The Chloroplast Protein Import System: from Algae to Trees☆

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Biochimica et Biophysica Acta 1833 (2013) 314–331

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Biochimica et Biophysica Acta

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Review The chloroplast protein import system: From algae to trees☆

Lan-Xin Shi, Steven M. Theg ⁎

Department of Plant Biology, University of California-Davis, One Shields Avenue, Davis, CA 95616, USA

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Article history: Chloroplasts are essential organelles in the cells of plants and algae. The functions of these specialized plas- Received 2 July 2012 tids are largely dependent on the ~3000 proteins residing in the organelle. Although chloroplasts are capable Received in revised form 7 September 2012 of a limited amount of semiautonomous protein synthesis – their genomes encode ~100 proteins – they must Accepted 1 October 2012 import more than 95% of their proteins after synthesis in the cytosol. Imported proteins generally possess an Available online 9 October 2012 N-terminal extension termed a transit peptide. The importing translocons are made up of two complexes in the outer and inner envelope membranes, the so-called Toc and Tic machineries, respectively. The Toc com- Keywords: Toc/Tic complex plex contains two precursor receptors, Toc159 and Toc34, a protein channel, Toc75, and a peripheral compo- Chloroplast nent, Toc64/OEP64. The Tic complex consists of as many as eight components, namely Tic22, Tic110, Tic40, Protein import Tic20, Tic21 Tic62, Tic55 and Tic32. This general Toc/Tic import pathway, worked out largely in pea chloroplasts, Protein conducting channel appears to operate in chloroplasts in all green plants, albeit with significant modifications. Sub-complexes of the Precursor receptor Toc and Tic machineries are proposed to exist to satisfy different substrate-, tissue-, cell- and developmental re- Evolution quirements. In this review, we summarize our understanding of the functions of Toc and Tic components, com- paring these components of the import machinery in green algae through trees. We emphasize recent findings that point to growing complexities of chloroplast protein import process, and use the evolutionary relationships between proteins of different species in an attempt to define the essential core translocon components and those more likely to be responsible for regulation. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids. © 2012 Elsevier B.V. All rights reserved.

1. Introduction endosymbiosis, two major events happened. First, the endosymbiont transferred the bulk of its genes to the host genome, and second, it de- The chloroplast is the major organelle in plant and algal cells respon- veloped protein import systems to translocate proteins from the host sible for photosynthesis. It is also the factory in which many other es- cytoplasm back into the endosymbiont. Two sources were used by the sential biosynthetic reactions occur, including synthesis of amino endosymbiont to construct the protein transport machinery; one native acids, fatty acids and terpenes. In plants, the chloroplast is only one of to the host cell and the other native to the prokaryotic endosymbiont a number of plastids that include proplastids, etioplasts, chromplasts, [3–11]. Eventually, two interacting protein conducting complexes were leucoplasts, amyloplasts, elaioplasts, proteinoplast or aleuronoplasts established in the envelope membranes; termed Toc (for Translocon at and gerontoplasts. These plastids display different morphologies, per- Outer envelope membrane of Chloroplast) and Tic (for Translocon at form specialized functions and store various biochemical compounds Inner envelope membrane of Chloroplast) [12]. Components originating during plant development. Under certain conditions, plastid types can with the prokaryotic endosymbiont include Toc75, Tic22, Tic21, Tic20, interconvert. Chloroplasts possess three membrane systems: the outer Tic55, Tic32 and Tic62, while those originating with the eukaryotic host envelope, the inner envelope and thylakoid membranes. These in turn are Toc159, Toc34, Tic110, Tic40 and Toc64/OEP64 (Table 1,andalso enclose three aqueous compartments: the intermembrane space, stro- see [3–7]). ma and thylakoid lumen. The structural and functional complexities of The genome of extant chloroplasts encodes only ~100 proteins. this organelle require the concerted action of some 3000 different pro- Accordingly, more than 95% of chloroplast proteins are nuclear teins [1]. encoded, synthesized in the cytoplasm as precursor proteins and Chloroplasts originated from endosymbiotic cyanobacteria, with the post-translationally imported into the plastids. Most chloroplast original symbiotic event estimated to have occurred approximately precursor proteins have a cleavable N-terminal extension, the transit 1.5 billion years ago [2]. In order to achieve a mutually beneﬁcial peptide, to direct them into chloroplasts and to target them to their ﬁnal destinations within plastids. The transit peptides are not conserved at the primary structural level. However, they share some ☆ This article is part of a Special Issue entitled: Protein Import and Quality Control in common structural and physical features, such as being rich in ser- Mitochondria and Plastids. ⁎ Corresponding author. Tel.: +1 530 752 0624; fax: +1 530 752 5410. ine, possessing a low abundance of acidic amino acids and having E-mail addresses: [email protected] (L.-X. Shi), [email protected] (S.M. Theg). lengths ranging from 20 to >100 residuals [13–16].Precursorsare

Table 1 Toc and Tic components from green algae to trees.

