|Danny J. Schnell
Professor, University of Massachusetts
Ph.D.: University of California, Davis, CA
Mechanisms of Protein Import into the Plant Chloroplast, Protein Targeting, Organelle Biogenesis
A fundamental element of all biological systems is the ability to generate organization and structure at the expense of chemical energy. In higher organisms, biological structure is reflected in the organization of cells into a number of membrane-delineated compartments, called organelles. The organelles serve to sequester and assemble the vast array of metabolic reactions within the cell, thereby insuring that these reactions proceed in a regulated and coordinated manner. The vast majority of the structural components and enzymes of organelles are encoded in nuclear DNA and synthesized in the cytosolic compartment of the cell. Therefore, the development and maintenance of cellular architecture relies on a number of elaborate trafficking systems that sort the newly synthesized proteins to their proper organelle (figure 1). In general terms, these trafficking systems are envisioned to consist of two components. One component is a molecular signal carried by the protein that marks it for delivery to the appropriate organelle. The second component is a signal recognition system at the organelle surface that decodes the signal. A match between signal and recognition system triggers a cascade of reactions that result in the transport of the protein across the membrane boundary into the organelle and its assembly into a functional complex. This general targeting principle appears to function in all organelles. However, the molecular mechanism of protein targeting has not been determined in any system. The long term goal of our research is to understand the molecular mechanism by which cytosolically synthesized proteins are targeted and assembled into their appropriate organelle.
Our model system for investigating protein trafficking is the process of protein import into the chloroplasts of higher plants. Chloroplasts are subdivided by three non-contiguous membrane systems into at least six suborganellar compartments that serve to segregate and organize a number of essential metabolic functions; most notably the reactions of photosynthesis. Although the chloroplast contains its own small genome, the vast majority of its protein components are encoded by nuclear genes and must be imported into the organelle after translation in the cytosol (figure 2). The intrinsic signal responsible for chloroplast targeting, called the transit sequence, has been defined and is carried within an amino-terminal extension on the newly synthesized protein. The initial, common step in the import of these precursor proteins is their recognition and transport across the boundary of the organelle through the double membrane of the chloroplast envelope. Little is known about the chloroplast envelope components that recognize this signal or mediate the subsequent membrane transport steps. Our current studies focus on identifying components of the chloroplast envelope that participate in import and defining the specific roles for these import components in transit sequence recognition and precursor protein transport.
Our major effort during the past six years has been the identification of protein import components of the chloroplast envelope. This strategy is motivated by the principle that an understanding of import mechanisms requires a detailed biochemical analysis of the import machinery. Our initial approaches took advantage of the well-established in vitro import assays using isolated chloroplasts from pea. The use of trapped import intermediates and covalent cross-linking strategies led us to the discovery of two unique membrane-bound translocation systems in the outer (Toc components) and inner (Tic components) envelope (figure 3). The Toc system contains a core complex of three integral membrane proteins, Toc159, Toc75, and Toc34. The integral outer membrane GTPase, Toc159, forms the primary contact with the targeting signals (transit sequences) of bound preproteins in vitro, and we hypothesize that it serves as the major import receptor. Toc159 and a second Toc GTPase, Toc34, form a complex with Toc75, a porin-like protein that appears to constitute a major component of the protein-conducting channel at the outer membrane. On the basis of the characteristics of the Toc components and an analysis of the early stages of import into isolated chloroplasts, we proposed that Toc159 and Toc34 form an integrated transit sequence recognition system at the chloroplast surface. GTP-binding/hydrolysis at the GTPases regulate the presentation of the preprotein to the translocation channel. As such, Toc159 and Toc34 would act as the gate to the translocation machinery. The precise role of these unique GTPases and their relationship to Toc75 are major areas of current investigation in my laboratory.
