Small GTPase Rab2B and Its Specific Binding Protein Golgi-associated Rab2B Interactor-like 4 (GARI-L4) Regulate Golgi Morphology

Aizawa, Megumi; Fukuda, Mitsunori

doi:10.1074/jbc.m115.669242

Cited by 56 publications

(51 citation statements)

References 51 publications

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“…Mammals each have two Rab2 isoforms, Rab2A and 2B (Aizawa and Fukuda, 2015). To examine whether Rab2 has an evolutionarily conserved role in autophagy, we deleted Rab2A and Rab2B using the CRISPR/Cas9 system in mouse embryonic fibroblasts (MEFs).…”

Section: Resultsmentioning

confidence: 99%

“…The following antibodies were used: mouse anti-fly Dlg1 (1:200; clone 4 F3; Developmental Studies Hybridoma Bank, Iowa City, IA; RRID:AB_528203), rabbit anti-mouse Atg16L1 ([Itoh et al, 2011]; RRID:AB_2631282), rabbit anti-mouse Rab2A (Aizawa and Fukuda, 2015) (1:1000; RRID:AB_2631288), rabbit anti-mouse Rab2B (Aizawa and Fukuda, 2015) (1:1,000; RRID:AB_2631289), rat anti-fly Atg8 (Takáts et al, 2013) (1:500; gift of G. Juhasz; RRID:AB_2568972), rabbit anti-Ref(2)P (Pircs et al, 2012) (1:500, also called p62; gift of G. Juhasz; RRID:AB_2569199), rabbit anti-Zormin B1 (Burkart et al, 2007) (1:500; B. Bullard; RRID:AB_2631283), guinea pig anti-fly Dor (1:500; gift of H. Krämer; RRID:AB_2569525), rabbit anti-human LC3 (1:50 for immuno-EM; 1:1000 for IF; 1:3000 for WB; PM036, MBL, Woburn, MA; RRID:AB_2274121), mouse anti-GFP JL-8 (1:25 for immuno-EM; Clontech, Mountain View, CA; RRID:AB_10013427), mouse anti-T7 (1:3,000; EMD Millipore Novagen, Danvers, MA; RRID:AB_11211744), mouse anti-FLAG (1:3,000; clone M2; Sigma, St. Louis, MO; RRID:AB_439685), mouse anti-Myc (1:1000 for IF and 1:3000 for WB; clone 9E10, Santa Cruz Biotechnology, Dallas, TX; RRID:AB_627268), rat anti-mouse Lamp1 (1:1,000; clone 1D4B, Santa Cruz Biotechnology, Dallas TX; RRID:AB_2134495), sheep anti-EGFR (1:2,000; #20-ES04, Fitzgerald Industries, Acton, MA; RRID:AB_231428) and mouse anti-β-actin (1:5,000; Applied Biological Materials, Canada; RRID:AB_2631287). …”

Section: Methodsmentioning

confidence: 99%

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Genetic screen in Drosophila muscle identifies autophagy-mediated T-tubule remodeling and a Rab2 role in autophagy

Fujita

Huang

Lin

et al. 2017

eLife

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101

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Transverse (T)-tubules make-up a specialized network of tubulated muscle cell membranes involved in excitation-contraction coupling for power of contraction. Little is known about how T-tubules maintain highly organized structures and contacts throughout the contractile system despite the ongoing muscle remodeling that occurs with muscle atrophy, damage and aging. We uncovered an essential role for autophagy in T-tubule remodeling with genetic screens of a developmentally regulated remodeling program in Drosophila abdominal muscles. Here, we show that autophagy is both upregulated with and required for progression through T-tubule disassembly stages. Along with known mediators of autophagosome-lysosome fusion, our screens uncovered an unexpected shared role for Rab2 with a broadly conserved function in autophagic clearance. Rab2 localizes to autophagosomes and binds to HOPS complex members, suggesting a direct role in autophagosome tethering/fusion. Together, the high membrane flux with muscle remodeling permits unprecedented analysis both of T-tubule dynamics and fundamental trafficking mechanisms.DOI: http://dx.doi.org/10.7554/eLife.23367.001

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Genetic screen in Drosophila muscle identifies autophagy-mediated T-tubule remodeling and a Rab2 role in autophagy