Component Gene in organism Transit peptide Proposed Origin C .reinhardtii P. patens A. thaliana P. sativum P. trichocarpa and Domain function Toc159 Cre17.g707500.t1.1 Pp1s123_61V6.1 At4g02510 AAF75761 POPTR_0004s17740.1 Acidic, Precursor Eukaryotic (8.3e−27) (0) (Toc159; 0) (0) GTPase , recognition; Cre17.g734300.t1.2 Pp1s123_63V6.1 At2g16640 POPTR_0008s22130.1 Membrane domain Translocation (9.5e−25) (0) (Toc132; 0) (0) driving motor Pp1s136_62V6.1 At3g16620 POPTR_0008s22140.1 (1.6e−180) (Toc120; 1.2e−178) (0) Pp1s136_59V6.1 At5g20300 POPTR_0009s13370.1 (3.8e−175) (Toc90; 4.9e−118) (0) At4g15810 POPTR_0009s13380.1 (Toc100; 2.8e−22) (6.8e−179) POPTR_0010s01800.1 (2.5e−136) POPTR_0006s06230.1 (1.1e−131) POPTR_0010s01820.1 (7.5e−123) POPTR_0025s00620.1 (1.5e−120) Toc34 Cre06.g252200.t1.1 Pp1s72_197V6.1 At5g05000 Q41009 POPTR_0002s18420.1 GTPase , Precursor Eukaryotic (2.5e−37) (6.1e−95) (Toc33, 1.3e−121) (9.3e−102) 1 TMHa recognition Pp1s54_12V6.2 At1g02280 POPTR_0014s10500.1 (2.3e−92) (Toc34, 2.2e−103) (3.7e−75) Pp1s6_441V6.1 (5.7e−65) Toc75 Cre03.g175200.t1.1 Pp1s2_62V6.1 (0) At3g46740 Q43715 POPTR_0001s12500.1 N−terminal Channel, Cyanobac- (3.7e−85) Pp1s23_111V6.1 (0) (Toc75−III, 0) (0) cleavable bipartite Precursor terial Pp1s317_51V6.1 (0) At4g09080 POPTR_0003s15670.1 transit peptide, recognition Pp1s44_230V6.1 (Truncated, 2.6e−139) (0) POTRA repeats, (2.6e−112) At1g35860 β−barrel (Pseudogene, 1.9e−128) OEP80 Cre02.g122700.t1.1 Pp1s1_532V6.1 At5g19620 P0C891 POPTR_0001s05640.1 POTRA repeats, Chloroplast Cyanobac- (1.1e-24) (2.6e−33) ( OEP80, Toc75−V,7.0e− (partial (1.8e−28) β−barrel biogenesis terial 29) sequence) POPTR_0003s20390.1 (7e−28) Toc64/ Cre18.g747150.t1.2 Pp1s167_52V6.2 AT3G17970 (0) Q9MUK POPTR_0012s04440.1 TMH, Receptor Eukaryotic OEP64 (AMI2, 6.7e-47) (9.1e−165) AT5G09420 (MtOM64, (0) TPR, Pp1s42_259V6.1 7e−154) POPTR_0015s04580.1 Amidase region (1.6e−161) AT1G08980 (AMI1, 1.2e− (0) Pp1s301_54V6.1 93) POPTR_0001s21240.1 (9.7e−153) (7.3e−153) Tic22 Cre14.g625750.t1.1 Pp1s282_30V6.1 At4g33350 Q9ZST9 POPTR_0002s12830.1 N−terminal transit Scaffold