The Toc components associate with three inner membrane proteins, Tic110, Tic22 and Tic20, in a Toc-Tic supercomplex to form functional import sites. In contrast to the Toc complex, we have not detected a stable complex consisting exclusively of Tic components. The assembly of a functional inner membrane translocon appears to be mediated by the direct or indirect association of Tic components with the Toc complex. Therefore, the Toc complex is likely to control the formation of translocation contact sites in addition to its roles in preprotein recognition and translocation. We originally identified Tic110 in an import-intermediate associated complex, and the laboratories of Keegstra, Blobel and Kessler have provided evidence that Tic110 serves as a docking site for stromal chaperones that bind to preproteins as they emerge from the import machinery. In this respect, the role of Tic110 appears to be analogous to that of Tim44 in recruiting chaperones to the import complex of the mitochondrial inner membrane. We have identified Tic20 and Tic22 as candidates for core components of the inner membrane translocon. Tic22 is peripherally associated with the outer face of the inner membrane, and it appears to be the first inner membrane protein that associates with preproteins upon insertion across the outer membrane. In contrast to Tic22, Tic20 is deeply integrated into the membrane with limited exposure of its structure to the stroma or intermembrane space. It cross-links with preproteins at the late stages in import. On the basis of these observations, we propose Tic20 as a candidate for a component of the protein-conducting machinery of the inner membrane translocon.
The analyses of the import components provide an emerging picture of two dynamic translocons incorporating regulatory and transport activities. Although the general features of targeting to chloroplasts resemble targeting to mitochondria, it is apparent from the most recent developments in both fields that the underlying mechanisms of protein import are distinct. With the exception of the involvement of molecular chaperones, the known import components in the two systems exhibit no apparent primary structural relationships. Although at first unexpected, these results are consistent with the need to maintain organelle identity during the endosymbiotic assimilation of two distinct organelles. In fact, the presence of the membrane GTPases recalls events in the co-translational targeting of ER proteins, and represents a unique example of the role of GTPases in regulating intracellular trafficking.
Inoue H, Wang F, Inaba T, Schnell DJ. 2011. Energetic manipulation of chloroplast protein import and the use of chemical cross-linkers to map protein-protein interactions. Methods Mol Biol.774:307-20. [PubMed]
Lee J, Wang F, Schnell DJ. 2009. Toc receptor dimerization participates in the initiation of membrane translocation during protein import into chloroplasts. J Biol Chem. 284:31130-41. [PubMed]
Huang J, Taylor JP, Chen JG, Uhrig JF, Schnell DJ, Nakagawa T, Korth KL, Jones AM. 2006. The Plastid Protein THYLAKOID FORMATION1 and the Plasma Membrane G-Protein GPA1 Interact in a Novel Sugar-Signaling Mechanism in Arabidopsis. Plant Cell. Mar 31; [Epub ahead of print]
Kessler F, Schnell DJ. 2006. The function and diversity of plastid protein import pathways: a multilane GTPase highway into plastids. Traffic. Mar;7(3):248-57.
Inaba, T., Alvarez-Huerta, M., Li, M., Bauer,B., Ewers, C., Kessler, F. and D.J. Schnell. 2005. atTic110 is essential for the assembly and function of the protein import machinery of plastids. Plant Cell. 17:1482-96.
Smith, M.D. and D.J. Schnell. 2004. Chloroplast protein targeting: multiple pathways for a complex organelle in Protein movement across membranes. J. Eichler, ed. Landes Biosciences, Georgetown, TX., pg. 1-18.
Tu S.-H., Chen, L.-J., Smith, M.D., Su, Y.S., Schnell, D.J. and H.-M. Li. 2004. Import pathways of chloroplast interior proteins and outer-membrane proteins converge at Toc75. Plant Cell 16: 2078-2088.
Kessler, F. and D.J. Schnell. 2004. Chloroplast protein import: solve the GTPase riddle for entry!. Trends Cell Biol. 14:334-338.
Ivanova, Y., Smith, M.D., Chen, K. and D. J. Schnell. 2004. Members of the Toc159 import receptor family represent distinct pathways for protein import into plastids. Mol. Biol. Cell, 15:3379-3392.
Smith, M.D, Rounds, C.M., Wang, F., Chen, K., Afitlhile, M. and D.J. Schnell. 2004. The cytoplasmic form of atToc159 is a selective transit peptide receptor for the import of nucleus-encoded chloroplast proteins. J. Cell Biol. 165: 323-334.
Wallas, T.R., Smith, M.D., Sanchez-Nieto, S. and D.J. Schnell. 2003. The roles of Toc34 and Toc75 in targeting the Toc159 preprotein receptor to chloroplasts. J. Biol. Chem. 278:44289-97.
Inaba, T., Li, M., Alvarez-Huerta, M., Kessler, F. and D.J. Schnell. 2003. atTic110 Functions as a Scaffold for Coordinating the Stromal Events of Protein Import into Chloroplasts. J. Biol. Chem. 278:38617-27.
