title: Nucleotide binding and dimerization at the chloroplast pre-protein import receptor, atToc33, are not essential in vivo but do increase import efficiency.
abstract: The atToc33 protein is one of several pre-protein import receptors in the outer envelope of Arabidopsis chloroplasts. It is a GTPase with motifs characteristic of such proteins, and its loss in the plastid protein import 1 (ppi1) mutant interferes with the import of photosynthesis-related pre-proteins, causing a chlorotic phenotype in mutant plants. To assess the significance of GTPase cycling by atToc33, we generated several atToc33 point mutants with predicted effects on GTP binding (K49R, S50N and S50N/S51N), GTP hydrolysis (G45R, G45V, Q68A and N101A), both binding and hydrolysis (G45R/K49N/S50R), and dimerization or the functional interaction between dimeric partners (R125A, R130A and R130K). First, a selection of these mutants was assessed in vitro, or in yeast, to confirm that the mutations have the desired effects: in relation to nucleotide binding and dimerization, the mutants behaved as expected. Then, activities of selected mutants were tested in vivo, by assessing for complementation of ppi1 in transgenic plants. Remarkably, all tested mutants mediated high levels of complementation: complemented plants were similar to the wild type in growth rate, chlorophyll accumulation, photosynthetic performance, and chloroplast ultrastructure. Protein import into mutant chloroplasts was also complemented to >50% of the wild-type level. Overall, the data indicate that neither nucleotide binding nor dimerization at atToc33 is essential for chloroplast import (in plants that continue to express the other TOC receptors in native form), although both processes do increase import efficiency. Absence of atToc33 GTPase activity might somehow be compensated for by that of the Toc159 receptors. However, overexpression of atToc33 (or its close relative, atToc34) in Toc159-deficient plants did not mediate complementation, indicating that the receptors do not share functional redundancy in the conventional sense.
authors.affiliation: Department of Plant and Environmental Sciences, University of Gothenburg, Box 461, SE-405 30 Gothenburg, Sweden.
authors.forename: Henrik
authors.initials: H
authors.lastname: Aronsson
authors.affiliation: Department of Biology, University of Leicester, University Road, Leicester LE1 7RH, UK.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Combe
authors.affiliation: Department of Biology, University of Leicester, University Road, Leicester LE1 7RH, UK.
authors.forename: Ramesh
authors.initials: R
authors.lastname: Patel
authors.affiliation: Laboratoire de Physiologie Végétale, Université de Neuchâtel, Rue Emile-Argand 11, 2007 Neuchâtel, Switzerland.
authors.forename: Birgit
authors.initials: B
authors.lastname: Agne
authors.affiliation: Laboratoire de Physiologie Végétale, Université de Neuchâtel, Rue Emile-Argand 11, 2007 Neuchâtel, Switzerland.
authors.forename: Meryll
authors.initials: M
authors.lastname: Martin
authors.affiliation: Laboratoire de Physiologie Végétale, Université de Neuchâtel, Rue Emile-Argand 11, 2007 Neuchâtel, Switzerland.
authors.forename: Felix
authors.initials: F
authors.lastname: Kessler
authors.affiliation: Department of Biology, University of Leicester, University Road, Leicester LE1 7RH, UK.
authors.forename: Paul
authors.initials: P
authors.lastname: Jarvis
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D002734:Chlorophyll / Q000032:analysis; D002736:Chloroplasts / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics; D017354:Point Mutation; D055503:Protein Multimerization; D021381:Protein Transport; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C114786:Toc33 protein, Arabidopsis; D002734:Chlorophyll; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases
references.citation: Agne, B., Infanger, S., Wang, F., Hofstetter, V., Rahim, G., Martin, M., Lee, D.W., Hwang, I., Schnell, D. and Kessler, F. (2009) A Toc159 import receptor mutant, defective in hydrolysis of GTP, supports preprotein import into chloroplasts. J. Biol. Chem. 284, 8670-8679.