Cyanobac- (4.5e−21) (1.4e−58) (2.0e−91) (9.6e−96) peptide between Toc terial Pp1s81_171V6.1 At3g23710 POPTR_0014s02990.1 and Tic (2.7e−37) (9.1e−37) (6.4e−92) POPTR_0002s23710.1 (3.5e−41) None Pp1s68_153V6.1 At5g62650 N/Ab POPTR_0017s00970.1 Predicted N− N/A N/A (1.7e−59) (Tic22−like) (6.4e−179) terminal transit POPTR_0004s07060.1 peptide (1.1e−156) Tic20 Cre08.g379650.t1.1 Pp1s54_101V6.1 At1g04940 Q9ZST8 POPTR_0002s03320.1 N−terminal transit Channel Cyanobac- (7.5e−10) (1.5e−61) (Tic20−I, 3.6e−82) (1.0e−83) peptide, terial Cre04.g225050.t1.2 Pp1s197_89V6.1 At4g03320 POPTR_0005s25300.1 4 TMHs (2.2e−8) (7.8e−61) (Tic20−IV, 5.6e−32) (1.2e−83) Cre01.g039150.t1.1 Pp1s5_404V6.1 At2g47840 POPTR_0019s13630.1 (2.7e−8) (2.6e−7) (Tic20−II, 1.1e−7) (3.1e−37) At5g55710 POPTR_0004s20320.1 (Tic20−V, 6.2e−6) (3.5e−5) POPTR_0013s08250.1 (9.27e−4) Tic21 Cre10.g454771.t1.1 Pp1s54_55V6.1 At2g15290/PIC1 ABG00264 POPTR_0001s30780.1 N−terminal transit Channel, Cyanobac- (1.4e−27) (1.7e−68) (1.5e−78) (7e−88) peptide, assembly terial Cre22.g763750.t1.2 Pp1s58_244V6.1 POPTR_0009s09920.1 4 TMHs factor of (5.1e−13) (1.3e−66) (2.3e−85) importing Cre01.g039450.t1.2 Pp1s257_95V6.1 complex, Cre01.g062500.t1.1 (6.4e−37) iron transport Pp1s113_77V6.1 (10e−29) Tic110 Cre10.g452450.t1.1 Pp1s26_23V6.1 At1g06950 (0) O24303 POPTR_0013s15040.1 N−terminal transit Channel; Eukaryotic (9.1e−46) (0) (0) peptide, Precursor and Pp1s509_22V6.2 POPTR_0019s14760.1 2 TMHs (or chaperone (0) (0) 6TMHs) docking Tic40 Cre12.g508000.t1.1 Pp1s159_2V6.1 At5g16620 Q8GT66 POPTR_0004s08560.1 N−terminal transit Co-chaperone Eukaryotic (7.7e−40) (4.0e−73) (2.8e−100) (7.4e−117) peptide, for import Pp1s110_13V6.1 POPTR_0017s02900.1 Ser/Pro−rich, motor(s) (2.5e−72) (1.7e−115) 1 TMH, TPR, Hip/Hop/Sti1 Tic55 None Pp1s153_103V6.1 At2g24820 (0) O49931 POPTR_0018s02890.1 N−terminal transit Redox sensor Cyanobac- (1.6e−156) (0) peptide, and regulator terial − Pp1s11_89V6.1 POPTR_0006s28310.1 Rieske [2Fe 2S] (1.4e−152) (0) cluster, Mononuclear iron− binding site, CxxC motif, (continued on next page) 316 L.-X. Shi, S.M. Theg / Biochimica et Biophysica Acta 1833 (2013) 314–331

