Modifications at the A-domain of the chloroplast import receptor Toc159

Two families of GTPases, the Toc34 and Toc159 GTPase families, take on the task of preprotein recognition at the translocon at the outer membrane of chloroplasts (TOC translocon). The major Toc159 family members have highly acidic N-terminal domains (A-domains) that are non-essential and so far have escaped functional characterization. But recently, interest in the role of the A-domain has strongly increased. The new data of three independent studies provide evidence that the Toc159 A-domain (I) participates in preprotein selectivity, (II) has typical features of intrinsically unfolded proteins and (III) is highly phosphorylated and possibly released from the rest of the protein by a proteolytic event. This hints at a complex regulation of A-domain function that is important for the maintenance of the preprotein selectivity at the TOC translocons.Key words: chloroplast, import, Toc159, acidic domain, kinase, protease     

Initial binding of preproteins involving the Toc159 receptor can be bypassed during protein import into chloroplasts

Two integral outer envelope GTPases, Toc34 and Toc86, are proposed to regulate the recognition and translocation of nuclear-encoded preproteins during the early stages of protein import into chloroplasts. Defining the precise roles of Toc86 and Toc34 has been complicated by the inability to distinguish their GTPase activities. Furthermore, the assignment of Toc86 function is rendered equivocal by recent reports suggesting that the standard protocol for the isolation of chloroplasts results in significant proteolysis of Toc86 (B. Bolter, T. May, J. Soll [1998] FEBS Lett 441: 59-62; G. Schatz [1998] Nature 395: 439-440). We demonstrate that Toc86 corresponds to a native protein of 159 kD in pea (Pisum sativum), designated Toc159. We take advantage of the proteolytic sensitivity of Toc159 to selectively remove its 100-kD cytoplasmic GTPase domain and thereby distinguish its activities from other import components. Proteolysis eliminates detectable binding of preproteins at the chloroplast surface, which is consistent with the proposed role of Toc159 as a receptor component. Remarkably, preprotein translocation across the outer membrane can occur in the absence of the Toc159 cytoplasmic domain, suggesting that binding can be bypassed. Translocation remains sensitive to GTP analogs in the absence of the Toc159 GTP-binding domain, providing evidence that Toc34 plays a key role in the regulation of translocation by GTP.     

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.     

Structural and guanosine triphosphate/diphosphate requirements for transit peptide recognition by the cytosolic domain of the chloroplast outer envelope receptor, Toc34

Toc34 is a transmembrane protein located in the outer envelope membrane of chloroplasts and involved in transit peptide recognition. The cytosolic region of Toc34 reveals 34% alpha-helical and 26% beta-strand structure and is stabilized by intramolecular electrostatic interaction. Toc34 binds both chloroplast preproteins and isolated transit peptides in a guanosine triphosphate- (GTP-) dependent mechanism. In this study we demonstrate that the soluble, cytosolic domain of Toc34 (Toc34deltaTM) functions as receptor in vitro and is capable to compete with the import of the preprotein of the small subunit (preSSU) of ribulose-1,5-bisphosphate carboxylase-oxygenase into chloroplasts in a GTP-dependent manner. We have developed a biosensor assay to study the interaction of Toc34deltaTM with purified preproteins and transit peptides. The results are compared with the interactions of both a full-size preprotein and the transit peptide of preSSU with the translocon of the outer envelope of chloroplasts (Toc complex) in situ. Several mutants of the transit peptide of preSSU were evaluated to identify amino acid segments that are specifically recognized by Toc34. We present a model of how Toc34 may recognize the transit peptide and discuss how this interaction may facilitate interaction and translocation of preproteins via the Toc complex in vivo.     

Toc64, a new component of the protein translocon of chloroplasts

A subunit of the preprotein translocon of the outer envelope of chloroplasts (Toc complex) of 64 kD is described, Toc64. Toc64 copurifies on sucrose density gradients with the isolated Toc complex. Furthermore, it can be cross-linked in intact chloroplasts to a high molecular weight complex containing both Toc and Tic subunits and a precursor protein. The 0 A cross-linker CuCl(2) yields the reversible formation of disulfide bridge(s) between Toc64 and the established Toc complex subunits in purified outer envelope membranes. Toc64 contains three tetratricopeptide repeat motifs that are exposed at the chloroplast cytosol interface. We propose that Toc64 functions early in preprotein translocation, maybe as a docking protein for cytosolic cofactors of the protein import into chloroplasts.     

