Danny J. Schnell

The major protein import receptor of plastids is essential for chloroplast biogenesis.

Light triggers the developmental programme in plants that leads to the production of photosynthetically active chloroplasts from non-photosynthetic proplastids. During this chloroplast biogenesis, the photosynthetic apparatus is rapidly assembled, mostly from nuclear-encoded imported proteins, which are synthesized in the cytosol as precursors with cleavable amino-terminal targeting sequences called transit sequences. Protein translocon complexes at the outer (Toc complex) and inner (Tic complex) envelope membranes recognize these transit sequences, leading to the precursors being imported. The Toc complex in the pea consists of three major components, Toc75, Toc34 and Toc159 (formerly termed Toc86). Toc159, which is an integral membrane GTPase, functions as a transit-sequence receptor. Here we show that Arabidopsis thaliana Toc159 (atToc159) is essential for the biogenesis of chloroplasts. In an Arabidopsis mutant (ppi2) that lacks atToc159, photosynthetic proteins that are normally abundant are transcriptionally repressed, and are found in much smaller amounts in the plastids, although ppi2 does not affect either the expression or the import of less abundant non-photosynthetic plastid proteins. These findings indicate that atToc159 is required for the quantitative import of photosynthetic proteins. Two proteins that are related to atToc159 (atToc120 and atToc132) probably help to maintain basal protein import in ppi2, and so constitute components of alternative, atToc159-independent import pathways.

The Plastid Protein THYLAKOID FORMATION1 and the Plasma Membrane G-Protein GPA1 Interact in a Novel Sugar-Signaling Mechanism in Arabidopsis

Mutations in genes encoding components of the heterotrimeric G-protein complex were previously shown to confer altered sensitivity to increased levels of d-glucose. This suggests that G-protein coupling may be a novel sugar-signaling mechanism in Arabidopsis thaliana. THYLAKOID FORMATION1 (THF1) is here demonstrated in vivo as a Gα interaction partner that functions downstream of the plasma membrane–delimited heterotrimeric G-protein (GPA1) in a d-glucose signaling pathway. THF1 is a plastid protein localized to both the outer plastid membrane and the stroma. Contact between root plastidic THF1 and GPA1 at the plasma membrane occurs at sites where the plastid membrane abuts the plasma membrane, as demonstrated by Förster resonance energy transfer (FRET). A probable role for THF1 in sugar signaling is demonstrated by both biochemical and genetic evidence. Root growth in the thf1-1 null mutant is hypersensitive to exogenous d-glucose, and THF1-overexpressing roots are resistant to inhibition of growth rate by high d-glucose. Additionally, THF1 levels are rapidly degraded by d-glucose but not l-glucose. The interaction between THF1 and GPA1 has been confirmed by in vitro and in vivo coimmunoprecipitation, FRET analysis, and genetic epistasis and provides evidence of a sugar-signaling mechanism between plastids and the plasma membrane.

atToc159 is a selective transit peptide receptor for the import of nucleus-encoded chloroplast proteins

The members of the Toc159 family of GTPases act as the primary receptors for the import of nucleus-encoded preproteins into plastids. Toc159, the most abundant member of this family in chloroplasts, is required for chloroplast biogenesis (Bauer, J., K. Chen, A. Hiltbunner, E. Wehrli, M. Eugster, D. Schnell, and F. Kessler. 2000. Nature. 403:203–207) and has been shown to covalently cross-link to bound preproteins at the chloroplast surface (Ma, Y., A. Kouranov, S. LaSala, and D.J. Schnell. 1996. J. Cell Biol. 134:1–13; Perry, S.E., and K. Keegstra. 1994. Plant Cell. 6:93–105). These reports led to the hypothesis that Toc159 functions as a selective import receptor for preproteins that are required for chloroplast development. In this report, we provide evidence that Toc159 is required for the import of several highly expressed photosynthetic preproteins in vivo. Furthermore, we demonstrate that the cytoplasmic and recombinant forms of soluble Toc159 bind directly and selectively to the transit peptides of these representative photosynthetic preproteins, but not representative constitutively expressed plastid preproteins. These data support the function of Toc159 as a selective import receptor for the targeting of a set of preproteins required for chloroplast biogenesis.

Arabidopsis Tic110 Is Essential for the Assembly and Function of the Protein Import Machinery of Plastids

The translocon at the inner envelope membrane of chloroplasts (Tic) plays a central role in plastid biogenesis by coordinating the sorting of nucleus-encoded preproteins across the inner membrane and coordinating the interactions of preproteins with the processing and folding machineries of the stroma. Despite these activities, the precise roles of known Tic proteins in translocation, sorting, and preprotein maturation have not been defined. In this report, we examine the in vivo function of a major Tic component, Tic110. We demonstrate that Arabidopsis thaliana Tic110 (atTic110) is essential for plastid biogenesis and plant viability. The downregulation of atTic110 expression results in the reduced accumulation of a wide variety of plastid proteins. The expression of dominant negative mutants of atTic110 disrupts assembly of Tic complexes and the translocation of preproteins across the inner envelope membrane. Together, these data suggest that Tic110 plays a general role in the import of nuclear-encoded preproteins as a common component of Tic complexes.

