Protein Import Mechanism in Chloroplast
Explore and order pathway-specific siRNAs, real-time PCR assays, and expression vectors. View pathway information and literature references for your pathway.
  • Click on your proteins of interest in the pathway image or review below
  • Select your genes of interest and click "add selection"
  • When you have finished your gene selection, click "Find Products" to find assays, arrays, or create custom products
Download Image Terms of Use Download PPT
Pathway Navigator
Protein Import Mechanism in Chloroplast

Chloroplasts represent the most prominent members of a diverse group of essential organelles referred to collectively as the plastids. Plastids are a heterogeneous family of organelles found ubiquitously in plant and algal cells. Chloroplasts perform a variety of biochemical functions within plant cells. They contain the green pigment chlorophyll and are responsible for the light-harvesting and carbon-fixation reactions of photosynthesis, as well as for the synthesis of many essential metabolites, such as fatty acids and amino acids. Other members of the plastid family include the Amyloplasts, which contain large quantities of starch and play important roles in energy storage and gravitropism, and the Chromoplasts, which accumulate the red-orange-yellow Carotenoid pigments and so act as attractants in flowers and fruits. The Chloroplast is an organelle of prokaryotic origin that is situated in a eukaryotic cellular environment. Plastids entered the eukaryotic lineage through endosymbiosis. They are thought to be of monophyletic origin, and to have evolved from an ancient photosynthetic prokaryote similar to present day Cyanobacteria. Due to their endosymbiotic origin, Chloroplasts have a relatively complex structure comprising six distinct suborganellar compartments: they have three different membranes (the two envelope membranes, Outer membrane and the Inner membrane, and the internal thylakoid membrane), and three discrete aqueous compartments (the Intermembrane space of the envelope, the Stroma and the thylakoid lumen). One of the consequences of this structural intricacy is that the internal routing of Chloroplast proteins is a surprisingly complex process (Ref.1).

Most Chloroplast proteins are nuclear encoded and translated in the Cytosol. These proteins are synthesized in precursor form — each bearing an amino-terminal targeting signal called a transit peptide — and are imported into the organelle by an active, post-translational targeting process. Genomic and proteomic analyses indicate that 2500–4000 different plastid proteins are imported into these organelles. Import mechanism is best studied in Pisum sativum. In Arabidopsis thaliana, this represents 10–15% of the entire nuclear genome. Import of the Chloroplast proteins is mediated by molecular machines in the Outer and Inner envelope membranes, referred to as TOC (Translocon at the Outer envelope membrane of Chloroplasts) and TIC (Translocon at the Inner envelope membrane of Chloroplasts) respectively. In addition, the TOC and TIC complexes interact with a set of molecular Chaperones in the exterior and interior of the organelle that appears to maintain preproteins in an unfolded import competent state, participate as part of the driving force for protein translocation, and assist in the folding and assembly of newly imported proteins. While envelope proteins may employ variations of the TOC/TIC import pathway to arrive at their final destination, proteins destined for the thylakoid membrane or lumen employ one of four distinct targeting pathways. Thylakoid membrane proteins are targeted by the SRP (Signal Recognition Particle)-dependent and Spontaneous Insertion pathways, whereas Lumenal proteins are targeted by the Sec and Tat (Twin-Arginine Translocation)  pathways.  Upon arrival in the Stroma, the transit peptide is removed and the protein either takes on its final conformation or is sorted to one of several internal compartments in a separate targeting process (Ref.2 & 4).

As Chloroplast protein import is a post-translational process, hence soluble, cytosolic factors facilitate the passage of precursors from the Ribosome to the Chloroplast surface. Phosphorylation of some transit peptides (e.g. of the small subunit of RubisCO (pSSU)) enhances the import rate presumably through interaction with 14-3-3 proteins. 14-3-3 proteins together with HSP70 (Heat Shock Protein-70) form a guidance complex for targeting phosphorylated preproteins to the Chloroplast surface. Cytosolic HSP70 is generally involved in the folding of newly synthesized proteins, and its association is required to keep the preproteins in an import competent unfolded state. Once preproteins arrive at the Chloroplast surface, translocation through the membrane-bound TOC/TIC import machinery begins. On the basis of energetic requirements, Chloroplast protein import can be divided into three distinct steps. In the first of these, the transit peptide makes reversible contacts with receptor components of the TOC complex; this step is referred to as energy-independent binding. In the second step, the preprotein becomes deeply inserted into the TOC complex and even makes contact with components of the TIC machinery. Progression to this early import intermediate stage is referred to as docking, requires low concentrations of ATP in the Intermembrane space, as well as GTP, and is irreversible. In the last step of import, the preprotein is completely translocated into the Stroma, and the transit peptide is cleaved by SPP (Stromal Processing Peptidase). Progression through this step requires high concentrations (~1 mM) of ATP in the Stroma. Translocation occurs through the Outer and Inner envelope membranes simultaneously, at locations called contact sites where the two membranes are held in close proximity (Ref.3, 4 & 5).

