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1. Details of Module and its Structure
Module Detail
Subject Name <BOTANY>
Paper Name <Cell Biology>
Module Name/Title <Chloroplast and mitochondria –Transport across membrane of chloroplast and mitochondria >
Module Id <10c>
Pre-requisites Basic knowledge about plant cells, their contents and basic idea about cell organelles
Objectives To make the students aware of the transport mechanisms across chloroplast and mitochondrial membranes.
Keywords Protein transport, Toc complex, Tic complex, Tom complex, Tim complex, SAM complex, presequences, MIA pathway, Mim1, tiny TIMs
2. Development Team
Role Name Affiliation
National Coordinator <NA>
Subject Coordinator <Dr. Sujata Bhargava>
Paper Coordinator <Dr. Nutan Malpathak>
Content Writer/Author (CW) <Dr. Gauri Abhyankar>
Content Reviewer (CR) < Dr. Nutan Malpathak>
Language Editor (LE) < Dr. Nutan Malpathak>
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TABLE OF CONTENTS 1. Introduction
2. Cytosolic components required for protein import into chloroplasts 3. Stages of envelope translocation
4. Toc apparatus 4.1 Toc159 4.2 Toc34 4.3 Toc75 4.4 Toc64
5. Crossing the intermembrane space 6. Tic apparatus
6.1 Tic110 6.2 Tic55
6.3 Tic20 and Tic22 6.4 Tic40
6.5 Hsp93 6.6 Hsp70
6.7 Tic62 and Tic32
7. Regulation of protein import
8. Protein transport pathways at the thylakoid membranes 8.1 SRP dependent transport
8.2 Spontaneous transport 8.3 Sec dependent transport 8.4 ΔpH/Tat dependent transport
9. Unique components of mitochondrial protein import apparatus 9.1 The outer membrane
a) TOM complex b) SAM complex 9.2 Intermembrane space 9.3 The inner membrane
a) TIM complex b) PRAT
c) Mitochondrial processing peptidase 10. Mitochondrial protein import pathways
10.1 Pre-sequence pathway 10.2 Carrier pathway
10.3 SAM pathway 10.4 MIA pathway 10.5 Mim1 pathway
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1. Introduction
Chloroplasts are specifically found in plants and they mainly function for carrying out photosynthesis. Photosynthesis was first supposed to be evolved in bacteria.
Chloroplasts originated from bacteria through engulfment of photosynthetic bacteria.
During evolution most of the chloroplast genes or bacterial genes were moved to plant nucleus and still the process is going on. Based on phylogenetic studies, it is approximated that many chloroplast import machinery units have originated from pre- existing bacterial proteins (Reumann & Keegstra, 1999). These bacterial units were accompanied by novel plant subunits like receptor proteins and chaperones. The plant subunits played a crucial role in supplementing the chloroplast units with specificity and directionality of transport into the chloroplasts. The targeting selectivity was observed at two levels – a) by addition of a specific targeting sequence to every protein intended to go to chloroplast, and b) use of surface receptor proteins. As of today, post-translational protein import into chloroplasts is a highly complex process.
Mitochondria also originated from bacteria and they still contain their own small genome that codes for very limited number of proteins like subunits of respiratory chain complexes and the F1Fo-ATPase. In course of evolution, mitochondrial genes have moved to plant nucleus and nearly all mitochondrial proteins are encoded by nuclear genes. They are synthesized as precursor proteins on cytosolic ribosomes and subsequently transported into mitochondria. Import of precursor proteins into mitochondria generally occurs in a post-translational manner that involves quite complicated cascades of receptors and transporters.
2. Cytosolic components required for protein import into chloroplasts –
Proteins are imported into chloroplasts after translation (Fig 1). So newly formed precursor proteins can fold in cytoplasm. Such unwanted folding can have certain unfavourable consequences – a) such a folded pre-protein may develop its biological activity in wrong place which might show serious effects, or b) it is favourable to move unfolded polypeptides across membranes. Now we know that cytosolic factors function for maintaining the import capability of many precursors and they also exert some regulatory effect on protein import (Jarvis & Soll, 2001). Chloroplast targeting sequences or transit peptides are phosphorylated which results in their binding to a cytosolic guiding complex.
The guiding complex consists of Hsp70 and 14-3-3 proteins. If pre-proteins lack phosphorylation site, they associate with Hsp70 only.
Large number of chloroplast targeted precursor proteins have ability to bind to Hsp70. It is a highly conserved chaperone and can assist in protein folding in ATP-
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dependent and also co-chaperone mediated manner (Mayer & Bukau, 2005). Around 80%
of chloroplast transit peptides have Hsp70 binding site, so that they bind in the N-terminal region of the precursors. But they can also bind to the mature part of pre-proteins (May &
Soll, 2000).
Along with Hsp70, other components were also involved in formation of guiding complex. 14-3-3 protein dimmers are one of them. They constitute a large family of proteins that are widely distributed amongst eukaryotes. They perform a variety of different regulatory functions by binding to phosphorylated peptide domains. The kinase responsible for the phosphorylation of these precursors belongs to a family of three homologous pants specific STY-kinases, containing a serine/threonine as well as a tryrosine phosphorylation domain (Martin et al, 2006). So proteins bound to 14-3-3 dimers are then handed over to Toc34.
Another chaperone involved in binding is Hsp90. It is mainly observed in guiding loosely folded precursors to the chloroplast. Hsp90 is also well studied chaperone in both prokaryotic and eukaryotic organisms. It helps in the folding of transcription factors and protein kinases. Its binding to precursor proteins prevents protein aggregation and also promotes docking of the bound precursors to an outer membrane protein, Toc64.
However, binding of Hsp90 is not universal but is plant specific (Schwenkert et al, 2011).