Toc,tic, and chloroplast protein import

The vast majority of chloroplast proteins are synthesized in precursor form on cytosolic ribosomes. Chloroplast precursor proteins have cleavable, N-terminal targeting signals called transit peptides. Transit peptides direct precursor proteins to the chloroplast in an organelle-specific way. They can be phosphorylated by a cytosolic protein kinase, and this leads to the formation of a cytosolic guidance complex. The guidance complex--comprising precursor, hsp70 and 14-3-3 proteins, as well as several unidentified components--docks at the outer envelope membrane. Translocation of precursor proteins across the envelope is achieved by the joint action of molecular machines called Toc (translocon at the outer envelope membrane of chloroplasts) and Tic (translocon at the inner envelope membrane of chloroplasts), respectively. The action of the Toc/Tic apparatus requires the hydrolysis of ATP and GTP at different levels, indicating energetic requirements and regulatory properties of the import process. The main subunits of the Toc and Tic complexes have been identified and characterized in vivo, in organello and in vitro. Phylogenetic evidence suggests that several translocon subunits are of cyanobacterial origin, indicating that today's import machinery was built around a prokaryotic core.     

Toc, Tic, and chloroplast protein import.

The vast majority of chloroplast proteins are synthesized in precursor form on cytosolic ribosomes. Chloroplast precursor proteins have cleavable, N-terminal targeting signals called transit peptides. Transit peptides direct precursor proteins to the chloroplast in an organelle-specific way. They can be phosphorylated by a cytosolic protein kinase, and this leads to the formation of a cytosolic guidance complex. The guidance complex--comprising precursor, hsp70 and 14-3-3 proteins, as well as several unidentified components--docks at the outer envelope membrane. Translocation of precursor proteins across the envelope is achieved by the joint action of molecular machines called Toc (translocon at the outer envelope membrane of chloroplasts) and Tic (translocon at the inner envelope membrane of chloroplasts), respectively. The action of the Toc/Tic apparatus requires the hydrolysis of ATP and GTP at different levels, indicating energetic requirements and regulatory properties of the import process. The main subunits of the Toc and Tic complexes have been identified and characterized in vivo, in organello and in vitro. Phylogenetic evidence suggests that several translocon subunits are of cyanobacterial origin, indicating that today's import machinery was built around a prokaryotic core.     

Molecular cloning and characterization of maize Toc34, a regulatory component of the protein import machinery of chloroplast

Several components of the chloroplast protein import machinery have been identified mostly in the C3 dicot plant pea. We describe here two homologous cDNA sequences of Toc34, a regulatory component of the import machinery, from maize, a C4 monocot plant. The deduced amino acid sequences of two maize Toc34 proteins, named ZmToc34-1 and ZmToc34-2, show 95% identity to each other and about 60% to those of previously identified Toc34 proteins. The GTP-binding motif and the presumed carboxyl terminal membrane anchor are also conserved in the ZmToc34 proteins. The maize Toc34, like that of pea, is tightly associated with the outer envelope membrane of chloroplast and exposed to the cytosol.     

Differential accumulation of plastid preprotein translocon components during spruce (Picea abies L. Karst.) needle development

We demonstrate that basic components of the plastid protein-import apparatus originally found in pea, Toc34, Toc159, and Tic110, are also conserved in evolutionarily younger gymnosperms. We show that multiple isoforms of the preprotein receptor Toc34 differentially accumulate in various stages of needle development, while the amounts of Toc159 drastically decrease during chloroplast morphogenesis. Spruce Toc34 and Toc159 receptors are able to recognise and interact with the angiosperm precursor of the Rubisco small subunit. Young proplastids found in closed buds contain a highly elevated number of protein translocation complexes equipped with only two types of outer envelope receptors, Toc159 and a 30-kDa Toc34-related protein. Photosystem II (PSII) can already be assembled in a fully functional complex at this very early stage of needle development, suggesting that no additional receptor isoforms are needed for translocation of all necessary PSII components. We conclude that the accumulation of evolutionarily conserved plastid preprotein translocation components is differentially regulated during spruce needle development.     

The Acidic A-Domain of Arabidopsis Toc159 Occurs as a Hyperphosphorylated Protein