references.citation: Aronsson, H. and Jarvis, P. (2002) A simple method for isolating import-competent Arabidopsis chloroplasts. FEBS Lett. 529, 215-220.
references.citation: Aronsson, H., Combe, J. and Jarvis, P. (2003) Unusual nucleotide-binding properties of the chloroplast protein import receptor, atToc33. FEBS Lett. 544, 79-85.
references.citation: Aronsson, H., Combe, J., Patel, R. and Jarvis, P. (2006) In vivo assessment of the significance of phosphorylation of the Arabidopsis chloroplast protein import receptor, atToc33. FEBS Lett. 580, 649-655.
references.citation: Aronsson, H., Boij, P., Patel, R., Wardle, A., Töpel, M. and Jarvis, P. (2007) Toc64/OEP64 is not essential for the efficient import of proteins into chloroplasts in Arabidopsis thaliana. Plant J. 52, 53-68.
references.citation: Bate, N., Spurr, C., Foster, G.D. and Twell, D. (1996) Maturation-specific translational enhancement mediated by the 5′-UTR of a late pollen transcript. Plant J. 10, 613-623.
references.citation: Bauer, J., Chen, K., Hiltbunner, A., Wehrli, E., Eugster, M., Schnell, D. and Kessler, F. (2000) The major protein import receptor of plastids is essential for chloroplast biogenesis. Nature, 403, 203-207.
references.citation: Bauer, J., Hiltbrunner, A., Weibel, P., Vidi, P.A., Alvarez-Huerta, M., Smith, M.D., Schnell, D.J. and Kessler, F. (2002) Essential role of the G-domain in targeting of the protein import receptor atToc159 to the chloroplast outer membrane. J. Cell Biol. 159, 845-854.
references.citation: Becker, T., Jelic, M., Vojta, A., Radunz, A., Soll, J. and Schleiff, E. (2004) Preprotein recognition by the Toc complex. EMBO J. 23, 520-530.
references.citation: Bölter, B., May, T. and Soll, J. (1998) A protein import receptor in pea chloroplasts, Toc86, is only a proteolytic fragment of a larger polypeptide. FEBS Lett. 441, 59-62.
references.citation: Bourne, H.R., Sanders, D.A. and McCormick, F. (1991) The GTPase superfamily: conserved structure and molecular mechanism. Nature, 349, 117-127.
references.citation: Chen, D. and Schnell, D.J. (1997) Insertion of the 34-kDa chloroplast protein import component, IAP34, into the chloroplast outer membrane is dependent on its intrinsic GTP-binding capacity. J. Biol. Chem. 272, 6614-6620.
references.citation: Chen, K., Chen, X. and Schnell, D.J. (2000) Initial binding of preproteins involving the Toc159 receptor can be bypassed during protein import into chloroplasts. Plant Physiol. 122, 813-822.
references.citation: Clough, S.J. and Bent, A.F. (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735-743.
references.citation: Constan, D., Patel, R., Keegstra, K. and Jarvis, P. (2004) An outer envelope membrane component of the plastid protein import apparatus plays an essential role in Arabidopsis. Plant J. 38, 93-106.
references.citation: Farnsworth, C.L. and Feig, L.A. (1991) Dominant inhibitory mutations in the Mg2+-binding site of RasH prevent its activation by GTP. Mol. Cell. Biol. 11, 4822-4829.
references.citation: Gutensohn, M., Schulz, B., Nicolay, P. and Flügge, U.I. (2000) Functional analysis of the two Arabidopsis homologues of Toc34, a component of the chloroplast protein import apparatus. Plant J. 23, 771-783.
references.citation: Gutensohn, M., Fan, E., Frielingsdorf, S., Hanner, P., Hou, B., Hust, B. and Klösgen, R.B. (2006) Toc, Tic, Tat et al.: structure and function of protein transport machineries in chloroplasts. J. Plant Physiol. 163, 333-347.
