3.A.9 The Chloroplast Envelope Protein Translocase (CEPT or Tic-Toc) Family
The vast majority of chloroplast proteins are encoded within the nucleus of the plant or algal cell. These proteins are made as preproteins on cytoplasmic ribosomes and are then targeted to the appropriate organellar subcompartment. Some of the proteins that comprise the translocation apparatus in the envelope and thylakoid membranes of the chloroplast have been identified. A single assembly is thought to catalyze translocation across the two-membrane envelope, and the protein components of the envelope that have been identified and sequenced appear to be unique. By contrast, translocation across the thylakoid membrane may occur via three or four pathways. One of these pathways involves ATP hydrolysis and proteins of the Type II general secretory pathway (IISP; TC #3.A.5). The SecY-dependent thylakoid membrane translocase is therefore not part of the translocase included under TC #3.A.9. A second thylakoid membrane translocase is GTP-dependent and includes proteins homologous to eukaryotic and bacterial signal recognition particles (SRP). This system probably uses the SecY system as described under TC #3.A.5. A third transthylakoid membrane translocation pathway is pmf-dependent but ATP- and GTP-independent. This is the Tat or VSP (TC #2.A.64) system. The basis for accurate targeting of multi-transmembrane-domain proteins to specific chloroplast membranes has been reviewed (Rolland et al. 2017).
Preproteins are normally recognized at the outer envelope and translocated in a single step across the outer and inner membranes through contact zones where the two membranes are maintained in close apposition. The targeting signal for envelope translocation is in the N-terminal bipartite 'transit sequence.' The N- and C-terminal portions of this sequence contain chloroplastic and intraorganellar targeting information, respectively. Translocation to the stroma occurs in at least two steps and requires the hydrolysis of both ATP and GTP. Subsequently, the transit sequence is removed in the stromal compartment. The determinants for stop-transfer and post-import pathways for protein targeting to the chloroplast inner envelope membrane have been analyzed (Viana et al., 2010). The metabolic redox state of the chloroplast regulates the import of preproteins (Balsera et al., 2010). The PetD subunit of the cytochrome b 6 f complex integrates into the thylakoid membrane in a post-translational and an SRP-dependent process that requires the formation of the cpSRP-cpFtsY-ALB3-PetD complex (Króliczewski et al. 2017).
In addition to chaperone proteins, four proteins of the outer membrane (TOC) and seven proteins in and associated with the inner membrane (TIC) of the chloroplast envelope import apparatus have been identified. The main Toc proteins (Toc(IAP)34/36), Toc(IAP)75 and Toc(IAP)86/159 or 160) are believed to form a recognition and translocation complex in the outer membrane (Becker et al., 2004; Tu et al., 2004; Wallas et al., 2003). IAP34/36 and IAP86/160 are related to each other in sequence and possess cytoplasmically exposed GTP-binding sites. They are receptor GTPases. IAP34/36 and IAP86/160 each represent a single gene product. The ratios of Toc75:Toc34:Toc159 are 3:3:1 (Kikuchi et al., 2006). The protein translocons of plastid envelopes in roots and leaves include constituents of both the Tics and the Tocs that are tissue specific (Vojta et al., 2004). While the Tic proteins appear to be relatively stable, several Toc subunits seem to have a high rate of turnover (Vojta et al., 2004). Chloroplast envelope biogenesis and cyanobacterial envelope biogenesis have been comopared (Day and Theg 2018).
A. thaliana has two homologues of Toc159 (atToc120 and atToc132) that may serve as receptors for alternative Toc159-independent import pathways. Toc75 (also called OEP75) forms an outer membrane porin consisting largely of β-structure that may serve as the outer membrane protein-conducting channel. It is distantly related to Gram-negative bacterial outer membrane proteins of the two-partner secretion (TPS) family (TC #1.B.20). Toc75 has one sequenced homologue in the blue green bacterium, Synechocystis (22% sequence identity). The Synechocystis protein is located in the outer membrane of the cyanobacterium and forms a voltage-gated, high conductance channel with high affinity for polyamines and peptides in reconstituted liposomes, and Toc34/36 and Toc86/160 have sequenced homologues from several plants. They also have distant homologues in bacteria and slime molds. Toc75 of the pea chloroplast and alr2269 of Nostoc PCC7120, which are homologous throughout their lengths, consist of two domains. The N-terminal domains function in recognition and complex assembly while the C-terminal domains provide the β-barrel pore. The pore is modulated by the N-terminal domain (Ertel et al., 2005).
The TOC core complex consists of Toc34, Toc75 and Toc159. Toc34 and Toc159 isoforms comprise a subfamily of the GTPase superfamily and act as GTP-dependent receptors at the chloroplast surface. Distinct members of each occur in defined, substrate-specific TOC complexes. Toc75 (BAM) is conserved from prokaryotes and functions as the unique protein-conducting channel at the outer membrane (Andrès et al., 2010).
The Tic proteins include Tic110, Tic44, Tic22 and Tic20. Tic110 (IEP110; IAP100) is a 996 aa protein with one or two probable, N-terminal, hydrophilic TMSs. They form oligomeric channels in the inner membrane. Tic110 is also called PIRAC (protein-import-related anion channel). It is a 50 picoSiemen anion channel that becomes inactivated during protein transport via a mechanism that requires both a functional transit peptide in the transported protein and stromal ATP. It is believed to be the protein import channel in the inner chloroplast membrane.