Fig 1: Cytosolic components of the import pathway. Precursors synthesized in cytosol are recognized by Hsp70, Hsp90 and 14-3-3 proteins. 14-3-3 binding precursors are initially phosphorylated and then guided to
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Toc34 whereas, Hsp90 binding precursors dock to Toc64 (Schwenkert et al, Biochim Biophys Acta 1808:901–911, 2011).
3. Stages of envelope translocation –
Once a precursor protein reaches the chloroplast surface, a highly specific recognition process is initiated. This recognition process ensures that proteins destined for other cellular compartments are not mistakenly imported into chloroplasts. At the same time, it is quite flexible to take into account wide structural diversity amongst chloroplast transit peptides.
On the basis of energy requirements, chloroplast protein import can be divided into three distinct steps (Fig 2). During first stage of import, precursors interact with protein components of the translocon. The translocon comprises protein complexes in the outer and inner envelope membranes called Toc (translocon at the outer envelope membrane of chloroplasts) and Tic (translocon at the inner envelope membrane of chloroplasts), respectively. The binding is energy independent. It is reversible and does not require ATP hydrolysis. In the second step, at low ATP concentration (<100 µM), the pre-protein becomes deeply inserted into the Toc complex and even makes contact with components of the Tic machinery. Formation of this early import intermediate stage requires GTP, and the process is irreversible. The stage remains stable till ATP concentrations remains low.
As ATP levels increase to more than 100 µM, the preprotein is completely translocated across the envelope into the stroma. In stroma transit peptide is cleaved by SPP (stromal processing peptidase). Progression through this step requires high concentrations (~1 mM) of ATP. During import, the Toc and Tic complexes come together at contact sites and the precursor protein passes through both membranes simultaneously.
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Fig 2: A working model for the chloroplast protein import mechanism. As chloroplast pre-proteins emerge from 80S ribosomes, they are bound by a cytosolic guidance complex which docks at Toc64 (stage 0); then they proceed directly to the core Toc complex (stage 1). Pre-proteins unload from the guidance complex and pass to a trimeric receptor complex comprising Toc159, Toc34 and Toc75. At stage 1 the pre-protein interacts reversibly with the heterotrimeric Toc receptor complex. Progression to stage 2 requires ATP at low concentrations in the inter-membrane space, and GTP. At this stage, the pre-protein is inserted across the outer envelope membrane and is in contact with components of the Tic apparatus. Stage 3 (complete translocation) requires high concentrations of ATP in the stroma. The pre-protein is translocated simultaneously across both envelope membranes at a contact site, the transit peptide is cleaved by the stromal processing peptidase (SPP) and the mature protein takes on its final conformation (Jarvis & Soll, Biochim Biophys Acta, 1541:64-79, 2001).
4. Toc apparatus –
The initial experimental work for studying chloroplast protein import was carried out in pea. Now Arabidopsis is routinely used as a model plant for different studies pertaining to transport. The Toc core complex is more than 500 kDa in size and is composed of
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Toc159, Toc34, Toc75 and Toc64, according to their predicted molecular masses. Let us see them in detail.
4.1 Toc 159 –
It is regarded as a chloroplast protein import receptor because it is a major point of contact for precursor proteins arriving at the translocon complex. Toc159 belongs to a unique class of GTP-binding proteins. It possesses characteristic GTP-binding site motifs within its central domain, but they are not homologous to other GTP-binding proteins. It is a highly labile, integral protein and was originally identified as an 86 kDa proteolytic fragment called Toc86. The complete protein shows three domains – N-terminal acidic domain, a central GTP-binding domain and a C-terminal membrane anchor domain (Chen et al, 2000). The N-terminal and central domains project into the cytosol. The N-terminal domain has a high proportion of acidic amino acids. Toc159 probably undergoes one or more rounds of GTP hydrolysis after energy independent-binding in order to transfer the pre-protein to other subunits of the Toc complex (Jarvis & Soll, 2001).
4.2 Toc 34 –
Toc34 belongs to the same class of GTP binding protein as Toc159. It shares substantial homology with Toc159. In Arabidopsis it shows 2 isoforms – AtToc33 and AtToc34 (Soll & Schleiff, 2004) (Fig 3). It is an integral membrane protein and attaches to the outer envelope membrane by a C-terminal membrane anchor. The protein has a single transmembrane domain close to its C-terminus. The majority of the protein is present into the cytosol. Even if precursor protein is not bound to the translocon, Toc34 forms a stable complex with Toc159 and other translocon components. AtToc33 is the most abundantly expressed and so it is considered as the true orthologue of psToc34.
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Fig 3: Model of the Arabidopsis thaliana TOC33 and TOC34 receptor cycles. The AtTOC33 and AtTOC34 receptors are activated by GTP binding. Activated AtTOC33/34 (AtToc33/34-GTP) binds the phosphorylated pre-protein with high affinity. The pre-protein activates the endogenous GTPase activity of AtTOC33/34, which results in the hydrolysis of GTP. AtTOC33/TOC34–GDP has a lower affinity for the pre-protein, so it is released to the next translocon subunit. After the release of GDP from AtTOC33/34, the receptor can be recharged with GTP and enter a new receptor cycle. AtTOC33 receptor is functional analogue of the Pisum sativum (Ps) TOC34 receptor that becomes non functional by phosphorylation. So phosphorylation in case of PsToc34 inhibits GTP binding. Dephosphorylation of AtTOC33 and PsTOC34 is required for re-activation through GTP binding. (Soll & Schleiff, Nat Rev Mol Cell Biol 5: 198 – 208, 2004).
Two models are currently proposed for translocation across the outer membrane.
