ARTICLES The deubiquitylase USP33 discriminates between RALB functions in autophagy and innate immune response Michal Simicek 1,2 , Sam Lievens 3,4 , Mathias Laga 3,4 , Dmytro Guzenko 5 , Vasily N. Aushev 6 , Peter Kalev 1,2 , Maria Francesca Baietti 1,2 , Sergei V. Strelkov 5 , Kris Gevaert 3,4 , Jan Tavernier 3,4 and Anna A. Sablina 1,2,7 The RAS-like GTPase RALB mediates cellular responses to nutrient availability or viral infection by respectively engaging two components of the exocyst complex, EXO84 and SEC5. RALB employs SEC5 to trigger innate immunity signalling, whereas RALB–EXO84 interaction induces autophagocytosis. How this differential interaction is achieved molecularly by the RAL GTPase remains unknown. We found that whereas GTP binding turns on RALB activity, ubiquitylation of RALB at Lys 47 tunes its activity towards a particular effector. Specifically, ubiquitylation at Lys 47 sterically inhibits RALB binding to EXO84, while facilitating its interaction with SEC5. Double-stranded RNA promotes RALB ubiquitylation and SEC5–TBK1 complex formation. In contrast, nutrient starvation induces RALB deubiquitylation by accumulation and relocalization of the deubiquitylase USP33 to RALB-positive vesicles. Deubiquitylated RALB promotes the assembly of the RALB–EXO84–beclin-1 complexes driving autophagosome formation. Thus, ubiquitylation within the effector-binding domain provides the switch for the dual functions of RALB in autophagy and innate immune responses. The RAS family of small GTPases function as molecular switches alternating between inactive GDP-bound and active GTP-bound states 1 . Once activated, RAS GTPases exert their effects through binding and activation of downstream effectors. Each RAS GTPase activates a diverse set of downstream effectors and may propagate multiple signalling pathways. However, the mechanisms that govern specificity of the interactions between RAS GTPases and their downstream effectors are mainly unknown. Two highly similar RAL proteins, RALA and RALB, constitute a family within the RAS branch of small GTPases. RALA and RALB share the same effector molecules and seem to function in distinct but inter-related biological processes that are largely connected through their interaction with the RALBP1 protein and the components of the hetero-octameric exocyst complex. Two components of the exocyst, SEC5 (EXOC2) and EXO84 (EXOC8), are effector molecules that mediate RAL regulation of dynamic secretory vesicle targeting and tethering processes 2–6 . RALA mobilizes SEC5 and EXO84 to regulate basolateral delivery of membrane proteins in polarized epithelial cells 4,5 and insulin- stimulated GLUT4 delivery to the plasma membrane 7,8 . The binding of 1 VIB Center for the Biology of Disease, VIB, 3000 Leuven, Belgium. 2 Department of Human Genetics, KU Leuven, Herestraat 49, 3000 Leuven, Belgium. 3 Department of Medical Protein Research, VIB, 9000 Ghent, Belgium. 4 Department of Biochemistry, Ghent University, Albert Baertsoenkaai 3, 9000 Ghent, Belgium. 5 Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Herestraat 49, 3000 Leuven, Belgium. 6 Institute of Carcinogenesis, N.N. Blokhin Russian Cancer Research Center, Kashirskoye Shosse 24, 115478 Moscow, Russia. 7 Correspondence should be addressed to A.A.S. (e-mail: Anna.Sablina@cme.vib-kuleuven.be) Received 14 March 2013; accepted 20 August 2013; published online 22 September 2013; DOI: 10.1038/ncb2847 RALBP1 to RALA is required for the appropriate mitochondrial fission and accurate distribution of mitochondria between daughter cells 9 . RALB has been demonstrated to mediate an entirely different set of cellular processes. Infection with double-stranded RNA (dsRNA) viruses promotes the formation of the RALB–SEC5 complex that activates the innate immunity signalling kinase TBK1 and the subsequent IRF3 transcription-factor-dependent interferon response 10 . On the other hand, nutrient starvation or double-stranded DNA triggers interaction between RALB and EXO84 driving the assembly of catalytically active ULK1 and the beclin-1–VPS34 autophagy initiation complex 11,12 . These data suggest that RALB and the components of the exocyst complex represent a regulatory hub through bifurcating activation of TBK1 and beclin-1–VPS34 that helps engage coordinated activation of the gene expression and organelle biogenesis responses supporting systemic pathogen recognition and clearance. The structural data of RAL–effector complexes indicate that the RAL downstream effectors bind overlapping residues of the RAL GTPases and compete for RAL binding 13–16 . It is unclear how specificity pertaining to effector recognition is achieved by the RAL GTPase. Here we found that RALB ubiquitylation at Lys 47, controlled by the 1220 NATURE CELL BIOLOGY VOLUME 15 | NUMBER 10 | OCTOBER 2013 © 20 13 M acmillan Publishers Limited. All rights reserved.