Preprotein recognition at the Chloroplast surface, and Outer Envelope translocation, are the two main functions of the TOC machine. The TOC core complex is more than 500 kDa in size and comprises three different proteins, each named for their molecular weight: TOC159, TOC34 and TOC75. Both TOC159 and TOC34 are exposed at the Chloroplast surface and interact with preproteins at the earliest stage of import. In the absence of added ATP and GTP, TOC34 and TOC159 can be chemically cross-linked to bound preproteins with TOC159 specifically interacting with the transit peptide. In the presence of external ATP and GTP, preproteins cross the outer membrane. At this stage, the mature regions of preproteins can be chemically cross-linked both to TOC159 and TOC75. TOC159 thus functions both as a primary preprotein receptor and in conjunction with the protein-conducting channel to promote membrane translocation. TOC34 also has been shown to directly bind to preproteins, supporting its role in preprotein recognition at the Chloroplast surface. The hallmark structural feature of the TOC159 and TOC34 receptors is a conserved GTP-binding domain (G-domain). TOC34 consists almost entirely of the G-domain, anchored at the surface of the Outer membrane by a stretch of hydrophobic amino acids at its C-terminus. TOC159 has a central G-domain flanked by an N-terminal acidic domain (A-domain) and C-terminal membrane-anchoring domain (M-domain). TOC75, on the other hand, is deeply embedded in the membrane and has a Beta-barrel structure (Ref. 5).

The mode of action of the TOC receptors is the subject of an on-going debate, and two models have now emerged. In the first model, the targeting hypothesis, the primary role of GTP is to regulate TOC GTPase dimerization and thereby act as a molecular switch that controls the fidelity of preprotein targeting to the translocon channel. The ‘targeting’ hypothesis proposes that TOC159-GTP serves as the primary preprotein receptor and that TOC159-GTP and TOC34-GTP interact co-operatively via their GTPase domains to form a GTP-regulated gate to the translocon. Preprotein binding activates GTP hydrolysis, converting the receptors into the GDP-bound state. Hydrolysis is proposed to induce a conformational change in the GTPase interaction that promotes insertion of the preprotein into the translocation channel. Preprotein translocation is driven by an ATP-dependent cycle at the Intermembrane space HSP70. The second model, ‘motor’ hypothesis proposes that TOC34 in its GTP-bound state acts as the initial receptor by binding to the phosphorylated form of the preprotein transit peptide. TOC34 is converted to its GDP-bound state by preprotein-stimulated GTP hydrolysis, resulting in transfer of the preprotein to TOC159-GTP. Following dephosphorylation, the preprotein is driven across the outer membrane through the TOC75 channel via a GTP-dependent TOC159 motor. Centrally located TOC159 rotate about its axis in order to accept preproteins from different TOC34 primary receptors, and act as a GTP-driven motor to push them through the TOC75 channels by a ‘sewing machine’-type mechanism. In both models, the TOC159 and -33 receptors are reset to their GTP-bound state and are ready for further translocation cycles (Ref.6 & 7).

After their passage through the TOC complex into the Intermembrane space, the nuclear-encoded Chloroplast proteins need the help of second transport machinery, notably the TIC complex, to get over the barrier of the Inner envelope membrane. Several putative components of the TIC complex have been identified — TIC110, TIC62, TIC55, TIC40, TIC32, TIC22 and TIC20 — but there is considerable disagreement about their roles in the import process. TIC22, which was identified as a soluble protein that is peripherally associated with the Inner envelope membrane from the Intermembrane space, is assumed to be the first TIC component interacting with the incoming precursor protein. TIC22 facilitates the passage of preproteins from TOC to TIC, functioning in association with HSP70 and the inwardly facing J-domain protein, TOC12. The integral membrane protein TIC20 is presumably involved in the formation of the protein translocation pore. The integral membrane component TIC110 is assumed to form at least part of the translocation pore, since it was shown to form a Cation selective channel. Moreover, TIC110 contains a large hydrophilic domain which extends into the Stroma where it might provide the docking site for both, precursor proteins and molecular chaperones. Several molecular Chaperones including HSP100 (Heat Shock Protein-100) and Cpn60 have been reported to interact from the stromal side with the TIC complex which is in line with the strict requirement for Stromal ATP to obtain complete translocation of precursor proteins. In addition to these components of the presumed basic TIC machinery, several putative additional factors have been identified. Among those is TIC40 which possesses a Stroma exposed domain that has homology to several co chaperones known to interact with HSP70. TIC32, TIC55, and TIC62, on the other hand, might be involved in the proposed redox regulation of Chloroplast protein import. While TIC55 carries a Rieske iron–sulphur cluster as well as a mononuclear iron-binding site, TIC32 and TIC62 both carry potential NAD(P) binding domains. In addition, TIC62 was shown to interact with ferredoxin NAD(P) oxidoreductase (FNR) located in the Stroma (Ref. 8, 9 & 10).