references.citation: Hajdukiewicz, P., Svab, Z. and Maliga, P. (1994) The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol. Biol. 25, 989-994.
references.citation: Hall, T.A. (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95-98.
references.citation: Hiltbrunner, A., Bauer, J., Alvarez-Huerta, M. and Kessler, F. (2001) Protein translocon at the Arabidopsis outer chloroplast membrane. Biochem. Cell Biol. 79, 629-635.
references.citation: Hiltbrunner, A., Grunig, K., Alvarez-Huerta, M., Infanger, S., Bauer, J. and Kessler, F. (2004) AtToc90, a new GTP-binding component of the Arabidopsis chloroplast protein import machinery. Plant Mol. Biol. 54, 427-440.
references.citation: Hirsch, S., Muckel, E., Heemeyer, F., von Heijne, G. and Soll, J. (1994) A receptor component of the chloroplast protein translocation machinery. Science, 266, 1989-1992.
references.citation: Inaba, T. and Schnell, D.J. (2008) Protein trafficking to plastids: one theme, many variations. Biochem. J. 413, 15-28.
references.citation: Inaba, T., Li, M., Alvarez-Huerta, M., Kessler, F. and Schnell, D.J. (2003) atTic110 functions as a scaffold for coordinating the stromal events of protein import into chloroplasts. J. Biol. Chem. 278, 38617-38627.
references.citation: Ivanova, Y., Smith, M.D., Chen, K. and Schnell, D.J. (2004) Members of the Toc159 import receptor family represent distinct pathways for protein targeting to plastids. Mol. Biol. Cell 15, 3379-3392.
references.citation: Jarvis, P. (2008) Targeting of nucleus-encoded proteins to chloroplasts in plants (Tansley Review). New Phytol. 179, 257-285.
references.citation: Jarvis, P., Chen, L.J., Li, H., Peto, C.A., Fankhauser, C. and Chory, J. (1998) An Arabidopsis mutant defective in the plastid general protein import apparatus. Science, 282, 100-103.
references.citation: Jelic, M., Sveshnikova, N., Motzkus, M., Horth, P., Soll, J. and Schleiff, E. (2002) The chloroplast import receptor Toc34 functions as preprotein-regulated GTPase. Biol. Chem. 383, 1875-1883.
references.citation: Jelic, M., Soll, J. and Schleiff, E. (2003) Two Toc34 homologues with different properties. Biochemistry, 42, 5906-5916.
references.citation: John, J., Sohmen, R., Feuerstein, J., Linke, R., Wittinghofer, A. and Goody, R.S. (1990) Kinetics of interaction of nucleotides with nucleotide-free H-ras p21. Biochemistry, 29, 6058-6065.
references.citation: Kessler, F. and Schnell, D.J. (2004) Chloroplast protein import: solve the GTPase riddle for entry. Trends Cell Biol. 14, 334-338.
references.citation: Kessler, F. and Schnell, D.J. (2006) The function and diversity of plastid protein import pathways: a multilane GTPase highway into plastids. Traffic, 7, 248-257.
references.citation: Kessler, F., Blobel, G., Patel, H.A. and Schnell, D.J. (1994) Identification of two GTP-binding proteins in the chloroplast protein import machinery. Science, 266, 1035-1039.
references.citation: Koenig, P., Oreb, M., Hofle, A., Kaltofen, S., Rippe, K., Sinning, I., Schleiff, E. and Tews, I. (2008) The GTPase cycle of the chloroplast import receptors Toc33/Toc34: implications from monomeric and dimeric structures. Structure, 16, 585-596.
references.citation: Kubis, S., Baldwin, A., Patel, R., Razzaq, A., Dupree, P., Lilley, K., Kurth, J., Leister, D. and Jarvis, P. (2003) The Arabidopsis ppi1 mutant is specifically defective in the expression, chloroplast import, and accumulation of photosynthetic proteins. Plant Cell, 15, 1859-1871.