According to the first model, a soluble, cytosolic form of Toc159 functions as an initial receptor for the transit peptide (Fig 4A). Once the pre-protein Toc159 complex is formed, it stops at Toc34in the outer membrane through a reciprocal GTPase domain interaction. During this interaction, it transfers the pre-protein to the Toc75 channel.
Toc159 receptor is now free to dissociate and it could then initiate another targeting cycle.
This model is supported with the fact that Toc159 and related proteins are present in cytosolic and membrane bound forms in equal amounts. Also the crystal structure of Toc34 suggests dimerization of Toc159 and Toc34 in vivo (Jarvis & Robinson, 2004).
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Fig 4A: Models of pre-protein recognition by Toc complex – In the first model, a newly synthesized pre- protein is bound by the central, GTPase domain of cytosolic Toc159. The preprotein–Toc159 complex then docks at the outer membrane through a homotypic interaction between the GTPase domains of Toc159 and Toc34, stimulating GTP hydrolysis by both proteins and leading to integration of Toc159 into the Toc complex and insertion of the pre-protein across the outer envelope membrane. Once translocation is complete, the two GTPases undergo GDP–GTP exchange, enabling Toc159 to dissociate from the complex (Jarvis & Robinson, Curr Biol, 14: R1064–R1077, 2004).
In the second model (Fig 4B), membrane-bound Toc34 acts as initial receptor for transit peptides. Structural studies of the purified Toc core complex disclosed a ring shaped surface containing four putative translocation channels surrounding a central finger-like domain (Schleiff et al, 2003). These four channels are each believed to correspond to one Toc75 and one Toc34 unit. The central region is made up of a single Toc159 molecule, which is thought to rotate about its axis. By doing so, it accepts pre- proteins from different Toc34 initial receptors, and act as a GTP-driven motor to push them through the Toc75 channels by a ‘sewing machine’-type mechanism.
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Fig 4B: In the second model, the transit peptide is first phosphorylated near its carboxyl end. It then binds with Toc34 that acts as the primary receptor. Due to transit peptide binding, Toc34 undergoes GTP hydrolysis. Along with GTP hydrolysis, dephosphorylation of the transit peptide takes place that leads to transfer of the pre-protein to Toc159. As transit peptide binds to Toc159, Toc159 undergoes GTP hydrolysis and results in a substantial conformational change in Toc159. This pushes the pre-protein through the translocation channel. More rounds of GTP hydrolysis by Toc159 complete the translocation process (Jarvis
& Robinson, Curr Biol, 14: R1064–R1077, 2004).
Studies also suggest substrate-specific protein import pathways. It is proposed that different isoforms of Toc proteins show a substantial degree of functional specialization.
They group together preferentially to form different Toc complexes with substrate specificity. AtToc159 associates preferentially with AtToc33 to form a Toc complex with specificity for highly abundant photosynthetic proteins. AtToc132 and/or AtToc120 (the other Toc159 isoforms) associate preferentially with AtToc34 to form a Toc complex with specificity for relatively low-abundance, housekeeping proteins. The existence of such substrate-specific complexes would prevent photosynthetic pre-proteins from out- competing much less abundant pre-proteins during the early stages of import (Jarvis &
Robinson, 2004).
4.3 Toc75 –
It is the most abundant protein of the chloroplast outer envelope. It is of prokaryotic origin and belongs to Omp85 family. Omp85 family members are responsible for
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integration of β barrel proteins into the outer membrane (Gentle et al, 2005). It is the only member of Omp85 family known to have N-terminal targeting sequence. This targeting sequence is of bipartite nature. The first part is the stromal targeting signal, whereas second part reaches only to intermembrane space and is cleaved there by a peptidase.
Toc75 is present in very close association with the pre-proteins during import. It joins stably with Toc159, Toc34 and other translocon components even in absence of precursor proteins. The larger part of the protein in embedded in the outer envelope membrane.
Toc75 shows a β barrel structure made up of 16 transmembrane β sheets (Jarvis & Soll, 2001). The typical structure of the protein suggests that it is involved in formation of channel. The channel size ranges between 20 and 25 A0. Arabidopsis shows four different Toc75 paralogues (Toc75-III, Toc75-IV, Toc75-V and Toc75-I), out of which Toc75-III is expressed at much higher level than others and is a part of Toc complex (Schwenkert et al, 2011).
4.4 Toc64 –
It was identified by its stable association with Toc159, Toc75 and Toc34 during sucrose density gradient centrifugation. Toc64 is an important element of the chloroplast outer envelope membrane. It is an integral membrane protein that shares homology with prokaryotic and eukaryotic amidases. But as it shows a mutation in a conserved residue of amidases, it does not show amidase activity. A major portion of C-terminus of Toc64 is exposed to the cytosol and it has three tetratricopeptide repeat (TPR) motifs. The TPR motifs mediate dynamic protein-protein interactions. It is suggested t hat Toc64 also serves as an import receptor for the guidance complex. In Arabidopsis two homologues of Toc64 exist.
5. Crossing the intermembrane space –
The incoming protein first comes into intermembrane space (IMS). Three proteins, viz. Toc12, Tic22 and IMS Hsp70 – form the translocation complex of IMS. Toc12 interacts with amidase domain of Toc64 and gets attached to the outer envelope membrane by a short hydrophobic domain in the N-terminus. Toc12 interacts with a Hsp70 isoform found in IMS (Becker et al, 2004). PsToc12 contains a conserved CXGXXC motif and it probably contributes to a regulatory disulfide bridge. This protein also contains a highly conserved tryptophan-rich motif in the C-terminus. Orthologues of Toc12 are also found in Arabidopsis, Zea, Medicago and Physcomitrella (Schwenkert et al, 2011). In Arabidopsis, Toc12 was indicated as a J-domain protein called as AtJ8 (Chen et al, 2010). IMS Hsp70 was shown to play a critical role in unfolding of chloroplast translocon (Ruprecht et al, 2010). The third component of the IMS complex is Tic22. It is of cyanobacterial origin and
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perhaps the rare evolutionary conserved Tic component. It might be functioning as a linker between the two translocation machineries. Cyanobacterial synTic22 is mainly found in the thylakoid lumen and a small amount is observed in the periplasmic space. It functions in protein transport and also electron transfer. Arabidopsis consists of two Tic22 isoforms:
Tic22-III and Tic22-IV, of which Tic22-IV is more closely related to pea protein.