Table 1 (continued) 2 TMHs

Tic55- Cre17.g724600.t1.1 Pp1s7_405V6.1 At3g44880 CAR82238 POPTR_0003s21190.1 N−terminal transit chlorophyll Cyanobac- related ( PAO2, 1.0e−32) (3.9e−45) (PAO, 3.3e−42) (PAO, (4.8e−43) peptide, breakdown terial Cre10.g450550.t1.2 Pp1s111_8V6.1 At4g25650 partial) POPTR_1851s00200.1 Rieske [2Fe−2S] (PAO) ( PAO3, 8.7e−32) (2.3e−43) (PTC52, 5.4e−39) (1.8e−38) cluster, Cre06.g305650.t1.1 Pp1s312_10V6.2 POPTR_0001s04290.1 Mononuclear iron− (1.7e−29) (3.7e−41) (5.4e−38) binding site, Cre17.g724700.t1.2 POPTR_0004s22680.1 CxxC motif, ( PAO1, 9.9e−31) (8.0e−38) 2 TMHs Cre13.g600650.t1.1 POPTR_0003s21150.1 (1.2e−18) (7.1e−37) Cre05.g231450.t1.1 POPTR_0001s04310.1 (3.4e−10) (8.2e−31) POPTR_0009s00950.1 (4.3e−28) POPTR_0906s00210.1 (1.3e−15) POPTR_0003s21200.1 (2.0e−9) Tic62 Cre06.g269050.t1.1 Pp1s51_261V6.1 At3g18890 Q8SKU2 POPTR_0009s11450.1 N−terminal transit Redox sensor Cyanobac- (1.0e−60) (1.4e−87) (Tic62, 1.3e−127) (6.4e−140) peptide, and regulator terial Cre07.g349700.t1.1 Pp1s53_232V6.1 AT2g34460 POPTR_0004s15750.1 hydrophobic patch, (3.2e−32) (4.5e−30) (2.6e−29) (7.7e−137) NADP(H)−binding, Cre22.g764000.t1.2 Pp1s189_34V6.1 AT3g46780 POPTR_0011s02760.1 Tic−binding, (2.1e−31) (6.4e−23) (1.1e−14) (9.6e−27) FNR−binding Cre13.g575850.t1.1 Pp1s230_36V6.1 POPTR_0001s25160.1 (1.5e−12 ) (5.6e−17) (6.9e−18) Cre02.g081250.t1.1 Pp1s64_41V6.1 POPTR_0009s04180.1 (9.03e−3) (2.1e−11) (1.0e−17) Pp1s15_424V6.1 (5.7e−5) Tic32 Cre02.g135400.t1.1 Pp1s337_5V6.1 AT4G23420 Q6RVV4 POPTR_0015s12880.1 NADP(H)−binding, Calcium and Cyanobac- (4.8e−43) (4.4e−86) (1.3e−127) (1.4e−131) calmodulin− redox sensor terial Cre16.g685400.t1.1 Pp1s198_42V6.1 AT4G23430 POPTR_0003s12880.1 binding and regulator (1.4e−27) (9.4e−84) (Tic32, 4.4e−127) (1.1e−128) Cre11.g480650.t1.1 Pp1s50_45V6.2 AT4G11410 POPTR_0012s12960.1 (7.5e−25) (1.6e−75) (9.8e−121) (5.5e−118) Pp1s392_35V6.1 AT5G02540 POPTR_0003s12890.1 (7.2e−67) (6.1e−99) (6.0e−118) Pp1s89_52V6.1 AT2G37540 POPTR_0012s12980.1 (2.0e−59) (2.4e−98) (4.2e−111) Pp1s475_14V6.1 AT5G50130 POPTR_0001s09510.1 (4.2e−48) (3.6e−69) (5.9e−111) Pp1s162_74V6.2 AT4G24050 POPTR_0006s08410.1 (1.2e−45) (3.3e−68) (2e−89) Pp1s3_218V6.1 AT1G64590 POPTR_0003s12900.1 (7.1e−37) (1.4e−61) (2.5e−71) Pp1s39_72V6.1 AT4G27760 POPTR_0015s09260.1 (1.0e−26) (4.7e−28) (5.6e−65) Pp1s30_362V6.1 AT5G53090 POPTR_0001s11070.1 (5.8e−24) (2.8e−26) (4.3e−61) Pp1s100_75V6.1 AT5G53100 POPTR_0003s14410.1 (1.8e−17) (1.2e−25) (2.1e−55) POPTR_0003s18040.1 (1.3e−27) POPTR_0015s05560.1 (1.0e−26) POPTR_0012s08970.1 (1.6e−24)

Sequences of the accession numbers in bold, most of which are from pea, were used in BLASTp to identify cognates in other species. Values in brackets are E-values.Blue, components of eukaryotic origin. Yellow, components of cyanobacterial origin.a: TMH: transmembrane helix.b: N/A: not available or not known.