The Function and Diversity of Plastid Protein Import Pathways: A Multilane GTPase Highway into Plastids

The photosynthetic chloroplast is the hallmark organelle of green plants. During the endosymbiotic evolution of chloroplasts, the vast majority of genes from the original cyanobacterial endosymbiont were transferred to the host cell nucleus. Chloroplast biogenesis therefore requires the import of nucleus‐encoded proteins from their site of synthesis in the cytosol. The majority of proteins are imported by the activity of Toc and Tic complexes located within the chloroplast envelope. In addition to chloroplasts, plants have evolved additional, non‐photosynthetic plastid types that are essential components of all cells. Recent studies indicate that the biogenesis of various plastid types relies on distinct but homologous Toc–Tic import pathways that have specialized in the import of specific classes of substrates. These different import pathways appear to be necessary to balance the essential physiological role of plastids in cellular metabolism with the demands of cellular differentiation and plant development.

atTic110 functions as a scaffold for coordinating the stromal events of protein import into chloroplasts.

The translocon of the inner envelope membrane of chloroplasts (Tic) mediates the late events in the translocation of nucleus-encoded preproteins into chloroplasts. Tic110 is a major integral membrane component of active Tic complexes and has been proposed to function as a docking site for translocation-associated stromal factors and as a component of the protein-conducting channel. To investigate the various proposed functions of Tic110, we have investigated the structure, topology, and activities of a 97.5-kDa fragment of Arabidopsis Tic110 (atTic110) lacking only the amino-terminal transmembrane segments. The protein was expressed both in Escherichia coli and Arabidopsis as a stable, soluble protein with a high α-helical content. Binding studies demonstrate that a region of the atTic110-soluble domain selectively associates with chloroplast preproteins at the late stages of membrane translocation. These data support the hypothesis that the bulk of Tic110 extends into the chloroplast stroma and suggest that the domain forms a docking site for preproteins as they emerge from the Tic translocon.

Protein trafficking to plastids: one theme, many variations

Plastids are a diverse group of essential organelles in plants that include chloroplasts. The biogenesis and maintenance of these organelles relies on the import of thousands of nucleus-encoded proteins. The complexity of plastid structure has resulted in the evolution of at least four general import pathways that target proteins into and across the double membrane of the plastid envelope. Several of these pathways can be further divided into specialty pathways that mediate and regulate the import of specific classes of proteins. The co-ordination of import by these specialized pathways with changes in gene expression is critical for plastid and plant development. Moreover, protein import is acutely regulated in response to physiological and metabolic changes within the cell. In the present review we summarize the current knowledge of the mechanism of import via these pathways and highlight the regulatory mechanisms that integrate the plastid protein-trafficking pathways with the developmental and metabolic state of the plant.

Import Pathways of Chloroplast Interior Proteins and the Outer-Membrane Protein OEP14 Converge at Toc75

Most chloroplast outer-membrane proteins are synthesized at their mature size without cleavable targeting signals. Their insertion into the outer membrane is insensitive to thermolysin pretreatment of chloroplasts and does not require ATP. It has therefore been assumed that insertion of outer-membrane proteins proceeds through a different pathway from import into the interior of chloroplasts, which requires a thermolysin-sensitive translocon complex and ATP. Here, we show that a model outer-membrane protein, OEP14, competed with the import of a chloroplast interior protein, indicating that the two import pathways partially overlapped. Cross-linking studies showed that, during insertion, OEP14 was associated with Toc75, a thermolysin-resistant component of the outer-membrane protein–conducting channel that mediates the import of interior-targeted precursor proteins. Whereas almost no OEP14 inserted into protein-free liposomes, OEP14 inserted into proteoliposomes containing reconstituted Toc75 with a high efficiency. Taken together, our data indicate that Toc75 mediates OEP14 insertion, and therefore plays a dual role in the targeting of proteins to the outer envelope membrane and interior of chloroplasts.

The targeting of the atToc159 preprotein receptor to the chloroplast outer membrane is mediated by its GTPase domain and is regulated by GTP

The multimeric translocon at the outer envelope membrane of chloroplasts (Toc) initiates the recognition and import of nuclear-encoded preproteins into chloroplasts. Two Toc GTPases, Toc159 and Toc33/34, mediate preprotein recognition and regulate preprotein translocation. Although these two proteins account for the requirement of GTP hydrolysis for import, the functional significance of GTP binding and hydrolysis by either GTPase has not been defined. A recent study indicates that Toc159 is equally distributed between a soluble cytoplasmic form and a membrane-inserted form, raising the possibility that it might cycle between the cytoplasm and chloroplast as a soluble preprotein receptor. In the present study, we examined the mechanism of targeting and insertion of the Arabidopsis thaliana orthologue of Toc159, atToc159, to chloroplasts. Targeting of atToc159 to the outer envelope membrane is strictly dependent only on guanine nucleotides. Although GTP is not required for initial binding, the productive insertion and assembly of atToc159 into the Toc complex requires its intrinsic GTPase activity. Targeting is mediated by direct binding between the GTPase domain of atToc159 and the homologous GTPase domain of atToc33, the Arabidopsis Toc33/34 orthologue. Our findings demonstrate a role for the coordinate action of the Toc GTPases in assembly of the functional Toc complex at the chloroplast outer envelope membrane.

Chloroplast biogenesis: diversity and regulation of the protein import apparatus.

The biogenesis of chloroplasts is dependent on the coordinate expression of genes encoded in both nuclear and plastid genomes. The chloroplast protein import machinery plays key roles in organelle biogenesis by mediating the import and assembly of thousands of nuclear-encoded proteins into the organelle. It is now apparent that multiple levels of control exist to integrate the activities of the protein import apparatus into the network of chloroplast-nuclear communication that is essential to maintain organelle homeostasis. The import apparatus has diversified into small, functionally specialized gene families to coordinate the import of distinct classes of differentially expressed proteins. Protein targeting to chloroplasts also has evolved regulatory mechanisms that respond to cellular developmental and physiological changes, including redox sensing, phosphorylation, and dual targeting. Recent studies also have revealed new components that could represent additional levels of control on the import process.