Other Tic components, Tic22, Tic20, and Tic55 may function in preprotein recognition/conduction via the Tic110 channel. Tic22 and Tic20 have homologues in various plants and in Synechocystis sp. (286 aas; gbD64003 and 160 aas; gbD90906, respectively). The driving force for inner membrane translocation may involve ATP hydrolysis by stromal chaperones, Hsp93, Hsp70, ClpC and Cpn60. Several other Tic subunits are believed to function in regulation. The 'redox regulon' (Tic62, Tic55, and Tic32) is involved in regulation of protein import via the metabolic redox status of the chloroplast. Regulation can additionally occur via thioredoxins (Tic110 and Tic55) or via the calcium/calmodulin network (Tic110 and Tic32) (Kovács-Bogdán et al., 2010).
The Tic and Toc complexes in the inner and outer membranes, respectively, may form independently, but they probably form a single supercomplex in the presence of a substrate preprotein by forming contact sites between the two membranes. Some evidence suggests that each complex can function in protein transport independently of the other when contact sites are disrupted. The complex is about one megadalton in size (Kikuchi et al., 2009). TOC receptor dimerization may participate in the initiation of membrane translocation during protein import into chloroplasts (Lee et al., 2009). Distant uncharacterized homologues in bacteria of several Tic and Toc proteins have been identified, suggesting a bacterial origin for some of these proteins, but the functions of the bacterial homologues are in general not known (B. Buyuktimkin and M. Saier, unpublished observations).
Cryptophytes, unicellular algae, evolved by secondary endosymbiosis and contain plastids surrounded by four membranes. In contrast to cyanobacteria and red algae, their phycobiliproteins do not assemble into phycobilisomes and are located within the thylakoid lumen instead of the stroma. Two gene families encode phycoerythrin α and light-harvesting complex proteins from an expressed sequence tag library of the cryptophyte, Guillardia theta (Gould et al., 2007). The proteins bear a bipartite topogenic signal responsible for the transport of nuclear encoded proteins via the ER into the plastid. More than half of them also carry an additional, third topogenic signal comprising a twin arginine motif, which is indicative of Tat (twin arginine transport-specific targeting signals). Import studies revealed different targeting properties of each individual part of the tripartite leader showing that phycoerythrin α is transported across the thylakoid membrane into the thylakoid lumen via the Tat pathway even if the 36 amino acid long bipartite topogenic signal precedes the actual twin arginine signal. Gould et al. (2007) provided the first experimental evidence of a protein being targeted across five biological membranes.
Tic20 forms a 1-megadalton complex at the inner membrane and directly interacts with translocating preproteins. Kikuchi et al. (2013) purified the 1-megadalton complex from Arabidopsis, comprising Tic20 and three other essential components, one of which is encoded by the enigmatic open reading frame ycf1 in the chloroplast genome. All four components, together with well-known TOC components, were found stoichiometrically associated with different translocating preproteins. When reconstituted into planar lipid bilayers, the purified complex formed a preprotein-sensitive channel. This complex is believed to constitute the general TIC translocon. Oh and Hwang 2014 summarized recent progress in the identification, functional characterization, and biogenesis of transporters and channels in the chloroplast envelope membranes.
The generalized transport reaction catalyzed by the chloroplast envelope protein import translocase is:
protein (cell cytoplasm) protein (chloroplast stroma)
Chloroplast envelope protein translocase (CEPT); outer membrane complex. The Tic20-like protein (acc # AUO68237.1; 413 aas, 6 TMSs in a 1 TMS (N-terminal) + 5 TMSs (C-terminal)), of the lower brown alga, Saccharina japonica, has been characterized (Chen et al. 2019).
Plants, algae (cyanobacteria)
The Toc (Translocase of the outer chloroplast membrane) complex of Pisum sativum (pea)
Toc75 (IAP75; OEP75) (16 β-strand porin) (O43715);
Toc(IAP)34/36 (receptor, GTP-binding protein and preprotein-activated GTPase);
Toc(IAP)86/159 (major import receptor; GTP-binding protein);
Toc(IAP)64 (surface docking protein)
The Tic (Translocase of the inner chloroplast membrane) complex of Pisum sativum
Tic110 (IAP100; IEP110) (recognizes preproteins; the probable channel; the C-terminal 700 residues form β-stranded cation-selective channels) (O24303);
Tic22 (peripheral intermembrane protein);
Tic20 (integral membrane protein);
Tic55 (possible redox sensor)
Tic62 (possible redox sensor)
Tic40 (stromal chaperone protein recruiter)
hsp70 (stromal ATPase chaperone)
The chloroplast envelope TIC translocase (TIC) complex. Tic21, Tic20-I, Tic214, Tic100 and Tic56 plus Toc components comprise a 1 Md complex in the membrane with each component being present in stoichiometric amounts (Teng et al. 2006; Kikuchi et al. 2013). A 1-megadalton complex consisting of Tic21, Tic20, Tic56, Tic100, and Tic214 has been identified in the chloroplast inner membrane (Nakai 2015).
The Tic complex of Arabidopsis thaliana
Tic214 (Ycf1) (1,786aas; 6 N-terminal TMSs) (P56785)
Tic100 (871aas) (Q8LPR8)
Tic56 (527aas) (Q7Y1Z4)
Tic20-I (274aas; 5 TMSs) (Q8GZ79)
Tic21 (296 aas; 4 TMSs) (Q9SHU7)
Tic40 (447aas) (Q9FMD5)
Tic110 (1,061aas; 2 TMSs) (Q8LPR9)
TicLTD (175aas) (GDC1) (Q8VY88)
ClpC1 Chloroplastic chaperone (929aas) (Q9FI56)
ClpC2 Chloroplastic chaperone (952aas) (Q9SXJ7)