6. Tic apparatus –
Once the chloroplast proteins cross the outer membrane and enter in IMS, they require help from second transport machinery (Tic apparatus) to cross the inner envelope membrane and reach the stroma. Many potential Tic proteins have been identified viz.
Tic110, Tic55, Tic40, Tic22 and Tic20 (Gutensohn et al, 2006) (Fig 5).
Fig 5: Model of the TIC translocon (Schwenkert et al, Biochim Biophys Acta 1808:901–911, 2011).
6.1 Tic110 –
After crossing the outer membrane, precursor protein comes in contact with Tic110.
It is considered to be the major import pore. Tic110 is the most abundant Tic component and was the first to be identified. It is encoded by a single copy gene in all plants except Physcomitrella patens and contains many conserved cysteine residues distributed throughout the sequence. Most of the cysteine residues are present on the stromal side.
Tic110 plays diverse functions like mediator for precursor binding, employing chaperones on stromal side of inner envelope and formation of protein translocation channel (Balsera et al, 2009). N-terminus of Tic110 consists of two hydrophobic α-helices. They help for membrane targeting and anchoring of the proteins. C-terminus of Tic110 alone can form a cation-selective, calcium sensitive channel.
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6.2 Tic55 –
It is an integral membrane protein and is partially exposed to the intermembrane space. Its protein sequence showed Rieske-type iron-sulfur cluster and a mononuclear iron-binding site. Rieske proteins are often involved in electron transfer chains. However, certain proteins may use their iron-sulfur cluster as biosensors. By doing so, they plausibly relate redox signals with changes in gene expression or programmed cell death (Jarvis &
Soll, 2001). Thus Tic55 participates in regulation during import by reacting to changes in redox status in the chloroplasts.
6.3 Tic20 and Tic22 –
Arabidopsis shows genes encoding two homologues of Tic22 and at least two Tic20 homologues. Tic20 is mainly hydrophobic and has 4 putative hydrophobic α-helices. It is deeply embedded within the inner envelope membrane. Tic22 is a hydrophilic protein with no predicted transmembrane domains. It is peripherally associated with the outer surface of the inner envelope membrane. It might function as a receptor for precursor proteins as they come out of the Toc complex. It is suggested that Tic22 possible can mediate association of Toc and Tic complexes at contact sites. In the absence of precursor proteins, both Tic20 and Tic22 can join together with major Toc proteins and Tic110 to form a Toc-Tic supercomplex. But if Toc components are lacking, then Tic20 and Tic22 do not connect with one another or with Tic110.
6.4 Tic40 –
Tic40 (formerly named Com44/Cim44 and Toc36) was first identified in Brassica napus. It is encoded by a single gene. Tic40 is found solely in the inner envelope membrane. It has a single membrane-spanning region at its extreme N-terminal end and a large part of the protein is exposed on the stromal surface of the inner envelope membrane.
6.5 Hsp93 –
Hsp93 is necessary for viability of plants. Arabidopsis shows two isoforms viz.
Hsp93-V and Hsp93-III. They belong to a superfamily of molecular chaperones that make use of ATP to mediate protein folding/unfolding. They either operate independently as chaperones or may act as regulatory associates of the ClpP protease complex. In maize
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chloroplasts, Hsp93 is observed to be active as a hexamer. Arabidopsis and pea mostly show the dimeric form in the stroma.
6.6 Hsp70 –
Two stromal Hsp70 isoforms have been shown to work in Physcomitrella for protein import. One isoform, Hsp70-2 is essential for viability and also for protein translocation.
Hsp70 and Hsp93 work together as chaperones for protein translocation.
6.7 Tic62 and Tic32 –
Tic62 and Tic32 belong to the extended and classical family of short chain dehydrogenases, respectively, and both bind to NADP(H). Association of Tic32 and Tic62 with Tic110 was dependent on the metabolic redox state in the stroma, i.e. ratio of NADP+ to NADPH. Oxidized environment favoured union of Tic32 and Tic62 with Tic110, whereas, more reduced conditions detached Tic32 and Tic62 from Tic110. Tic32 also binds to calmodulin. Tic62 is a multifaceted protein. It has a two-domain structure with a conserved NADP(H) binding-site in the N-terminus and a new FNR binding domain in the C-terminal part. Tic62 shows triple localization in chloroplasts – a) at the inner envelope membrane, b) in the stroma and, c) at the thylakoid membrane. Thylakoid-associated Tic62 functions for storing of FNR in high molecular-weight complexes.
7. Regulation of protein import –
As plants require adapting to changing environmental conditions, regulation of protein import in chloroplasts is very much essential. There are several levels of regulation that affect either the activity of Toc or Tic. The first mode of regulation is mediated by GTP at outer envelope (Fig 2).
A second mode of regulation is thiol mediated regulation seen at the Toc complex (Fig 6). Formation of intermolecular disulfide bridges decreases import activity, probably by blocking binding sites at receptor components as well as the channel. Breaking of the disulfide bridges results in effective import.
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Fig 6: Thiol-dependent regulation of the TOC complex. Under reduced conditions, cysteines in Toc components contain free thiol groups making the translocon active. Oxidation of cysteines to inter- or intra- molecular disulfide bridges results in decrease of transport activity (Schwenkert et al, Biochim iophys Acta 1808:901–911, 2011).