imported through the Toc and Tic complexes at the expense of inter- chloroplasts have been reported. An example of one such pathway is nal ATP [17], likely driven by stromal Hsp70 [18,19] and Hsp93 that suggested for the substrate-dependent import of the precursor to [20,21]. During or shortly after the precursors are translocated through the NADPH-dependent protochlorophyllide oxidoreductase isoform A the Toc and Tic machineries, the transit peptide is cleaved off by the (prPORA) [30,31], although there are reports of conﬂicting results in stroma processing peptidase (SPP). From this point, many proteins are the literature [2]. Another non-canonical pathway has been suggested folded by chaperonin (Hsp60) and function in the stroma. Those des- for chloroplast proteins that do not appear to have cleavable transit tined for the thylakoid membrane or lumen are further targeted peptides [32], with Tic32 being an example; see below for details. A through one of the four pathways: the cpTat, cpSec, cpSRP and sponta- third unusual pathway taken by proteins to the chloroplast interior in- neous pathways (see the reviews and references within [22–25]). Some volves passage through the endomembrane system [33–36].These proteins are also targeted back into the inner envelope membrane after non-canonical protein import pathways have been discussed in recent passing into the stroma, although other proteins take up residence in reviews elsewhere [16,28,29]. this membrane by a so-called ‘stop-transfer’ mechanism targeting of The components of the Toc and Tic complexes were identiﬁed main- these proteins, and others residing in the outer envelope membrane, ly using chloroplasts from Pisum sativum (peas). The Toc complex con- are not addressed here, and interested readers are referred to a number sists of three core proteins: Toc159 and Toc34, both receptors with of excellent reviews on those subjects [16,26–29,25]. GTPase activity, and Toc75, a protein conducting channel. A fourth In addition to this general import pathway utilizing the Toc/Tic ma- non-essential component, Toc64/OEP64 is often associated with this chineries, other non-canonical routes through which proteins enter complex (Fig. 1A). The core Tic components include Tic20, Tic22, L.-X. Shi, S.M. Theg / Biochimica et Biophysica Acta 1833 (2013) 314–331 317

Precursor Protein

TPR A G POTRA G

Toc64/ Toc75 Toc34 Toc159 OEP64 M

55 32 40 110 20 21

TPRT Hsp70sp JDP

CGE Sti1 Hsp93 ATP Hsp70

ATP ADP+Pi

ADP+Pi Cpn60

SPP

Fig. 1. The Toc and Tic translocons. A. The Toc complex. Shown are the three core subunits, Toc75, Toc159 and Toc34, and the non-essential Toc64/OEP64. Cytoplasmic proteins involved in protein import are not shown in this drawing. Toc75 is of prokaryotic origin and has two domains. The POTRA domain is located on the cytoplasmic side (Cyt) of the outer envelope membrane (OM) and is proposed to interact with precursor proteins to facilitate insertion or translocation. The β-barrel domain of Toc75 is embedded in the membrane and serves as a protein channel. Toc75 is conserved from cyanobacteria through higher plants. The receptors Toc159 and Toc34 are GTPases (G) and both have membrane domains. Most members of the Toc159 family possess a third acidic domain (A), which varies in length depending on species and Toc159 subgroup. The two GTPases are of eukaryotic origin and are present in most Viridiplantae species. Toc159 is absent in Cyanophoro paradoxa, a basally diverging alga. Toc64/OEP64 is depicted with its TPR domain on the cytoplasmic side of the membrane. B. The Tic complex. After or during translocation across the outer envelope membrane, the precursor protein interacts with Tic22 in the intermembrane space (IMS) and is engaged in the Tic complex. Three Tic components, Tic110, Tic20 and Tic21, are each proposed to be protein channels. Tic40 and the stromal portion of Tic110 recruit chaperones, Hsp70 and Hsp93, from stroma (Str) to drive the precursor protein across the inner envelope membrane (IM) at the expense of ATP. Tic55, Tic62 and Tic32 are components which are proposed to regulate import through redox and Ca2+ signaling. The transit peptide of the precursor protein is removed by the stromal processing peptidase (SPP) and may be folded by the chaperonin Cpn60. Tic22, Tic 20, Tic21, Tic55 and Tic32 are found in cyanobacteria through higher plants. Different domains of Tic62 likely originate from prokaryotic and eukaryotic genes. Tic110 and Tic40 are of eukaryotic origin, and Tic40 is absent in C. paradoxa. CGE is a nucleotide exchange factor and JDP is a J-domain protein, both serving as co-chaperones for Hsp70.