references.citation: Kubis, S., Patel, R., Combe, J. et al. (2004) Functional specialization amongst the Arabidopsis Toc159 family of chloroplast protein import receptors. Plant Cell, 16, 2059-2077.
references.citation: Kubis, S.E., Lilley, K.S. and Jarvis, P. (2008) Isolation and preparation of chloroplasts from Arabidopsis thaliana plants. Methods Mol. Biol. 425, 171-186.
references.citation: Lee, K.H., Kim, S.J., Lee, Y.J., Jin, J.B. and Hwang, I. (2003) The M domain of atToc159 plays an essential role in the import of proteins into chloroplasts and chloroplast biogenesis. J. Biol. Chem. 278, 36794-36805.
references.citation: Lee, J., Wang, F. and Schnell, D.J. (2009) Toc receptor dimerization participates in the initiation of membrane translocation during protein import into chloroplasts. J. Biol. Chem. 284, 31130-31141.
references.citation: Li, H., Shen, J.J., Zheng, Z.L., Lin, Y. and Yang, Z. (2001) The Rop GTPase switch controls multiple developmental processes in Arabidopsis. Plant Physiol. 126, 670-684.
references.citation: Li, H.M., Kesavulu, M.M., Su, P.H., Yeh, Y.H. and Hsiao, C.D. (2007) Toc GTPases. J. Biomed. Sci. 14, 505-508.
references.citation: Ma, Y., Kouranov, A., LaSala, S.E. and Schnell, D.J. (1996) Two components of the chloroplast protein import apparatus, IAP86 and IAP75, interact with the transit sequence during the recognition and translocation of precursor proteins at the outer envelope. J. Cell Biol. 134, 315-327.
references.citation: Olsen, L.J. and Keegstra, K. (1992) The binding of precursor proteins to chloroplasts requires nucleoside triphosphates in the intermembrane space. J. Biol. Chem. 267, 433-439.
references.citation: Olsen, L.J., Theg, S.M., Selman, B.R. and Keegstra, K. (1989) ATP is required for the binding of precursor proteins to chloroplasts. J. Biol. Chem. 264, 6724-6729.
references.citation: Porra, R.J., Thompson, W.A. and Kriedemann, P.E. (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim. Biophys. Acta, 975, 384-394.
references.citation: Rahim, G., Bischof, S., Kessler, F. and Agne, B. (2009) In vivo interaction between atToc33 and atToc159 GTP-binding domains demonstrated in a plant split-ubiquitin system. J. Exp. Bot. 60, 257-267.
references.citation: Reddick, L.E., Vaughn, M.D., Wright, S.J., Campbell, I.M. and Bruce, B.D. (2007) In vitro comparative kinetic analysis of the chloroplast Toc GTPases. J. Biol. Chem. 282, 11410-11426.
references.citation: Rodriguez, C.M., Freire, M.A., Camilleri, C. and Robaglia, C. (1998) The Arabidopsis thaliana cDNAs coding for eIF4E and eIF(iso)4E are not functionally equivalent for yeast complementation and are differentially expressed during plant development. Plant J. 13, 465-473.
references.citation: Schleiff, E., Soll, J., Sveshnikova, N., Tien, R., Wright, S., Dabney-Smith, C., Subramanian, C. and Bruce, B.D. (2002) Structural and guanosine triphosphate/diphosphate requirements for transit peptide recognition by the cytosolic domain of the chloroplast outer envelope receptor, Toc34. Biochemistry, 41, 1934-1946.
references.citation: Schleiff, E., Jelic, M. and Soll, J. (2003) A GTP-driven motor moves proteins across the outer envelope of chloroplasts. Proc. Natl Acad. Sci. USA, 100, 4604-4609.
references.citation: Schnell, D.J., Kessler, F. and Blobel, G. (1994) Isolation of components of the chloroplast protein import machinery. Science, 266, 1007-1012.
references.citation: Schnell, D.J., Blobel, G., Keegstra, K., Kessler, F., Ko, K. and Soll, J. (1997) A consensus nomenclature for the protein-import components of the chloroplast envelope. Trends Cell Biol. 7, 303-304.