The Tic translocon is regulated from inside of the chloroplast. It has been shown that the NADP/NADPH ratio in stroma influences the import rate of pre-proteins (Fig 7).
An oxidized metabolic redox state, represented by a low NADPH/NADP ratio, within the stroma leads to attachment of regulatory subunits as Tic62 and Tic32 to the core translocon and makes the complex more active. The channel protein Tic110 can be modulated by thioredoxins, where the reduced form of Tic110 is likely to be more active.
When the stroma becomes more reduced, the redox regulon members detach from the translocon, resulting in decreased import activity for a certain subset of precursor proteins.
Fig 7: Redox regulation of the TIC complex. (Schwenkert et al, Biochim Biophys Acta 1808:901–911, 2011).
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8. Protein transport pathways at the thylakoid membranes –
Protein transport into or across the thylakoid membrane takes place by at least four independent pathways viz. Sec-dependent, SRP-dependent, ΔpH/Tat-dependent, and spontaneous (Albiniak et al, 2012). Each of them operates with a unique mechanism and is specific for a distinct subset of thylakoid proteins.
8.1 SRP dependent transport –
SRP-dependent transport in chloroplasts offers the major pathway for integration of polytopic thylakoid membrane proteins. The main representative of SRP-dependent transport is the apoprotein of the light-harvesting complex associated with photosystem II (LHCP). LHCP has a transit peptide with exclusively stroma targeting function. So it is completely removed by a stromal processing peptidase. Thylakoid targeting of LHCP depends on three stromal factors – a) cpSRP54, b) cpSRP43, and c) cpFtsY. In addition to these stromal factors, a thylakoid membrane protein (Alb3) is required. Protein transport through SRP-dependent pathway requires energy that is acquired by GTP hydrolysis and by the trans thylakoidal proton gradient (Gutensohn et al, 2006).
8.2 Spontaneous transport –
Spontaneous protein insertion into thethylakoid membrane (Schleiff and Kloesgen, 2001) is restricted to a specific class of membrane proteins like Cfo-II (nuclear-encoded component of chloroplast ATP synthase), photosystem II subunits PsbW, PsbX, and PsbY.
It represents a pathway used for membrane proteins having two topologies. The single membrane pass of protein is always found close to the amino terminus of the mature polypeptide located in the thylakoid lumen. Membrane insertion of these proteins is strictly dependent on the presence of two hydrophobic domains flanking the hydrophilic amino terminal segment translocated across the membrane. One hydrophobic domain is shared by the membrane anchor of the mature protein while the other one is provided by transit peptide.
8.3 Sec dependent transport –
Sec systems are evolutionarily conserved protein translocation machineries observed in the eukaryotic as well as prokaryotic systems. Sec-dependent protein transport depends on nucleoside triphosphates and a stromal homolog of bacterial SecA protein. It does not require the proton gradient across the thylakoid membrane. Here substrates are transported in an unfolded conformation through a protein-conducting
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channel. Typical substrates of the thylakoidal Sec-dependent pathway are plastocyanin, the photosystem I subunit PsaF, and the 33 kDa protein of the oxygen evolving system. In chloroplasts, homologues to E. coli SecA (cpSecA), SecY (cpSecY), and SecE (cpSecE) have been identified. Sec translocon consists of SecA, SecE, and SecY. After translation, the signal peptide of the pre-protein binds to the SecA ATPase in cytoplasm and forms SecA-preprotein complex (Fig 8). The complex binds with the Sec core components (Sec Y & Sec E) that form the channel. SecA directs translocation of the precursor through the SecYE channel by means of a cycle of ATP binding hydrolysis and release.
Fig 8: Basic structure of thylakoid Sec systems. A common feature of all Sec systems is that the substrates are transported in an unfolded conformation through a protein-conducting channel. In thylakoids the Sec transport involves cpSecA ATPase (a dimer) and the membrane-bound cpSecE and cpSecY subunits.
cpSecA ATPase activity provides the energy to drive the translocation of proteins through the SecE/Y pore.
After translocation, the signal peptides (represented as grey rectangles) of thylakoid Sec substrates are removed by processing peptidases (represented as scissors). (Albiniak et al, J Exp Bot, 63: 1689 – 1698, 2012).
8.4 ΔpH / Tat dependent transport –
Proteins like the 16 and 23 kDa subunits of the oxygen evolving system are translocated by the ΔpH/Tat-dependent pathway across the thylakoid membrane. Proteins transported through Tat pathway contain a thylakoid targeting signal peptides that carry a twin pair of arginine residues upstream of the hydrophobic core domain. The Tat pathway
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shows many unique features. It does not require soluble factors and nucleoside triphosphates. It obtains energy solely through proton gradient generated
across the thylakoid membrane by the photosynthetic electron flow. Tat pathway can translocate fully folded proteins or protein domains across the thylakoid membrane. The chloroplast Tat translocase consists of three membrane proteins called as Tha4 (TatA), Hcf106 (TatB), and cpTatC (TatC). Hcf106 and Tha4 are single-spanning membrane proteins containing an N-terminal transmembrane domain followed by a short amphipathic a-helix and an unstructured C-terminal domain. cpTatC is predicted to contain six transmembrane domains and both N- and C- termini are located in the stroma.
Nearly 50% of thylakoid lumen proteins are translocated either by the cpSec or the cpTat pathway. All thylakoid cpTat targeting signals are similar to classical bacterial signal peptides in overall structure. They contain N-terminal basic region, a hydrophobic central core and a polar C-terminal region ending in an Ala-X-Ala terminal processing site (Aldridge et al, 2009). Plant Tat signal peptides have a shorter consensus sequence (RRXXΦ) where Φ is typically leucine, phenylalanine, valine or methionine, and X can be any amino acid (Stanley et al., 2000).