Tic110 and Tic40, with Tic21, Tic55, Tic62 and Tic32 representing facil- have become available. It is often the case that a particular Toc or Tic itating or regulatory components (Fig. 1B). component is encoded by more than one gene in a plant's genome. The Toc and Tic complexes appear to form the general translocation Some of the paralogs appear to impart specificity for different precursor pathway through which almost all chloroplast proteins are imported, proteins, tissues or developmental stages. In this review, we summarize with possible exceptions noted above. Our understanding of this gener- the known membrane-associated Toc and Tic components. Stromal al pathway has expanded recently as the genomes of various plants chaperones that act as ATP-dependent motors and play an integral part 318 L.-X. Shi, S.M. Theg / Biochimica et Biophysica Acta 1833 (2013) 314–331 in the import mechanism are reviewed by Flores-Perez and Jarvis [37] in the other GTPase, Toc34, in recognition of the precursor proteins [51]. this special issue. We further compare the Toc and Tic components from The M-domain of Toc159 is believed to function as the membrane an- five organisms; the green alga Chlamydomonas reinhardtii, the moss chor, although other additional roles have been suggested. Transient ex- Physcomitrella patens, two small model angiosperms, P. sativum and pression of M- and G-domains of Toc159 in Toc159 null ppi2 mutant Arabidopsis thaliana and a tree model plant Populus trichocarpa plants indicates that the M-domain confers the minimal structure re- (Table 1), and use their evolutionary relationships to sort the core quired for the protein import function, while the G-domain plays a reg- from the regulatory subunits of the respective translocons. ulatory role [52]. In addition to its receptor role, it has been proposed that Toc159 can provide a driving force for precursor translocation across the 2. The Toc complex outer membrane [53]. It was shown that interaction of Toc34 with Toc75/Toc159 is GTP-dependent and is stimulated by precursor pro- 2.1. Toc159 teins [48]. This study supports a model in which Toc159 is a part of the complete import motor (see below). 2.1.1. Discovery of Toc159 The targeting of Toc159 into the outer envelope membrane requires Toc159 is one of two GTPases in the Toc complex. It was originally the Toc machinery. The putative protein transport channel Toc75 is es- identified as an 86 kDa fragment in contact with importing precursor sential for targeting of Toc159 [54]. In addition, association of the proteins in the pea chloroplast outer envelope membrane by several GTPase domains in the two receptors, Toc159 and Toc34, contributes laboratories in early 1990s [38–41]. Based on this, it was suggested to Toc159 targeting [54,55]. Both Toc159 insertion into the membrane to function as a receptor for precursor proteins [39,41–44]. The pro- and assembly into the Toc complex are GTP-dependent processes and tein was known under several names, including OEP86, IAP86 and require GTPase activity of Toc159 itself [56]. In addition, it was reported the 86-kDa protein until 1998, when Soll and his colleagues discovered that the extreme C-terminal tail of the M-domain in Toc159 homologs that it was in fact a C-terminal proteolytic fragment of a larger receptor, processes transit peptide-like properties and can direct Toc159s to the now known as Toc159 in A. thaliana and pea [45]. Non-hydrolyzable outer membrane [57]. Thus, three elements in Toc159 are required for GTP analogs inhibit protein import [40],reflecting the essential nature its biogenesis: the C-terminal tail containing sorting information, the of at least one of the two Toc GTPases for protein import. Using a G-domain for insertion and assembly and the M-domain for membrane cross-linking strategy, it was shown that in the absence of exogenous anchoring. energy, this was one of the proteins associated with precursors bound to the plastid surface, indicating that it is involved in precursor recognition prior to translocation [43,46]. 