A Novel Membrane Sensor Controls the Localization and ArfGEF Activity of Bacterial RalF


The intracellular bacterial pathogen Legionella pneumophila (Lp) evades destruction in macrophages by camouflaging in a specialized organelle, the Legionella-containing vacuole (LCV), where it replicates. The LCV maturates by incorporating ER vesicles, which are diverted by effectors that Lp injects to take control of host cell membrane transport processes. One of these effectors, RalF, recruits the trafficking small GTPase Arf1 to the LCV. LpRalF has a Sec7 domain related to host ArfGEFs, followed by a capping domain that intimately associates with the Sec7 domain to inhibit GEF activity. How RalF is activated to function as a LCV-specific ArfGEF is unknown. We combined the reconstitution of Arf activation on artificial membranes with cellular expression and Lp infection assays, to analyze how auto-inhibition is relieved for LpRalF to function in vivo. We find that membranes activate LpRalF by about 1000 fold, and identify the membrane-binding region as the region that inhibits the Sec7 active site. It is enriched in aromatic and positively charged residues, which establish a membrane sensor to control the GEF activity in accordance with specific lipid environments. A similar mechanism of activation is found in RalF from Rickettsia prowazekii (Rp), with a different aromatic/charged residues ratio that results in divergent membrane preferences. The membrane sensor is the primary determinant of the localization of LpRalF on the LCV, and drives the timing of Arf activation during infection. Finally, we identify a conserved motif in the capping domain, remote from the membrane sensor, which is critical for RalF activity presumably by organizing its active conformation. These data demonstrate that RalF proteins are regulated by a membrane sensor that functions as a binary switch to derepress ArfGEF activity when RalF encounters a favorable lipid environment, thus establishing a regulatory paradigm to ensure that Arf GTPases are efficiently activated at specific membrane locations.


Vyšlo v časopise: A Novel Membrane Sensor Controls the Localization and ArfGEF Activity of Bacterial RalF. PLoS Pathog 9(11): e32767. doi:10.1371/journal.ppat.1003747
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1003747

Souhrn

The intracellular bacterial pathogen Legionella pneumophila (Lp) evades destruction in macrophages by camouflaging in a specialized organelle, the Legionella-containing vacuole (LCV), where it replicates. The LCV maturates by incorporating ER vesicles, which are diverted by effectors that Lp injects to take control of host cell membrane transport processes. One of these effectors, RalF, recruits the trafficking small GTPase Arf1 to the LCV. LpRalF has a Sec7 domain related to host ArfGEFs, followed by a capping domain that intimately associates with the Sec7 domain to inhibit GEF activity. How RalF is activated to function as a LCV-specific ArfGEF is unknown. We combined the reconstitution of Arf activation on artificial membranes with cellular expression and Lp infection assays, to analyze how auto-inhibition is relieved for LpRalF to function in vivo. We find that membranes activate LpRalF by about 1000 fold, and identify the membrane-binding region as the region that inhibits the Sec7 active site. It is enriched in aromatic and positively charged residues, which establish a membrane sensor to control the GEF activity in accordance with specific lipid environments. A similar mechanism of activation is found in RalF from Rickettsia prowazekii (Rp), with a different aromatic/charged residues ratio that results in divergent membrane preferences. The membrane sensor is the primary determinant of the localization of LpRalF on the LCV, and drives the timing of Arf activation during infection. Finally, we identify a conserved motif in the capping domain, remote from the membrane sensor, which is critical for RalF activity presumably by organizing its active conformation. These data demonstrate that RalF proteins are regulated by a membrane sensor that functions as a binary switch to derepress ArfGEF activity when RalF encounters a favorable lipid environment, thus establishing a regulatory paradigm to ensure that Arf GTPases are efficiently activated at specific membrane locations.


Zdroje

1. AktoriesK (2011) Bacterial protein toxins that modify host regulatory GTPases. Nat Rev Microbiol 9: 487–498.

2. HubberA, RoyCR (2010) Modulation of host cell function by Legionella pneumophila type IV effectors. Annu Rev Cell Dev Biol 26: 261–283.

3. IsbergRR, O'ConnorTJ, HeidtmanM (2009) The Legionella pneumophila replication vacuole: making a cosy niche inside host cells. Nat Rev Microbiol 7: 13–24.

4. NagaiH, KaganJC, ZhuX, KahnRA, RoyCR (2002) A bacterial guanine nucleotide exchange factor activates ARF on Legionella phagosomes. Science 295: 679–682.

5. NagaiH, CambronneED, KaganJC, AmorJC, KahnRA, et al. (2005) A C-terminal translocation signal required for Dot/Icm-dependent delivery of the Legionella RalF protein to host cells. Proc Natl Acad Sci U S A 102: 826–831.

6. TilneyLG, HarbOS, ConnellyPS, RobinsonCG, RoyCR (2001) How the parasitic bacterium Legionella pneumophila modifies its phagosome and transforms it into rough ER: implications for conversion of plasma membrane to the ER membrane. J Cell Sci 114: 4637–4650.

7. KaganJC, RoyCR (2002) Legionella phagosomes intercept vesicular traffic from endoplasmic reticulum exit sites. Nat Cell Biol 4: 945–954.

8. HardimanCA, McDonoughJA, NewtonHJ, RoyCR (2012) The role of Rab GTPases in the transport of vacuoles containing Legionella pneumophila and Coxiella burnetii. Biochem Soc Trans 40: 1353–1359.

9. D'Souza-SchoreyC, ChavrierP (2006) ARF proteins: roles in membrane traffic and beyond. Nat Rev Mol Cell Biol 7: 347–358.

