The authors suggest the following model complexes has been unclear inhibits autophagy through for activation of autophagy. Under nutrient-rich conditions, an exocyst sub- (Mehrpour et al., 2010). The complex containing the Sec5 protein associates with the Unc-51 like kinase inactivation of the ULK com- (ULK) and phosphatidylinositol-3 kinase (PI3K) complexes at the perinuclear direct interaction of these plex (Mehrpour et al., 2010), region forming an autophagy-inactive complex. Induction of autophagy (e.g., complexes with the exocyst is present only in the auto- in response to starvation) leads to the activation of the Ras-like small GTPase, complex may provide this RalB. The activated RalB interacts with the exocyst, promoting the replace- phagy-inactive complex. ment of Sec5 by another exocyst component, Exo84, and formation of an missing link. The work of Bod- The cellular localization of active autophagy complex that includes the ubiquitin-like (Ubl) conjugation emann and colleagues shows complex components under system and the ubiquitin-like molecule LC3. This autophagy-active complex that all of these complexes different conditions suggests localizes in cytosolic dots that could correspond to the isolation membrane. assemble on a common scaf- The Exo84 exocyst subcomplex may bring together complexes of the core that the transition from an autophagic machinery or facilitate their concerted action. The exact subunit fold, a recurring theme ex- autophagy-inactive to an compositions of the autophagy-inactive and autophagy-active exocyst sub- ploited by other signaling autophagy-active complex complexes remain unknown. PI3P, phosphatidylinositol 3-phosphate; PI, systems (Shaw and Filbert, phosphatidylinositol. WIPI-1 is a WD40 repeat autophagy protein that inter- may involve a change in local- acts with phosphoinositides such as PI3P. 2009),suggesting that the exo- ization. Bodemann et al. ﬁnd cyst may coordinate molecular that, under nutrient-rich con- events in autophagy. ditions, RalB associates with The primary known function an exocyst subcomplex containing PI3K complex). These results suggest of the exocyst is the targeting and tethering Sec5, and components of the PI3K au- that activation of RalB and recruitment of post-Golgi vesicles to plasma mem- tophagy complex colocalize with RalB in of Exo84 may trigger translocation of the brane domains. By analogy, the exocyst perinuclear regions of the cell. Upon star- autophagy complex to sites of autopha- may also provide a targeting site for the au- vation and activation of RalB, Exo84 gosome membrane formation. tophagy machinery, perhaps tethering this replaces Sec5. Under these conditions Epistasis analysis also supports an early machinery to the isolation membrane for (starvation), the authors observe the role for RalB in triggering autophagy autophagosome expansion. Exo84 exocyst subcomplex at cytosolic upstream of the initial nucleation step dots, along with the PI3K autophagy mediated by the ULK complex. Further- ACKNOWLEDGMENTS complex, components of the Ubl com- more, depletion of several components of plex, and phosphatidylinositol 3-phos- the active exocyst complex (Sec3, Sec8, Our autophagy expertise is made possible by NIH phate (PI3P, the product of an active Exo70, Exo84, but not Sec5) suppresses grant GM069373 to S.S.

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