references.citation: Schünemann, D. (2007) Mechanisms of protein import into thylakoids of chloroplasts. Biol. Chem. 388, 907-915.
references.citation: Seedorf, M., Waegemann, K. and Soll, J. (1995) A constituent of the chloroplast import complex represents a new type of GTP-binding protein. Plant J. 7, 401-411.
references.citation: Smith, M.D. (2006) Protein import into chloroplasts: an ever-evolving story. Can. J. Bot. 84, 531-542.
references.citation: Smith, M.D., Hiltbrunner, A., Kessler, F. and Schnell, D.J. (2002) The targeting of the atToc159 preprotein receptor to the chloroplast outer membrane is mediated by its GTPase domain and is regulated by GTP. J. Cell Biol. 159, 833-843.
references.citation: Smith, M.D., Rounds, C.M., Wang, F., Chen, K., Afitlhile, M. and Schnell, D.J. (2004) atToc159 is a selective transit peptide receptor for the import of nucleus-encoded chloroplast proteins. J. Cell Biol. 165, 323-334.
references.citation: Soll, J. and Schleiff, E. (2004) Protein import into chloroplasts. Nat. Rev. Mol. Cell Biol. 5, 198-208.
references.citation: Strasser, R.J., Srivastava, A. and Tsimilli-Michael, M. (2004) Analysis of the chlorophyll a fluorescence transient. In Advances in Photosynthesis and Respiration: Chlorophyll Fluorescence, a Signature of Photosynthesis (Papageorgiou, G. and Govindjee, eds). Dordrecht, The Netherlands: Kluwer Academic Publishers, pp. 321-362.
references.citation: Sun, C.W., Chen, L.J., Lin, L.C. and Li, H.M. (2001) Leaf-specific upregulation of chloroplast translocon genes by a CCT motif-containing protein, CIA 2. Plant Cell, 13, 2053-2061.
references.citation: Sun, Y.J., Forouhar, F., Li Hm, H.M., Tu, S.L., Yeh, Y.H., Kao, S., Shr, H.L., Chou, C.C., Chen, C. and Hsiao, C.D. (2002) Crystal structure of pea Toc34, a novel GTPase of the chloroplast protein translocon. Nat. Struct. Biol. 9, 95-100.
references.citation: Sveshnikova, N., Soll, J. and Schleiff, E. (2000) Toc34 is a preprotein receptor regulated by GTP and phosphorylation. Proc. Natl Acad. Sci. USA, 97, 4973-4978.
references.citation: Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673-4680.
references.citation: Wallas, T.R., Smith, M.D., Sanchez-Nieto, S. and Schnell, D.J. (2003) The roles of Toc34 and Toc75 in targeting the Toc159 preprotein receptor to chloroplasts. J. Biol. Chem. 278, 44289-44297.
references.citation: Wang, F., Agne, B., Kessler, F. and Schnell, D.J. (2008) The role of GTP binding and hydrolysis at the atToc159 preprotein receptor during protein import into chloroplasts. J. Cell Biol. 183, 87-99.
references.citation: Weibel, P., Hiltbrunner, A., Brand, L. and Kessler, F. (2003) Dimerization of Toc-GTPases at the chloroplast protein import machinery. J. Biol. Chem. 278, 37321-37329.
references.citation: Yeh, Y.H., Kesavulu, M.M., Li, H.M., Wu, S.Z., Sun, Y.J., Konozy, E.H. and Hsiao, C.D. (2007) Dimerization is important for the GTPase activity of chloroplast translocon components atToc33 and psToc159. J. Biol. Chem. 282, 13845-13853.
references.citation: Young, M.E., Keegstra, K. and Froehlich, J.E. (1999) GTP promotes the formation of early-import intermediates but is not required during the translocation step of protein import into chloroplasts. Plant Physiol. 121, 237-244.