The contribution of individual domains of chloroplast protein AtDeg2 to its chaperone and proteolytic activities

Przemysław Jagodzik, Robert Luciński, Lucyna Misztal, Grzegorz Jackowski

Abstract


The thylakoid protease AtDeg2 is a non-ATP hydrolyzing chloroplast protease/chaperone peripherally connected with stromal side of thylakoid membrane. Its linear structure consists of protease domain and two PDZ domains. To unveil the significance of individual domains, chaperone and proteolytic activities of AtDeg2, its mutated recombinant versions have been developed and their ability to suppress protein aggregation and resolubilization of protein aggregates as well as the ability to degrade substrate protein was examined in vitro. Our work reveals for the first time that AtDeg2 is able not only to suppress aggregation of denatured proteins, but to resolubilize existing protein aggregates as well. We show that PDZ2 domain contributes significantly to both chaperone and protease activities of AtDeg2, whereas PDZ1 is required for chaperone but superfluous for proteolytic activity. Protease domain – but not S-268 in its catalytic site – contributes to chaperone activities of AtDeg2. These results show an entirely new function of AtDeg2 chaperone/protease (i.e., disaggregation of protein aggregates) and allow to identify structural motifs required for “old” and new functions of AtDeg2.

Keywords


AtDeg2 chloroplast protein; Arabidopsis thaliana; PDZ domain; protease domain; chaperone activity; aggregation; disaggregation; protease activity

Full Text:

PDF

References


Lipińska B, Sharma S, Georgopoulos C. Sequence analysis and regulation of the htr A gene of Escherichia coli: a sigma32-independent mechanism of heat-inducible transcription. Nucleic Acids Res. 1988;16(21):10053–10067. https://doi.org/10.1093/nar/16.21.10053

Strauch KL, Beckwith J. An Escherichia coli mutation preventing degradation of abnormal periplasmic proteins. Proc Nat Acad Sci USA. 1988;85(5):1576–1580. https://doi.org/10.1073/pnas.85.5.1576

Ortega J, Iwanczyk J, Jomaa A. Escherichia coli DegP: a structure-driven functional model. J Bacteriol. 2009;191(15):4705–4713. https://doi.org/10.1128/JB.00472-09

Schuhmann H, Huesgen P, Adamska I. The family of Deg/Htr proteases in plants. BMC Plant Biol. 2012;12:52. https://doi.org/10.1186/1471-2229-12-52

Haussühl K, Andersson B, Adamska I. A chloroplast DegP2 protease performs the primary cleavage of the photodamaged D1 protein in plant photosystem II. EMBO J. 2001;20(4):713–722. https://doi.org/10.1093/emboj/20.4.713

Chassin Y, Kapri-Pardes E, Sinvany G, Arad T, Adam Z. Expression and characterization of the thylakoid lumen protease DegP1 from Arabidopsis. Plant Physiol. 2002;130(2):857–864. https://doi.org/10.1104/pp.007922

Schubert M, Petersson UA, Haas BJ, Funk C, Schröder WP, Kieselbach T. Proteome map of the chloroplast lumen of Arabidopsis thaliana. J Biol Chem. 2002;277(10):8354–8365. https://doi.org/10.1074/jbc.M108575200

Tanz SK, Castleden I, Hooper CM, Small I, Millar AH. Using the SUBcellular database for Arabidopsis proteins to localize the Deg protease family. Front Plant Sci. 2014;5:396. https://doi.org/10.3389/fpls.2014.00396

Sun XW, Peng L, Guo J, Chi W, Ma J, Lu C, et al. Formation of DEG5 and DEG8 complexes and their involvement in the degradation of photodamaged photosystem II reaction center D1 protein in Arabidopsis. Plant Cell. 2007;19(4):1347–1361. https://doi.org/10.1105/tpc.106.049510

Stroher E, Dietz KJ. The dynamic thiol-disulphide redox proteome of the Arabidopsis thaliana chloroplast as revealed by differential electrophoretic mobility. Physiol Plant. 2008;133(3):566–583. https://doi.org/10.1111/j.1399-3054.2008.01103.x

Sun R, Fan H, Gao F, Lin Y, Zhang L, Gong W, et al. Crystal structure of Arabidopsis Deg2 protein reveals an internal PDZ ligand locking the hexameric resting state. J Biol Chem. 2012;287(44):37564–37569. https://doi.org/10.1074/jbc.M112.394585