Chloroplast Tat components are organized into two distinct complexes in the membrane. A) A large, 700 kDA Hcf106-cpTatC complex and B) Tha4 is observed as a separate homo-oligomer. It is considered that cpTatC targets to thylakoids via a stromal intermediate and cpSecY or Alb3 are not essential for cpTatC membrane integration or for assembly into the cpTat receptor complex. The current models (Fig 9) for the overall translocation process involves three main phases –
a) binding of precursor protein to the Hcf106-cpTatC complex b) coalescence of this complex with the Tha4 complex and
c) translocation of the folded substrate protein through the membrane.
However, the late stages of translocation are not fully understood.
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Fig 9: Current model for the Tat translocation process. (1) The precursor protein binds through the signal peptide to a cpTatC-Hcf106 receptor complex in the thylakoid membrane. (2) Precursor binding in the presence of the proton gradient triggers assembly of Tha4 oligomers with the precursor–receptor complex and the putative translocase is formed. (3a) The precursor protein is then transported across the thylakoid membranes. The signal peptide is cleaved by the TPP and the mature protein is released into the lumen.
After protein transport, Tha4 dissociates from the receptor complex and the system is reset. (3b) The translocation reversal process (Albiniak et al, J Exp Bot, 63: 1689 – 1698, 2012).
9. Unique components of mitochondrial protein import apparatus –
Mitochondrial proteins contain an N-terminal pre-sequence that is specifically recognised by receptors on the outer membrane of mitochondria and translocated through the TOM complex. Precursor protein targeting to mitochondria and sorting to specific mitochondrial sub-compartments necessitates presence of specific import signals within the transported polypeptides (Fig 10). The most frequently found mitochondrial import signal is an N-terminal extension known as pre-sequence. They are amphipathic α-helical segments with a net positive charge and usually vary in length from 15 to 55 amino acids (Dudek et al, 2013). These pre-sequences are cleaved by mitochondrial processing peptidase after they reach the mitochondrial matrix.
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Fig 10: Different targeting signals direct nuclear encoded precursor proteins on specific transport routes to their final localization within mitochondria. After translocation of precursors through the general translocase of the outer membrane (TOM complex), distinct downstream import pathway diverge in the inter-membrane space (IMS): Biogenesis of β-barrel proteins of the outer membrane (OM) requires the small Tim chaperones of the IMS and the sorting and assembly machinery (SAM). Proteins of the IMS that contain cysteines-rich signals (CxnC) are imported via the mitochondrial inter-membrane space import and assembly (MIA) pathway. Carrier proteins of the inner membrane (IM) are transported with the help of the small Tims and the translocase of the inner membrane 22 (TIM22 complex). Pre-sequence containing proteins are inserted into the inner membrane or imported into the matrix by the translocase of the inner membrane 23 (TIM23 complex; pre-sequence translocase). Matrix translocation requires the activity of the pre-sequence translocase-associated import motor (PAM). Pre-sequences are proteolytically removed by the mitochondrial processing peptidase (MPP) upon import. Δψ, membrane potential across the inner mitochondrial membrane (Dudek et al, Biochim Biophys Acta 1833: 274–285, 2013).
9. The outer membrane – a) Tom complex –
The TOM complex is the major outer membrane import complex and most of the mitochondrial proteins are thought to pass through it (Duncan et al, 2013) (Fig 11). It
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facilitates the transport of different types of precursors with highly diverse import signals across the outer membrane. The TOM complex consists of eight subunits in yeast and plants, the cytosolic facing receptor subunits Tom20, Tom9, Tom70, and the twin-pore- forming Tom40 channel, which spans the outer membrane and is associated with small Tom5, Tom6, and Tom7 (Murcha et al, 2014).
Fig 11: The mitochondrial protein import apparatus of plants. Conserved components found in all eukaryotic lineages are shown in yellow. Plant specific components are shown in green and include — TOM20, TOM9, OM64 and the C-terminal regions of TIM17. Components which have been shown to be essential for normal plant development are outlined in red and pre-sequence interacting domains are shown in blue (Duncan et al, Biochim Biophys Acta 1833: 304–313, 2013).
Tom20 is a tetratricopeptide repeat (TPR)-containing protein. Tom20 receptor specifically recognizes precursor proteins having N-terminal targeting signals and facilitates protein – protein interactions. It has ability to distinguish between vast array of mitochondrial and non-mitochondrial proteins. The Arabidopsis genome contains four Tom20 genes of which three are functional and expressed.
Tom complex shows one novel plant specific outer membrane receptor called as OM64 (outer-membrane protein 64). It is a paralogue of Toc64, a component of Toc complex. Once transferred from Tom20, precursors interact with Tom22 receptor protein and begin transfer of precursors to the Tom40 channel. Tom22 is essential for viability in yeast and is involved in the assembly of the TOM complex itself. Tom22 consists of a receptor domain and an extended IMS domain. The IMS domain is concerned with the transfer of pre-proteins from Tom40 pore to the TIM17:23 complex. Arabidopsis Tom22 is
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unique. It is 50 amino acids shorter than yeast or human Tom22 and shows absence of cytosolic domain. This is considered as a mechanism to prevent mis-targeting of chloroplast precursor proteins to plant mitochondria. Tom22 in plants is termed as Tom9 (Cerrie et al, 2010). Tom9 was first identified as the small 8 kDa protein from both potato and Arabidopsis.
Several other small proteins, viz. Tom5, Tom6 and Tom7 have also been isolated with the TOM complex. In yeast, they participate in transfer of pre-proteins from the receptors to the Tom40 channel and in the assembly and maintenance of the TOM complex itself (Becker et al, 2011). The small Toms are conserved throughout plant species.