In contrast to the general route for protein import into the Chloroplast Stroma, protein transport into or across the thylakoid membrane takes place by at least four independent pathways which are designated as Sec-dependent, SRP-dependent, DpH/Tat-dependent, or ‘Spontaneous’. Each of them operates with a unique mechanism and is specific for a distinct subset of thylakoid proteins. All four pathways have originally been identified for nuclear-encoded proteins, although it appears likely that they all are capable also of mediating thylakoid transport of proteins encoded by the plastid genome. Although all four thylakoid transport pathways were shown to accept membrane proteins as substrates, two of them, notably the ‘Spontaneous’ and the SRP-dependent transport, are exclusively targeting integral membrane proteins and are thus specific for the insertion of proteins into the thylakoid membrane. SRP-dependent transport in Chloroplasts presumably provides the major pathway for the integration of polytopic thylakoid membrane proteins, in analogy to its role in the bacterial system. The SRP pathway mediates insertion of the major light-harvesting chlorophyll binding protein, LHCP. LHCP is synthesized with a presequence, but unlike those of Lumenal proteins, this presequence specifies targeting only to the Stroma and it is structurally and functionally indistinguishable from those of Stromal proteins. The insertion of LHCP into the thylakoid membrane requires SRP (cpSRP54 and cpSRP43) and its partner protein, FtsY, both of which hydrolyse GTP during their mode of action. Once at the membrane surface, insertion depends on the integral membrane protein Alb3. Direct or ‘Spontaneous’ protein insertion into the thylakoid membrane is restricted to a specific class of membrane proteins with strikingly similar structure and membrane topology. It was first described for CFo-II, the only nuclear-encoded component of the CFo-membrane assembly of Chloroplast ATP synthase. Meanwhile, the same transport mechanism was found to operate also for the Photosystem-II subunits PsbW, PsbX, and PsbY demonstrating that it represents a mainstream pathway for bitopic membrane proteins. The single membrane span is always found close to the NH2- terminus of the respective mature polypeptide (Ref.9 & 11).

The other two protein transport pathways operating at the thylakoid membrane, notably the Sec and DpH/Tat-dependent pathways are responsible for the translocation of hydrophilic proteins into the thylakoid lumen. Nuclear-encoded proteins targeted by either of the two pathways are synthesized in the Cytosol with bipartite transit peptides carrying two translocation signals in tandem, an NH2-terminal envelope transit signal for the import of the protein into the Chloroplast  stroma followed by a second transport signal which mediates translocation across the Thylakoid membrane and is generally removed after transport by a membrane-bound endopeptidase executing its function on the Lumenal side of the membrane i.e TPP (Thylakoidal Processing Peptidase). A subset of Lumenal proteins is transported by a Sec-type system that resembles the well-characterized Sec systems in bacterial inner membranes. In most bacteria, the core elements of these systems are SecA, an ATP-driven translocation motor, and the membrane bound SecYEG translocation channel. Additional components in the membrane (such as SecDE) play ancillary, but rather ill-defined roles. The thylakoidal Sec system transports lumenal proteins such as Plastocyanin and the 33 kDa Photosystem-II protein, and nucleus-encoded SecA, SecY and SecE homologs have been identified and shown to be involved in thylakoid protein transport processes.  The alternative means of transport across the thylakoid membrane is provided by the Tat pathway. Substrates for this pathway also bear cleavable amino-terminal signal peptides but these targeting signals are recognized by a translocase that has only recently been characterized in detail. The system derives its name from a twin-arginine motif, located in the amino-terminal region of the signal peptide, which is essential for translocation by this pathway. Curiously, these signal peptides are otherwise very similar to Sec-type signal peptides: they contain clearly identifiable N-, H- and C-domains and similarly end with the Ala-Xaa-Ala consensus motif that specifies cleavage by TPP. Translocation is wholly dependent on the thylakoidal ∆pH but not reliant on any form of nucleoside triphosphate. This system is capable of transporting proteins in a folded state, and this appears to be the defining attribute of this system. Three components of the thylakoidal DpH/Tat transport machinery have been identified to date, called TatA (or Tha4), TatB (or Hcf106), and TatC (or cpTatC). In Pea thylakoids, these subunits form multimeric complexes of approximately 560 and 620 kDa upon transport of a precursor protein. More recently, the binding of transport substrates to an approximately 700 kDa TatB/C complex lacking TatA was reported. TatA apparently joins this preformed TatB/C complex only transiently in the presence of both, substrate and trans thylakoidal proton gradient, and is assumed to provide the transport pore. However, little is known about the actual translocation mechanism at present. Recently, most components of the Chloroplast protein import apparatus have been identified and attention has turned to the assignment of specific functions to individual components, and to the elucidation of mechanistic details of the import process. Significant progress has been made in this direction, but it is clear — as evidenced by the conflicting models for preprotein recognition, and disagreement concerning the functions of putative TIC complex components — that further work will be required before a complete and accurate picture can be formed (Ref. 12 & 13).