Fujita

Huang

Lin

et al. 2017

eLife

Self Cite

101

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“…Rab1 is responsible for transporting vesicles from the ER to the Golgi. Disruption of Rab1 activity, by knockdown or overexpression of the dominant-negative Rab1S25N, results in fragmentation of the Golgi apparatus (36,37). We tested the effect of overexpression of nonphosphorylatable T75A and phosphomimetic T75E Rab1 on Golgi structure to determine if the T75 site was critical for Rab1 function.…”

Section: Resultsmentioning

confidence: 99%

Innate immunity kinase TAK1 phosphorylates Rab1 on a hotspot for posttranslational modifications by host and pathogen

Levin

Hertz

Burlingame

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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TGF-β activated kinase 1 (TAK1) is a critical signaling hub responsible for translating antigen binding signals to immune receptors for the activation of the AP-1 and NF-κB master transcriptional programs. Despite its importance, known substrates of TAK1 are limited to kinases of the MAPK and IKK families and include no direct effectors of biochemical processes. Here, we identify over 200 substrates of TAK1 using a chemical genetic kinase strategy. We validate phosphorylation of the dynamic switch II region of GTPase Rab1, a mediator of endoplasmic reticulum to Golgi vesicular transport, at T75 to be regulated by TAK1 in vivo. TAK1 preferentially phosphorylates the inactive (GDP-bound) state of Rab1. Phosphorylation of Rab1 disrupts interaction with GDP dissociation inhibitor 1 (GDI1), but not guanine exchange factor (GEF) or GTPaseactivating protein (GAP) enzymes, and is exclusive to membranelocalized Rab1, suggesting phosphorylation may stimulate Rab1 membrane association. Furthermore, we found phosphorylation of Rab1 at T75 to be essential for Rab1 function. Previous studies established that the pathogen Legionella pneumophila is capable of hijacking Rab1 function through posttranslational modifications of the switch II region. Here, we present evidence that Rab1 is regulated by the host in a similar fashion, and that the innate immunity kinase TAK1 and Legionella effectors compete to regulate Rab1 by switch II modifications during infection.

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“…The molecular functions of RAB2B-binding GARI family members remain unknown at present. Because GARI family members are involved in antiviral innate immune responses and maintenance of Golgi morphology (Aizawa and Fukuda, 2015), their targets and mode of action should be clarified. In future studies, we will address these points to increase our understanding of RAB2B-dependent biological processes that occur on the Golgi apparatus.…”

Section: Discussionmentioning

confidence: 99%

The RAB2B-GARIL5 Complex Promotes Cytosolic DNA-Induced Innate Immune Responses

et al. 2017

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Summary Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that induces the IFN antiviral response. However, the regulatory mechanisms that mediate cGAS-triggered signaling have not been fully explored. Here, we show the involvement of a small GTPase RAB2B and its effector protein Golgi-associated RAB2B interactor-like 5 (GARIL5) in the cGAS-mediated IFN response. RAB2B-deficiency affects the IFN response induced by cytosolic DNA. Consistent with this, RAB2B-deficiency enhances replication of vaccinia virus, a DNA virus. After DNA stimulation, RAB2B colocalizes with stimulator of interferon genes (STING), the downstream signal mediator of cGAS, on the Golgi apparatus. GTP-binding activity of RAB2B is required for its localization on the Golgi apparatus and for recruitment of GARIL5. GARIL5 deficiency also affects the IFN response induced by cytosolic DNA and enhances replication of vaccinia virus. These findings indicate that the RAB2B-GARIL5 complex promotes IFN responses against DNA viruses by regulating the cGAS-STING signaling axis.

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Small GTPase Rab2B and Its Specific Binding Protein Golgi-associated Rab2B Interactor-like 4 (GARI-L4) Regulate Golgi Morphology

Cited by 56 publications

References 51 publications

Genetic screen in Drosophila muscle identifies autophagy-mediated T-tubule remodeling and a Rab2 role in autophagy

Genetic screen in Drosophila muscle identifies autophagy-mediated T-tubule remodeling and a Rab2 role in autophagy

Innate immunity kinase TAK1 phosphorylates Rab1 on a hotspot for posttranslational modifications by host and pathogen

The RAB2B-GARIL5 Complex Promotes Cytosolic DNA-Induced Innate Immune Responses

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