Schnell D.J. and D.N. Hebert. 2003. Protein translocons: multifunctional mediators of protein translocation across membranes. Cell 112:491-505
Bauer, J., Hiltbrunner, A., Weibel, P., Vidi, P.A., Alvarez-Huerta, M., Smith, M.D., Schnell, D.J. and F. Kessler. 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.
Smith, M.D., Hiltbrunner, A., Kessler, F. and D.J. Schnell. 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.
Kessler, F. and D.J. Schnell. 2002. A GTPase gate for protein import into chloroplasts. Nat. Struct. Biol. 9:81-83
Chen, X., Smith, M.D., Fitzpatrick, L. and D.J. Schnell. 2002. In vivo analysis of the role of atTic20 in protein import into chloroplasts. Plant Cell 14:641-654
Smith, M.D., Fitzpatrick, L., Keegstra, K. and D.J. Schnell. 2002. In vitro analysis of chloroplast protein import. In Current Protocols in Cell Biology, J. S. Bonifacino, M. Dasso, J. B. Harford, J. Lippincott-Schwartz, and K.M. Yamada, eds. NY: John Wiley & Sons Inc.
Smith, M.D. and D.J. Schnell. 2001. Peroxisomal protein import. the paradigm shifts. Cell 105:293-296.
Chen, K., Chen, X. and D.J. Schnell. Initial binding of preproteins involving the Toc159 receptor can be bypassed during protein import into chloroplasts. Plant Physiol., in press.
Bauer,B., Chen, K., Hiltbunner, A., Wehrli, E., Eugster, M., Schnell, D.J. and F. Kessler. 2000. The major protein import receptor of plastids is essential for chloroplast biogenesis. Nature 403:203-207.
Kouranov, A., Wang, H. and D.J. Schnell. 1999. Tic22 is targeted to the intermembrane space of chloroplasts by a novel pathway. J. Biol. Chem. 274: 25181-25186.
Kessler F., Schnell, D.J. and G. Blobel. 1999. Isolation and molecular characterization of chloroplast plastoglobules: the plastoglobule associated protein PG40 is conserved from cyanobacteria to higher plants. Planta 208: 107-113.
Schnell, D.J. 1999. Functions and origins of the chloroplast protein import machinery. Essays in Biochem., in press.
Chen, X. and D.J. Schnell. 1999. Protein import into chloroplasts. Trends in Cell Biol. 9: 222-223.
Kouranov, A., X. Chen, B. Fuks, and D.J. Schnell. 1998. Tic20 and Tic22 are new components of the protein import apparatus at the chloroplast inner envelope membrane. J. Cell Biol.143: 991-1002.
Schnell, D.J. 1998. Protein targeting to the thylakoid membrane. Annu. Rev. Plant Phys. Plant Mol. Biol. 49: 97-126.
Kouranov, A., and D.J. Schnell. 1997. Analysis of the interactions of preproteins with the import machinery over the course of protein import into chloroplasts. J. Cell Biol. 139: 1677-1685.
Chen, D. and D.J. Schnell. 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.
Schnell, D.J., G. Blobel, K. Keegstra, F. Kessler, K. Ko, J. Soll. 1997. A consensus nomenclature for the protein-import components of the chloroplast envelope. Trends in Cell Biol. 7: 303-304.
Fuks, B. and D.J. Schnell. 1997. Mechanism of protein transport across the chloroplast envelope. Plant Physiol. 114: 405-410.
Ma, Y., A. Kouranov, S.E. LaSala and D.J. Schnell. 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.
Kouranov, A. and D. J. Schnell. 1996. Protein translocation at the envelope and thylakoid membranes of chloroplasts. J. Biol. Chem. 271: 31009-31012.
Schnell, D.J. 1995. Shedding light on the chloroplast protein import machinery. Cell 83: 521-524.
Tian, F., Y. Ma, A. Kouranov, S.E. LaSala, and D.J. Schnell. 1995. Molecular dissection of the mechanism of protein import into chloroplasts. In: Cold Spring Harbor Symposia on Quantitative Biology, Protein Kinesis: The dynamics of protein trafficking and stability. Volume 60. 629-636.
Schnell, D.J., F. Kessler and G. Blobel. 1994. Isolation of componentsof the chloroplast protein import machinery. Science 266: 1007-1012.
Kessler, F., G. Blobel, H.A. Patel and D.J. Schnell. 1994. Identification of two GTP-binding proteins in the chloroplast protein import machinery. Science 266: 1035-1039.