Zienkiewicz M, Ferenc A, Wasilewska W, Romanowska E. High light stimulates Deg1-dependent cleavage of the minor LHCII antenna proteins CP26 and CP29 and PsbS protein in Arabidopsis thaliana. Planta. 2012;235(2):279–288. https://doi.org/10.1007/s00425-011-1505-x

Kapri-Pardes E, Naveh L, Adam Z. The thylakoid lumen protease Deg1 is involved in the repair of photosystem II from photoinhibition in Arabidopsis. Plant Cell. 2007;19(3):1039–1047. https://doi.org/10.1105/tpc.106.046573

Kato Y, Sun XW, Zhang L, Sakamoto W. Cooperative D1 degradation on the photosystem II repair mediated by chloroplastic proteases in Arabidopsis. Plant Physiol. 2012;159(4):1428–1439. https://doi.org/10.1104/pp.112.199042

Huesgen PF, Schuhmann H, Adamska I. Deg/HtrA proteases as components of a network for photosystem II quality control in chloroplasts and cyanobacteria. Res Microbiol. 2009;160(9):726–732. https://doi.org/10.1016/j.resmic.2009.08.005

Luciński R, Misztal L, Samardakiewicz S, Jackowski G. The thylakoid protease Deg2 is involved in stress-related degradation of the photosystem II light-harvesting protein Lhcb6 in Arabidopsis thaliana. New Phytol. 2011;192(1):74–86. https://doi.org/10.1111.j.1469-8137.2011.03782.x

Luciński R, Misztal L, Samardakiewicz S, Jackowski G. Involvement of Deg5 proteasae in wounding-related disposal of PsbF apoprotein. Plant Physiol Biochem. 2011;4(3):311–320. https://doi.org/10.1016/j/plaphy.2011.01.001

Baranek M, Wyka T, Jackowski G. Downregulation of chloroplast protease AtDeg5 leads to changes in chronological progression of ontogenetic stages, leaf morphology and chloroplast ultrastructure in Arabidopsis. Acta Soc Bot Pol. 2015;84(1):59–70. https://doi.org/10.5586/asbp.2015.001

Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227(5259):680–685. https://doi.org/10.1038/22768a0

Ansorge W. Fast and sensitive detection of protein and DNA bands by treatment with potassium permanganate. J Biochem Biophys Methods. 1985;11(1):13–20. https://doi.org/10.1016/0165-022X(85)90037-5

Sun X, Ouyang M, Guo J, Ma J, Lu C, Adam Z, et al. The thylakoid protease Deg1 is involved in photosystem-II assembly in Arabidopsis thaliana. Plant J. 2010;62(2):240–249. https://doi.org/10.1111/j.1365-313X.2010.04140.x

Moore M, Goforth RL, Mori H, Henry R. Functional interaction of chloroplast SRP/FtsY with the ALB3 translocase in thylakoids: substrate not required. J Cell Biol. 2003;162(7):1245–1254. https://doi.org/10.1083/jcb.200307067

Schünemann D. Structure and function of the chloroplast signal recognition particle. Curr Genet. 2004;44(6):295–304. https://doi.org/10.1007/s00294-003-0450-z

Jaru-Ampornpan P, Shen K, Lam VQ, Ali M, Doniach S, Jia TZ, et al. ATP-independent reversal of a membrane protein aggregate by a chloroplast SRP subunit. Nat Struct Mol Biol. 2010;17(6):696–702. https://doi.org/10.1038/nsmb.1836

Nguyen TX, Jaru-Ampornpan P, Lam VQ, Cao P, Piszkiewicz S, Hess S, et al. Mechanism of an ATP-independent protein disaggregase: I. Structure of a membrane protein aggregate reveals a mechanism of recognition by its chaperone. J Biol Chem. 2013;288(19):13420–13430. https://doi.org/10.1074/jbc.M113.462812

Spiess C, Beil A, Ehrmann M. A temperature-dependent switch from chaperone to protease in a widely conserved heat shock protein. Cell. 2004;97(3):339–347. https://doi.org/10.1016/S0092-8674(00)80743-6

Jiang J, Zhang X, Chen Y, Wu Y, Zhou ZH, Chang Z, et al. Activation of DegP chaperone-protease via formation of large cage-like oligomers upon binding to substrate proteins. Proc Natl Acad Sci USA. 2008;105(33):11939–11944. https://doi.org/10.1073/pnas.0805464105