2.1.3. Functional studies Toc159 homologs in A. thaliana are grouped by phylogenetic anal- 2.1.2. Biochemical properties and targeting ysis into three subtypes, namely AtToc159, AtToc132/AtToc120 and Toc159 is an integral membrane protein that is accessible to external Toc90 [57,58]. The Arabidopsis Toc159 null mutant ppi2 has an albino proteases added to isolated chloroplasts [40,41]. Thermolysin treatment phenotype and is seedling lethal [59]. The mutant plastids are similar yields a 50–52 kDa degradation product that is protected by the chloro- to undifferentiated proplastids with a limited paracrystalline internal plast outer membrane [41,45]. The nature of the membrane anchor is un- membrane structure and a prolamellar body, indicating that the devel- known and is not detected by prediction programs [40].Itwasreported opment of chloroplasts is arrested [59]. Remarkably, it was shown that that Toc159 exists in two forms: one soluble in the cytosol and the other atToc159 is required for import of photosynthetic proteins, whereas, associated with the outer membrane [47]. However, it was subsequently deletion of Toc159 has little effect on the accumulation of non- demonstrated that the seemingly soluble form of Toc159 was actually lo- photosynthetic proteins [59]. In vivo targeting of photosynthetic cated in small particles/vesicles that originated by membrane shredding and constitutive house-keeping precursor proteins into ppi2 plastids and which could be pelleted with lipids only by centrifugation at supports this, in that Toc159 is important for the former group of 600,000×g for 2 h [48]. Whether these Toc159-containing shreds are proteins [60]. In addition, Toc159 specifically recognizes and binds all produced during cell fractionation or are at least partially due to a nat- transit peptides of photosynthetic precursor proteins [60].Indeed, ural phenomenon in plants is not clear at present. If the latter case is cor- multiple sequence motifs in the transit peptide of the small subunit rect, it remains to be elucidated whether this fraction of Toc159 arises as of Rubisco were identified and were shown to contribute to the part of its functional cycle or is en route for degradation. Toc159-dependent import mechanism [61]. Toc159 has three discrete regions: an acidic N-terminal domain Studies on different types of cells demonstrated that plastids in roots (A-domain), a middle GTPase domain (G-domain) and a C-terminal and guard cells from wild type, ppi1 (AtToc33 mutant) and the ppi2 mu- membrane domain (M-domain). The A-domain is the least conserved tant have similar morphology and can differentiate into chloroplasts among Toc159 homologs (see below) and is dispensable for the recog- [62]. This led to the suggestion that AtToc159 and AtToc33 are not es- nition and translocation of precursors. It may, however, be involved in sential in the root and guard cells, prompting Li and her colleagues to substrate specificity, as the diverse sequences of A-domains in dif- propose that the Toc complexes are cell-type specific [62]. In line with ferent Toc159 isoforms appear to contribute major determinants this hypothesis, it was reported that in the ppi2 mutant, shoot apical for distinct import pathways [49]. The A-domain of AtToc159 can meristems show normal morphology, however there are fewer meso- be hyperphosphorylated and can exist as a separate soluble protein phyll cells and larger intercellular spaces [63]. [50], although the significance of these forms has not been elucidat- A somewhat different view of Toc159 was derived from a proteomic ed. Selective removal of cytoplasmic A- and G-domains blocks precursor study of ppi2 mutant plants. In these mutants some chloroplast-targeted binding to the chloroplast surface, confirming the role of this protein as a precursor proteins have apparently been N-terminal acetylated and ac- receptor [51]. However, under such conditions, translocation of the pre- cumulated outside the plastids. These modified precursors have diverse cursor across the outer membrane can still occur, suggesting some role of functions, which would be consistent with the idea that Toc159 serves