The translocon at the outer membrane of the chloroplast assists the import of a large class of preproteins with amino-terminal transit sequences. The preprotein receptors Toc159 and Toc33 in Arabidopsis (Arabidopsis thaliana) are specific for the accumulation of abundant photosynthetic proteins. The receptors are homologous GTPases known to be regulated by phosphorylation within their GTP-binding domains. In addition to the central GTP-binding domain, Toc159 has an acidic N-terminal domain (A-domain) and a C-terminal membrane-anchoring domain (M-domain). The A-domain of Toc159 is dispensable for its in vivo activity in Arabidopsis and prone to degradation in pea (Pisum sativum). Therefore, it has been suggested to have a regulatory function. Here, we show that in Arabidopsis, the A-domain is not simply degraded but that it accumulates as a soluble, phosphorylated protein separated from Toc159. However, the physiological relevance of this process is unclear. The data show that the A-domain of Toc159 as well as those of its homologs Toc132 and Toc120 are targets of a casein kinase 2-like activity.The Toc and Tic complexes cooperate to import nuclear-encoded chloroplast preproteins from the cytosol (Jarvis, 2008; Kessler and Schnell, 2009). Initially, incoming preproteins encounter the receptors Toc159 and Toc34 at the chloroplast surface. Both are GTP-binding proteins and share sequence homology in their G-domains. While Toc34 is anchored in the outer membrane by a short hydrophobic C-terminal tail, the triple-domain Toc159 is inserted via a largely hydrophilic 52-kD M-domain. In addition to the G- and M-domains, Toc159 has a large acidic A-domain covering the N-terminal half of the protein. Arabidopsis (Arabidopsis thaliana) encodes two isoforms of Toc34 (Toc33 and Toc34) and four of Toc159 (Toc159, Toc132, Toc120, and Toc90; Jackson-Constan and Keegstra, 2001). The Toc159 isoforms have a similar domain structure, but they differ from each other in length and sequence of their A-domain (Hiltbrunner et al., 2001a). However, Toc90 does not have an acidic domain at all and only consists of the G- and M-domains (Hiltbrunner et al., 2004). It has been demonstrated that the A-domain of AtToc159 and AtToc132 have properties of intrinsically disordered proteins (Hernández Torres et al., 2007; Richardson et al., 2009), suggesting an involvement of the A-domain in transient and multiple protein-protein interactions possibly with the transit peptides of preproteins. Toc34 and Toc159 together with the Toc75 channel constitute the Toc-core complex (Schleiff et al., 2003) and are required for the accumulation of highly abundant photosynthesis-associated proteins in the chloroplast. The Arabidopsis deletion mutants of Toc33 (ppi1; Jarvis et al., 1998) and Toc159 (ppi2; Bauer et al., 2000) have indicative phenotypes of their role in chloroplast biogenesis, respectively pale green and albino. Complementation experiments of the ppi2 mutant have established that the G- and M-domains have essential functions whereas the A-domain is dispensable (Lee et al., 2003; Agne et al., 2009). In preceding studies, possibly influenced by the model organism and experimental tools, Toc159 occurred in different forms. Initially, Toc159 was identified in pea (Pisum sativum) as an 86-kD protein lacking the entire A-domain (Hirsch et al., 1994; Bolter et al., 1998). In addition to its membrane-associated form, Arabidopsis Toc159 has been found as a soluble protein (Hiltbrunner et al., 2001b). However, the function and the fate of the A-domain as well as that of soluble Toc159 remain unknown and a matter of debate.Not only GTP binding and hydrolysis by the Toc GTPases but also phosphorylation is known as a regulatory mechanism of chloroplast protein import at the Toc complex level (Oreb et al., 2008b). First, some precursor proteins, such as the small subunit of Rubisco, may be phosphorylated in their transit sequence by a cytosolic kinase (Martin et al., 2006). Phosphorylation promotes binding to a 14-3-3 protein and cytosolic Hsp70 in the guidance complex that delivers the phosphorylated preprotein to the Toc complex (May and Soll, 2000). Second, both Toc159 and Toc34 are known to be phosphorylated and independently so by distinct kinases, OEK70 and OEK98, respectively (Fulgosi and Soll, 2002). These two kinase activities have been located to the outer envelope membrane, but their molecular identification is still pending. Phosphorylation of the Toc GTPases may occur in the GTP-binding domains (Oreb et al., 2008a). For Toc34, data on the site (Ser-113 in pea and Ser-181 in Arabidopsis) and effects of phosphorylation are available (Jelic et al., 2002, 2003). It imposes a negative regulation on the Toc complex by inhibiting GTP and preprotein binding to Toc34, reducing its ability to bind Toc159 and to assemble into the Toc complex (Oreb et al., 2008a). The in vivo mutational analysis in Arabidopsis indicated that phosphorylation at Toc34 represents a nonessential mechanism (Aronsson et al., 2006; Oreb et al., 2007). Despite the 86-kD proteolytic fragment of Toc159 being a major phosphoprotein in the pea outer chloroplast membrane (Fulgosi and Soll, 2002), little is known of the molecular and regulatory mechanisms of Toc159 phosphorylation. In this study, we report that the A-domain of Toc159 can be purified as a stable fragment. Moreover, it is hyperphosphorylated, hinting at an important and highly regulated functional role. Our data suggest that Toc159 is the target of casein kinase 2 (CK2)-like and membrane-associated kinase activities.     