10. HutagalungAH, NovickPJ (2011) Role of Rab GTPases in membrane traffic and cell physiology. Physiol Rev 91: 119–149.

11. CasanovaJE (2007) Regulation of Arf activation: the Sec7 family of guanine nucleotide exchange factors. Traffic 8: 1476–1485.

12. BalrajP, RenestoP, RaoultD (2009) Advances in rickettsia pathogenicity. Ann N Y Acad Sci 1166: 94–105.

13. AmorJC, SwailsJ, ZhuX, RoyCR, NagaiH, et al. (2005) The structure of RalF, an ADP-ribosylation factor guanine nucleotide exchange factor from Legionella pneumophila, reveals the presence of a cap over the active site. J Biol Chem 280: 1392–1400.

14. AlixE, ChesnelL, BowzardBJ, TuckerAM, DelpratoA, et al. (2012) The Capping Domain in RalF Regulates Effector Functions. PLoS Pathog 8: e1003012.

15. CherfilsJ, ZeghoufM (2013) Regulation of Small GTPases by GEFs, GAPs, and GDIs. Physiol Rev 93: 269–309.

16. Pasqualato S, Renault L, Cherfils J (2004) The GDP/GTP cycle of Arf proteins. Structural and biochemical aspects. In: Richard A Kahn, editor. The ARF Book. Kluwer Academic Publishers. pp. 23–48.

17. ZeehJC, ZeghoufM, GrauffelC, GuibertB, MartinE, et al. (2006) Dual specificity of the interfacial inhibitor brefeldin a for arf proteins and sec7 domains. J Biol Chem 281: 11805–11814.

18. MargaritSM, SondermannH, HallBE, NagarB, HoelzA, et al. (2003) Structural evidence for feedback activation by Ras.GTP of the Ras-specific nucleotide exchange factor SOS. Cell 112: 685–695.

19. StalderD, BarelliH, GautierR, MaciaE, JacksonCL, et al. (2011) Kinetic studies of the Arf activator Arno on model membranes in the presence of Arf effectors suggest control by a positive feedback loop. J Biol Chem 286: 3873–3883.

20. RichardsonBC, McDonoldCM, FrommeJC (2012) The Sec7 Arf-GEF is recruited to the trans-Golgi network by positive feedback. Dev Cell 22: 799–810.

21. LomizeAL, PogozhevaID, LomizeMA, MosbergHI (2007) The role of hydrophobic interactions in positioning of peripheral proteins in membranes. BMC Struct Biol 7: 44.

22. GoldbergJ (1998) Structural basis for activation of ARF GTPase: mechanisms of guanine nucleotide exchange and GTP-myristoyl switching. Cell 95: 237–248.

23. RenaultL, GuibertB, CherfilsJ (2003) Structural snapshots of the mechanism and inhibition of a guanine nucleotide exchange factor. Nature 426: 525–530.

24. DiNittoJP, DelpratoA, Gabe LeeMT, CroninTC, HuangS, et al. (2007) Structural basis and mechanism of autoregulation in 3-phosphoinositide-dependent Grp1 family Arf GTPase exchange factors. Mol Cell 28: 569–583.

25. LemmonMA (2008) Membrane recognition by phospholipid-binding domains. Nat Rev Mol Cell Biol 9: 99–111.

26. AntonnyB (2011) Mechanisms of membrane curvature sensing. Annu Rev Biochem 80: 101–123.

27. AizelK, BiouV, NavazaJ, DuarteLV, CampanacciV, et al. (2013) Integrated Conformational and Lipid-Sensing Regulation of Endosomal ArfGEF BRAG2. PLoS Biol 11: e1001652.

28. BergerKH, IsbergRR (1993) Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila. Mol Microbiol 7: 7–19.

29. FeeleyJC, GibsonRJ, GormanGW, LangfordNC, RasheedJK, et al. (1979) Charcoal-yeast extract agar: primary isolation medium for Legionella pneumophila. J Clin Microbiol 10: 437–441.

30. FrancoM, ChardinP, ChabreM, ParisS (1995) Myristoylation of ADP-ribosylation factor 1 facilitates nucleotide exchange at physiological Mg2+ levels. J Biol Chem 270: 1337–1341.

31. Béraud-DufourS, RobineauS, ChardinP, ParisS, ChabreM, et al. (1998) A glutamic finger in the guanine nucleotide exchange factor ARNO displaces Mg2+ and the beta-phosphate to destabilize GDP on ARF1. Embo J 17: 3651–3659.

32. AntonnyB, Beraud-DufourS, ChardinP, ChabreM (1997) N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipids upon GDP to GTP exchange. Biochemistry 36: 4675–4684.

33. KabschW (2010) Xds. Acta Crystallogr D Biol Crystallogr 66: 125–132.

34. McCoyAJ, Grosse-KunstleveRW, AdamsPD, WinnMD, StoroniLC, et al. (2007) Phaser crystallographic software. J Appl Crystallogr 40: 658–674.

35. BlancE, RoversiP, VonrheinC, FlensburgC, LeaSM, et al. (2004) Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT. Acta Crystallogr D Biol Crystallogr 60: 2210–2221.

36. EmsleyP, CowtanK (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60: 2126–2132.

37. BiouV, AizelK, RoblinP, ThureauA, JacquetE, et al. (2010) SAXS and X-ray crystallography suggest an unfolding model for the GDP/GTP conformational switch of the small GTPase Arf6. J Mol Biol 402: 696–707.

38. LomizeMA, PogozhevaID, JooH, MosbergHI, LomizeAL (2012) OPM database and PPM web server: resources for positioning of proteins in membranes. Nucleic Acids Res 40: D370–376.

39. ArasakiK, RoyCR (2010) Legionella pneumophila promotes functional interactions between plasma membrane syntaxins and Sec22b. Traffic 11: 587–600.

Štítky
Hygiena a epidemiológia Infekčné lekárstvo Laboratórium

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