Metaxin is also an outer membrane protein. In its absence, plants were shown to develop more slowly, remain smaller than wild type, display lesions on developing and mature leaves and accumulate higher levels of starch than wild type plants (Lister et al, 2007). The cytosolic domain of metaxin interacts directly and specifically with the pre- sequences.
b) SAM complex –
β-barrel proteins are exclusively found in the outer membranes of eukaryotic mitochondria. Porin, Tom40, Sam50 and Mdm10 are β-barrel proteins of the outer mitochondrial membrane. Tom complex receptors recognize β-barrel precursors and direct them through the Tom40 pore. They pass through a series of hydrophobic binding sites which prevent precursor aggregation (Dudek et al, 2013). Proteins with different trans- membrane topologies are integrated in the outer mitochondrial membrane via different mechanisms. They are necessary for biogenesis of β barrel proteins in the outer membrane (Fig 12).
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Fig 12: Multiple mechanisms for integration of proteins into the outer mitochondrial membrane (OM). A few α-helical proteins of the outer membrane, like Fis1, seem to insert without the help of proteinaceous translocases. OM proteins with an N-terminal α-helical membrane anchor, like Tom20 or Tom70, and multi- spanning α-helical OM proteins, like Ugo1, depend on Mim1 for membrane integration. Tom22 is recognized by the TOM complex receptors and subsequently inserted into the OM via the SAM complex. The SAM complex also mediates the membrane integration of β-barrel OM proteins, which are handed over from the TOM to the SAM complex by the small Tim chaperones Tim9–Tim10. Mdm10 has a dual function in the SAM complex and in the endoplasmic reticulum (ER)-mitochondria encounter structure (ERMES). Further ERMES proteins have been implicated in the biogenesis of β-barrel proteins as well. IMS, intermembrane space.
(Dudek et al, Biochim Biophys Acta 1833: 274–285, 2013).
10. Intermembrane space –
The space between mitochondrial outer and inner membrane is called as intermembrane space (IMS). The space accommodates many proteins involved in various import pathways. The tiny TIM proteins (TIM8, 9, 10 and 13) are involved in import of mitochondrial carrier proteins into the inner membrane and β-barrel proteins into the outer membrane (Chacinska et al, 2009). This space also contains proteins involved in the Mitochondrial Import and Assembly (MIA) pathway that is responsible for oxidative folding and maturation of IMS proteins through the formation of disulphide bonds between cysteine residues.
Tiny TIMs belong to a highly conserved protein family and consist of TIM8, 9, 10 and 13. In yeast, TIM9 and 10 play a very crucial role in viability. They form a hexameric chaperone complex that directs precursor proteins from TOM complex to the inner membrane. TIM8 and TIM13 also form a chaperone complex involved in import of TIM23.
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11. The inner membrane –
Proteins destined for the inner membrane or the mitochondrial matrix are transferred to the TIM22 or TIM17:23 complexes of the inner membrane. The TIM22 and TIM17:23 complexes consist of channel forming pores and associated proteins involved in the mechanisms of translocation and assembly.
a) TIM complex –
The TIM17:23 complex is the major translocation pore through which most of the mitochondrial proteins are imported. There are 2 different forms of this complex in yeast (Chacinska et al, 2009). One form called as TIM23-SORT consists of TIM23, TIM17, TIM50 and TIM21. It identifies the precursors from TOM complex and inserts them into the inner membrane. TIM23-SORT forms super-complexes with TOM40 channel or with complex III and complex IV of the mitochondrial respiratory chain through TIM21. These super-complexes use proton motive force for increasing translocation at sites of contacts.
The second form is called as TIM23-PAM, which is involved in protein transfer to mitochondrial matrix. TIM23-PAM contains TIM17, TIM23, TIM50 and the pre-sequence translocase associated motor (PAM) complex. PAM complex includes mtHSP70, TIM44 and its associated cochaperones - PAM16, PAM17 and PAM18 (Bolender et al, 2008).
Plants show orthologues for all TIM17:23 translocase components in yeast. These proteins also show certain plant specific features. Plant TIM17 protein shows C-terminal extension of 143 amino acids as compared to yeast. But the exact role for this C-terminal extension is still largely unknown.
The TIM22 complex is one more complex that functions for movement of multi- spanning membrane proteins through and into the inner membrane. Yeast TIM22 complex includes four subunits, the TIM22 channel and accessory proteins (TIM54, TIM18, TIM12).
In Arabidopsis, two identical genes on different chromosomes encode the homologue for TIM22.
b) PRAT –
In Arabidopsis, TIM17, TIM23 and TIM22 belong to a large gene family known as preprotein and amino acid transporters (PRAT). There are 17 members of the family in Arabidopsis (Murcha et al, 2007). The PRAT proteins typically show four transmembrane regions connected with a characteristic motif - [G/A]X2[F/Y}X10RX3Dx6[G/A/S]GX3G, (where X is any amino acid) by three short hydrophilic loops. The 17 genes encode 16 different PRAT proteins (two proteins are 100% identical). These 16 PRAT proteins
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demonstrate substantial diversity in size, structure and location. Out of 16 PRAT proteins, 11 were shown to be mitochondrial proteins which are classified into three subfamilies viz.
TIM17, TIM23 and TIM22. Three genes encode TIM17 (At1g20250, At2g37410 and At5g11690), 3 genes encode TIM23 (At1g72750, At1g17530 and At3g04800), and two genes encode TIM22 (At3g10110 and At1g18320).
c) Mitochondrial processing peptidase –
Mitochondria contain a large number of peptidases involved in protein turnover.