The GTPase cycle of the chloroplast import receptors Toc33/Toc34: implications from monomeric and dimeric structures

Transport of precursor proteins across chloroplast membranes involves the GTPases Toc33/34 and Toc159 at the outer chloroplast envelope. The small GTPase Toc33/34 can homodimerize, but the regulation of this interaction has remained elusive. We show that dimerization is independent of nucleotide loading state, based on crystal structures of dimeric Pisum sativum Toc34 and monomeric Arabidopsis thaliana Toc33. An arginine residue is--in the dimer--positioned to resemble a GAP arginine finger. However, GTPase activation by dimerization is sparse and active site features do not explain catalysis, suggesting that the homodimer requires an additional factor as coGAP. Access to the catalytic center and an unusual switch I movement in the dimeric structure support this finding. Potential binding sites for interactions within the Toc translocon or with precursor proteins can be derived from the structures.     

Targeting of an abundant cytosolic form of the protein import receptor at Toc159 to the outer chloroplast membrane

Chloroplast biogenesis requires the large-scale import of cytosolically synthesized precursor proteins. A trimeric translocon (Toc complex) containing two homologous GTP-binding proteins (atToc33 and atToc159) and a channel protein (atToc75) facilitates protein translocation across the outer envelope membrane. The mechanisms governing function and assembly of the Toc complex are not yet understood. This study demonstrates that atToc159 and its pea orthologue exist in an abundant, previously unrecognized soluble form, and partition between cytosol-containing soluble fractions and the chloroplast outer membrane. We show that soluble atToc159 binds directly to the cytosolic domain of atToc33 in a homotypic interaction, contributing to the integration of atToc159 into the chloroplast outer membrane. The data suggest that the function of the Toc complex involves switching of atToc159 between a soluble and an integral membrane form.     

Preprotein recognition by the Toc complex

The Toc core complex consists of the pore-forming Toc75 and the GTPases Toc159 and Toc34. We confirm that the receptor form of Toc159 is integrated into the membrane. The association of Toc34 to Toc75/Toc159 is GTP dependent and enhanced by preprotein interaction. The N-terminal half of the pSSU transit peptide interacts with high affinity with Toc159, whereas the C-terminal part stimulates its GTP hydrolysis. The phosphorylated C-terminal peptide of pSSU interacts strongly with Toc34 and therefore inhibits binding and translocation of pSSU into Toc proteoliposomes. In contrast, Toc159 recognises only the dephosphorylated forms. The N-terminal part of the pSSU presequence does not influence binding to the Toc complex, but is able to block import into proteoliposomes through its interaction with Toc159. We developed a model of differential presequence recognition by Toc34 and Toc159.     

Toc Receptor Dimerization Participates in the Initiation of Membrane Translocation during Protein Import into Chloroplasts

The post-translational import of nucleus-encoded preproteins into chloroplasts occurs through multimeric translocons in the outer (Toc) and inner (Tic) membranes. The high fidelity of the protein import process is maintained by specific recognition of the transit peptide of preproteins by the coordinate activities of two homologous GTPase Toc receptors, Toc34 and Toc159. Structural and biochemical studies suggest that dimerization of the Toc receptors functions as a component of the mechanism to control access of preproteins to the membrane translocation channel of the translocon. We show that specific mutations that disrupted receptor dimerization in vitro reduced the rate of protein import in transgenic Arabidopsis compared with the wild type receptor. The mutations did not affect the GTPase activities of the receptors. Interestingly, these mutations did not decrease the initial preprotein binding at the receptors, but they reduced the efficiency of the transition from preprotein binding to membrane translocation. These data indicate that dimerization of receptors has a direct role in protein import and support a hypothesis in which receptor-receptor interactions participate in the initiation of membrane translocation of chloroplast preproteins as part of the molecular mechanism of GTP-regulated protein import.     

