Interaction of Actin and the Chloroplast Protein Import Apparatus

Actin filaments are major components of the cytoskeleton and play numerous essential roles, including chloroplast positioning and plastid stromule movement, in plant cells. Actin is present in pea chloroplast envelope membrane preparations and is localized at the surface of the chloroplasts, as shown by agglutination of intact isolated chloroplasts by antibodies to actin. To identify chloroplast envelope proteins involved in actin binding, we have carried out actin co-immunoprecipitation and co-sedimentation experiments on detergent-solubilized pea chloroplast envelope membranes. Proteins co-immunoprecipitated with actin were identified by mass spectrometry and by Western blotting and included the Toc159, Toc75, Toc34, and Tic110 components of the TOC-TIC protein import apparatus. A direct interaction of actin with Escherichia coli-expressed Toc159, but not Toc33, was shown by co-sedimentation experiments, suggesting that Toc159 is the component of the TOC complex that interacts with actin on the cytosolic side of the outer envelope membrane. The physiological significance of this interaction is unknown, but it may play a role in the import of nuclear-encoded photosynthesis proteins.Actin is a ubiquitous protein of eukaryotic cells. Actin microfilaments are formed from polymerization of actin monomers and are a major component of the cytoskeleton. In plant cells, actin microfilaments are arranged in longitudinal arrays of thick actin bundles with randomly oriented thin actin filaments extending from the bundles (1). Chloroplasts are either aligned along the actin bundles or closely associated with the fine filaments and are surrounded by baskets of actin microfilaments (1, 2). A direct interaction of chloroplasts with the actin cytoskeleton has been postulated to anchor chloroplasts at appropriate intracellular positions (3). Chloroplast movement depends on cytosolic actin filaments and is stimulated by high light intensity (4). A chloroplast envelope protein involved in blue light-dependent chloroplast repositioning has been identified by the analysis of the Arabidopsis chup1 (chloroplast unusual positioning 1) mutant, which was unable to relocate its chloroplasts under high light stimulation (5). CHUP1 is a protein exclusively targeted to the chloroplast outer envelope membrane that is essential for chloroplast anchorage to the plasma membrane (6). CHUP1 interacts with actin and profilin, a modulator of actin polymerization, and it may play a regulatory role in actin polymerization during chloroplast photo-relocation (7).The interaction of amyloplasts with the actin cytoskeleton has been implicated in gravity perception and signal transduction. Several models for the role of the actin cytoskeleton have been proposed (8), but the nature of the interaction is not known. However, disruption of the actin cytoskeleton enhanced sedimentation of amyloplasts and promoted gravitropism (9, 10), and a role for myosin has been proposed on the basis of inhibitor experiments (11).The actin cytoskeleton and myosin have also been implicated in plastid stromule movement. Stromules (stroma-filled tubules) are highly dynamic tubular structures extending from the surface of all plastid types (12, 13). Stromules are delimited by the inner and outer plastid envelope membranes, which are closely associated (for a review, see Refs. 12 and 13). Experiments with inhibitors of microfilament- and microtubule-based movement suggested that stromules move along actin microfilaments powered by the ATPase activity of myosin motors (14). Physical connection between the envelope membranes seems likely to be required to provide a means of coordinating the movement of the inner envelope membrane with the microfilament-associated outer envelope membrane. There is evidence for direct connection of the inner and outer envelope membranes at contact sites, which support protein translocation through the protein import apparatus (15, 16). This apparatus consists of two membrane protein complexes that associate to allow translocation of nucleus-encoded proteins from the cytoplasm to the interior stromal compartment (for a review, see 17). The translocon at the outer envelope membrane of chloroplasts (TOC complex)² mediates the initial recognition of preproteins and their translocation across the outer membrane (18). The translocon at the inner envelope membrane of chloroplasts (TIC complex) physically associates with the TOC complex and provides the membrane translocation channel for the inner membrane. In addition, the TOC and TIC complexes interact with a set of molecular chaperones, which assist the transfer of imported proteins (19–21).With the aim of identifying components involved in the interaction of the chloroplast envelope with the actin cytoskeleton, we have used actin co-immunoprecipitation and co-sedimentation experiments with detergent-solubilized pea chloroplast envelope membranes. Components of the TOC-TIC protein import apparatus have been identified by mass spectrometry and Western blotting, and a direct interaction of Escherichia coli-expressed Toc159 with actin was demonstrated by co-sedimentation. This interaction may have a so far unrecognized physiological role in chloroplast protein import.