These peptidases carry out various roles in protein import pathway such as removal of target peptide, formation of peptide intermediates or degradation of the signal peptide after cleavage. The mitochondrial processing peptidase (MPP) is an ATP independent protease that functions for cleavage of the targeting peptide from the translocating precursor protein. Yeast MPP contains MPPα and MPPβ (two structurally related components) that are present in mitochondrial matrix. In Arabidopsis, both MPPα and MPPβ are integrated into the cytochrome bc1 complex and so are located on the inner mitochondrial membrane. In Arabidopsis, another protease known as pre-sequence protease (PreP) was observed in both mitochondria and chloroplasts and is involved in degradation of the pre- sequence peptide signal after its import.
10. Mitochondrial protein import pathways –
Mitochondria are the organelles that contain a complete genetic and protein synthesis system. Still, 99% of mitochondrial proteins are encoded by nuclear genes and precursor proteins are translated in the cytoplasm. These proteins have targeting signals for getting imported into the mitochondria. Earlier it was assumed that precursor proteins are imported into mitochondria via a general pathway. However, recent studies have disclosed five different protein import pathways. The import machinery of all pathways do not function in isolation, but cooperate with each other and are connected to other systems.
10.1 Pre-sequence pathway –
The classical mitochondrial targeting signals are termed as pre-sequences. Pre- sequences are positively charged peptide extensions of about 10–60 amino acid residues and are present the amino-terminal end of the proteins. Pre-proteins containing target signals are recognized by TOM20 and TOM22 receptors in the outer mitochondrial membrane and then are transferred across the outer membrane by TOM40 channel.
After passing through TOM40 channel, precursor proteins connect with TIM23 complex of inner mitochondrial membrane with the help of TIM50 and TIM21 (Fig 13). TIM23 is
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activated by the membrane potential, which directs translocation of positively charged pre- sequences across the inner mitochondrial membrane. Protein transport into mitochondrial matrix requires more energy and so one more source of energy (PAM) is utilized. PAM is pre-sequence translocase associated motor that receives energy from ATP. PAM contains mitochondrial heat shock protein 70 (mtHsp70) – a molecular chaperone, as its major component (Chacinska et al, 2009). The pre-sequences are cleaved off by the mitochondrial processing peptidase (MPP). Large number of inner membrane proteins that contain pre-sequences, are laterally released from the TIM23 complex. Other precursor proteins are first received by the matrix and then they are exported to inner membrane through Oxa1 and get integrated there.
Fig 13: The two classical pathways for mitochondrial protein import. (a) The presequence pathway (b) The carrier pathway (Becker et al, Trends in Biochem Sci 37: 85 – 91, 2012).
10.2 Carrier pathway –
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Certain inner membrane proteins like metabolite carriers do not have pre- sequences that can be cleaved after their translocation. But these proteins show many internal target signals. Such carrier proteins are attached to cytosolic chaperones and are identified by TOM70. Both carrier precursors and pre-sequence-carrying precursors make use of TOM40 to pass outer mitochondrial membrane. The hydrophobic carrier precursors then pass through the intermembrane space with the help of Tim9-Tim10 chaperone complex and get inserted into the inner membrane by the carrier translocase TIM22 complex (Fig 13).
10.3 SAM pathway (β barrel pathway) –
The mitochondrial outer membrane contains two classes of proteins – β-barrel proteins and proteins with α-helical transmembrane segments. Precursor proteins enter mitochondria through TOM complex, but they require some pathway for insertion of these proteins into the outer membrane. In the β-barrel pathway, β-barrel proteins are inserted into the outer membrane via the SAM complex (Fig 14). First, the precursors of β-barrel proteins are transported across the outer membrane by TOM complex. Once the precursor proteins are in IMS, small TIM chaperone complexes bind to these precursors and guide them to the SAM complex. SAM complex consists of Sam50 (also known as Tob55) and Sam35 (Tob38). These two are core components of SAM complex. The SAM complex can also connect with Mdm10 to form a larger complex. The larger complex helps for easy import of α-helical precursor of Tom22. Small TIM chaperones further help the precursor proteins for their insertion into the outer membrane.
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Fig 14: Three new pathways for mitochondrial protein import. (a) The β-barrel pathway (b) The mitochondrial intermembrane space assembly pathway (MIA) (c) α-Helical insertion (Mim1) Pathway (Becker et al, Trends in Biochem Sci 37: 85 – 91, 2012).
10.4 MIA pathway –
Many IMS proteins use the mitochondrial intermembrane space assembly pathway (MIA) for their transport (Fig 14). Large numbers of these proteins are cysteine-rich proteins containing disulfide bonds. The precursor proteins are transported from cytosol via TOM complex in reduced state. As soon as they reach IMS, Mia40 binds to cysteines containing signal of precursor protein. This binding forms a temporary disulfide bond. The sulfhydryl oxidase Erv1 catalyzes the formation of disulfide bonds in Mia40. Mia40 catalyzes the oxidative folding of the proteins. Mia40, Erv1 and the substrate protein form a ternary complex that facilitates efficient transfer of disulfide bonds (disulfide channeling).
10.5 Mim1 pathway –
This is the most recently defined import pathway into mitochondria (Fig 14). Mim1 is the core of the pathway. It contains one α-helical transmembrane segment and forms a large oligomeric complex. This complex is the functional unit that interacts with the
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precursor proteins. Majority of the TOM proteins and multi-spanning outer membrane proteins use Mim1 as their insertion pathway. Precursors for these proteins are not channeled through TOM40. These proteins are recognized by Tom70 and transferred to the Mim1 complex for membrane insertion. Mim1 also supports membrane insertion of several proteins with a single transmembrane helix into the outer membrane. It can transiently associate with the SAM complex to help assembly of small (α-helical) TOM proteins with Tom40.
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