Stable megadalton TOC–TIC supercomplexes as major mediators of protein import into chloroplasts

Preproteins are believed to be imported into chloroplasts through membrane contact sites where the translocon complexes of the outer (TOC) and inner (TIC) envelope membranes are assembled together. However, a single TOC–TIC supercomplex containing preproteins undergoing active import has not yet been directly observed. We optimized the blue native polyacrylamide gel electrophoresis (PAGE) (BN‐PAGE) system to detect and resolve megadalton (MD)‐sized complexes. Using this optimized system, the outer‐membrane channel Toc75 from pea chloroplasts was found in at least two complexes: the 880‐kD TOC complex and a previously undetected 1‐MD complex. Two‐dimensional BN‐PAGE immunoblots further showed that Toc75, Toc159, Toc34, Tic20, Tic56 and Tic110 were all located in the 880‐kD to 1.3‐MD region. During active preprotein import, preproteins were transported mostly through the 1‐MD complex and a smaller amount of preproteins was also detected in a complex of 1.25 MD. Antibody‐shift assays showed that the 1‐MD complex is a TOC–TIC supercomplex containing at least Toc75, Toc159, Toc34 and Tic110. Results from crosslinking and import with Arabidopsis chloroplasts suggest that the 1.25‐MD complex is also a supercomplex. Our data provide direct evidence supporting that chloroplast preproteins are imported through TOC–TIC supercomplexes, and also provide the first size estimation of these supercomplexes. Furthermore, unlike in mitochondria where translocon supercomplexes are only transiently assembled during preprotein import, in chloroplasts at least some of the supercomplexes are preassembled stable structures.     

In vitro targeting of the Toc36 component of the chloroplast envelope protein import apparatus involves a complex set of information

Toc36 is a family of 44-kDa envelope polypeptides previously identified as components of the chloroplast protein import apparatus. Toc36 exists as multiple outer and inner envelope membrane forms. One member, Toc36B (formerly Bce44B), is targeted to the envelope without the typical maturation event. Targeting and assembly into the envelope is thus likely to involve a complex interplay of indigenous signals. These signals were examined by testing the effects of truncations and chimeric fusions on the targeting of Toc36B. The targeting ability of Toc36B appeared unaffected by carboxyl truncations of up to 80% of the protein, but was abolished by N-terminal deletions. The N-terminal 39 residues of Toc36B conferred the same targeting profile to mouse dihydrofolate reductase as that displayed by unaltered Toc36B. However, removal of 18 residues from the carboxyl end of the N-terminal 39-amino acid segment abolished targeting to the chloroplast. Additional information in the remaining Toc36B segment was also apparent based on the import results of chimeric fusions between the transit peptide of the small subunit of ribulose-1,5-bisphosphate carboxylase and Toc36B. The targeting of Toc36B to various destinations in the chloroplast envelope appears to be influenced by information from at least two segments of the protein.     

Analysis of the Interactions of Preproteins with the Import Machinery over the Course of Protein Import into Chloroplasts

We have investigated the interactions of two nuclear-encoded preproteins with the chloroplast protein import machinery at three stages in import using a label-transfer crosslinking approach. During energy-independent binding at the outer envelope membrane, preproteins interact with three known components of the outer membrane translocon complex, Toc34, Toc75, and Toc86. Although Toc75 and Toc86 are known to associate with preproteins during import, a role for Toc34 in preprotein binding previously had not been observed. The interaction of Toc34 with preproteins is regulated by the binding, but not hydrolysis of GTP. These data provide the first evidence for a direct role for Toc34 in import, and provide insights into the function of GTP as a regulator of preprotein recognition. Toc75 and Toc86 are the major targets of cross-linking upon insertion of preproteins across the outer envelope membrane, supporting the proposal that both proteins function in translocation at the outer membrane as well as preprotein recognition. The inner membrane proteins, Tic(21) and Tic22, and a previously unidentified protein of 14 kD are the major targets of crosslinking during the late stages in import. These data provide additional support for the roles of these components during protein translocation across the inner membrane. Our results suggest a defined sequence of molecular interactions that result in the transport of nuclear-encoded preproteins from the cytoplasm into the stroma of chloroplasts.     

Toc159- and Toc75-independent import of a transit sequence-less precursor into the inner envelope of chloroplasts

Chloroplast envelope quinone oxidoreductase (ceQORH) is an inner plastid envelope protein that is synthesized without cleavable chloroplast transit sequence for import. In the present work, we studied the in vitro-import characteristics of Arabidopsis ceQORH. We demonstrate that ceQORH import requires ATP and is dependent on proteinaceous receptor components exposed at the outer plastid surface. Competition experiments using small subunit precursor of ribulose-bisphosphate carboxylase/oxygenase and precursor of ferredoxin, as well as antibody blocking experiments, revealed that ceQORH import does not involve the main receptor and translocation channel proteins Toc159 and Toc75, respectively, which operate in import of proteins into the chloroplast. Molecular dissection of the ceQORH amino acid sequence by site-directed mutagenesis and subsequent import experiments in planta and in vitro highlighted that ceQORH consists of different domains that act concertedly in regulating import. Collectively, our results provide unprecedented evidence for the existence of a specific import pathway for transit sequence-less inner plastid envelope membrane proteins into chloroplasts.