The Role of Tic Components in Protein Import into Chloroplasts

University Of Massachusetts Amherst, Amherst MA

Investigators

Abstract

Abstract Organelle identity and development rely on a complex set of intracellular protein trafficking systems that mediate the specific targeting of nuclear-encoded proteins to their proper subcellular compartment. Although protein targeting systems have been described for all organelles, the mechanisms of signal recognition and protein translocation at boundary membranes remain major topics of investigation in cell biology. The long-term goal of the research is to understand the mechanism of protein import into plant chloroplasts as a model for protein targeting and organelle biogenesis in eukaryotic cells. 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, and aspects of fatty acid and amino acid metabolism. 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 cytoplasm. As a consequence, protein import plays a central role in the differentiation and maintenance of chloroplasts during plant growth and development. The import of the majority of nuclear-encoded proteins is directed by an intrinsic, N-terminal transit sequence, and is mediated by the coordinate action of protein translocon complexes in the outer (Toc apparatus) and inner (Tic apparatus) envelope membranes of the chloroplast. This project focuses on the roles of the Tic machinery in preprotein translocation at the inner envelope membrane. The Tic apparatus performs three key functions. First, it physically associates with the Toc apparatus to facilitate the direct translocation of preproteins from the cytoplasm to the stroma. Second, it forms a selectively permeable protein-conducting channel across the inner membrane. Third, it coordinates the association of soluble stromal factors (e.g. molecular chaperones) with the translocon to facilitate the processing and folding of newly imported proteins. Three inner membrane proteins (Tic20, Tic22, and Tic110) have been identified which directly or indirectly associate with preproteins during import across the envelope. Tic22 is a peripheral membrane protein that is bound to the outer face of the inner membrane. Tic22 can be chemically cross-linked to preproteins as they emerge from the outer membrane translocon and thus might coordinate the association of the Toc and Tic machinery by guiding the preprotein to the Tic machinery. The availability of Arabidopsis mutants in which expression of Tic22 is disrupted permits direct testing of this hypothesis in vivo, with isolated chloroplasts and at the biochemical level. Similar approaches will determine whether Tic20, an integral inner membrane protein is a component of the protein conducting channel of the inner membrane. In addition, oligomeric complexes containing Tic20 from pea chloroplast envelope membranes will be purified and analyzed with the goal of identifying additional components of the Tic machinery. The third Tic component, Tic110, is an integral inner membrane protein with a large (~90 kDa) stromal domain. Potential roles of this protein in concentrating the ATP-dependent chaperone ClpC at import sites in the stroma and in stabilizing the insertion of preproteins across the inner membrane will be tested through in vitro binding studies of Tic110 to preproteins and ClpC. In addition, the preprotein and ClpC binding domains of Tic110 will be mapped using deletion analysis. As with the other proteins, the function of Tic110 will also be examined in vivo through characterization of plants in which its expression has been blocked. Overall, the synergistic use of biochemical and genetic approaches in this project should provide new insights into how cells assemble their organelles.