Fig. 2. Two new Toc159 members identiﬁed in Chlamydomonas reinhardtii and Arabidopsis thaliana. A. Alignment of CrToc159-1 (Cre17.g707500, 880 amino acids) and CrToc159-2 (Cre17.g734300, 967 amino acids) from C. reinhardtii. The two protein sequences were aligned using a web-based program LALIGN (Version 2.1) [229] (http://www.ch.embnet.org/ software/LALIGN_form.html) to identify local similarities. The N-terminal regions of the two Toc159s share 31.1% identity in their 530 amino acid overlap (5–523:17–487). B and C, align- ments of AtToc159 (At4g02510, 1503 amino acids) and AtToc100 (AT4G15810, 918 amino acids). Two conserved regions are shown. B. Regions in the G-domain of the two Toc159s share 37.3% identity in their 166 amino acid overlap (764–927:536–701). C. Regions in the M-domain share 32.3% identity in their 62 amino acid overlap (1056–1117:731–792). L.-X. Shi, S.M. Theg / Biochimica et Biophysica Acta 1833 (2013) 314–331 319

C 320 L.-X. Shi, S.M. Theg / Biochimica et Biophysica Acta 1833 (2013) 314–331 as a receptor for non-photosynthetic as well as photosynthetic proteins only share 18% identity at the overall amino acid level. When local align- [64]. ment using Lalign (http://www.ch.embnet.org/software/LALIGN_form. A mutant version of AtToc159 protein with reduced GTPase activity, html) was performed, two regions of greater similarity were found, A864R, can rescue the albino phenotype of ppi2 plants and increases one in the G-domain (37.3% identity in 166 amino acid overlap, protein import rates by stabilizing the formation of GTP-dependent Fig. 2B) and the other in the M-domain (32.3% identity in 62 amino binding intermediates [65]. Another Toc159 mutant, K868R,lacksGTP acid overlap, Fig. 2C). Compared with AtToc159, AtToc100 has shorter binding and hydrolysis activity. Similar to the A864R mutant, the but more acidic A-domain. Interestingly, the M-domain of AtToc100 is K868R protein can rescue the lethal phenotype of ppi2 and support pro- only roughly one third the length of that in AtToc159. Accordingly, tein import into chloroplasts [66]. It appears that GTP hydrolysis by this protein has lower hydrophobicity than other Toc159s, which sug- Toc159 is not strictly required for protein import and that another gests that it may be loosely associated with the outer envelope mem- GTPase contributes to protein translocation [66]. brane of chloroplasts or might be even located in the cytoplasm. Studies on mutants in which AtToc132 and AtToc120 are deleted sug- We searched for expression data for Toc100 in three online gest that these two Toc159 family members constitute a different import databases: Genevestigator (https://www.genevestigator.com/gv/plant. pathway from that using AtToc159 [67]. AtToc132 and AtToc120 single jsp), Tair (http://www.arabidopsis.org/index.jsp) and Gene Expression mutants do not have a strong visible phenotype, but the double mutant Atlas (http://www.ebi.ac.uk/gxa/). We found that AtToc100 is expressed is pale, similar to the AtToc159 mutant, indicating a functional redundan- in many experiments, organs/tissues and developmental stages. The cy between AtToc132 and AtToc120 [58]. The double mutant has abnor- highest expression levels are in seeds and embryos, although the overall mal plastids in its roots, which presumably reflects the involvement of expression of the gene is in the low to medium range as defined by these two proteins in the import of non-photosynthetic proteins [58]. Genevestigator. Blast searching against the NCBI EST database using Moreover, it has been reported that Toc132 plays a role in A. thaliana the mRNA sequence of AtToc100 (NM_117673.5) retrieved only one root gravitropism [68]. EST sequence with an identity of 99%. Considering the relatively low AtToc90 is a fourth Toc159 member (Table 1) which has only M- and expression levels and the single EST hit, it cannot be totally excluded G-domains, and lacks the A-domain. An AtToc90 mutant, named ppi4, that AtToc100 is a pseudogene. Clearly, the question of the function of has no phenotype. However, when it is deleted in the background of this potential new member of the Toc159 family awaits further ppi2, the double mutant plants are paler than the ppi2 single mutant experimentation. [69]. AtToc90 appears to participate in the biogenesis of photosynthetic proteins in chloroplasts, such as LHCP. In addition, AtToc90 interacts 2.2. Toc34 with other Toc components, Toc75 and Toc34, in vitro [69].Infangeret al. [70] showed that overexpression of AtToc90 in the ppi2 mutant back- 2.2.1. A second GTPase receptor of the Toc complex ground (Toc90/ppi2) could cause partial rescue of the ppi2 albino pheno- Toc34, initially named IAP34, was identified as a component of the type and resultant import defect. Consistent with this, photosynthetic early import intermediate in pea chloroplasts along with other Toc–Tic proteins accumulate in the Toc90/ppi2 plant [70].Thesefindings suggest proteins, such as Toc75, Toc159 and Tic110 [39,40,43,76].Toc34isan that AtToc90 has a similar function to that of AtToc159 in protein import, integral membrane protein with a transmembrane domain in its with a preference for photosynthetic proteins. C-terminal region and an N-terminal portion sensitive to externally added protease [40,76]. Similar to Toc159, Toc34 is a GTP-binding 2.1.4. Evolutionary considerations protein and possesses GTPase activity [76].Inaddition,Toc34shares Toc159 is of eukaryotic origin [4,7]. Through hydrophobic cluster sequence similarity to Toc159 outside of the GTPase domain [40]. analysis, it has been proposed that the G-, M- and A-domains of This receptor associates with precursor proteins in a manner regu- Toc159 originated from tandem duplication of a GTP-binding domain lated by GTP-binding, but not GTP hydrolysis [46].Later,itwasalso [71]. During the course of evolution, the A-domain has become more suggested that hydrolysis of GTP by Toc34 is stimulated by precursor acidic. proteins [77]. Several Toc and Tic components, including Toc159, Toc34, Tic110, were detected in a gymnosperm [72]. Interestingly, there are at least 2.2.2. Targeting two Toc34 isoforms which have distinct expression profiles during Toc34 does not contain a cleavable transit peptide. Studies of the needle development [72]. dependence on nucleotides or proteinaceous factors for its insertion A survey of 31 Viridiplantae genomes conducted at the Phytozome into the outer envelope membrane have produced conflicting results. (http://www.phytozome.net/search.php, version 8.0) [73] shows that In one report, Toc34 was shown to be integrated into the outer enve- all species except the green alga Volvox carteri contain more than one lope membrane in a spontaneous manner, requiring neither ATP nor Toc159 homolog. Although it has been reported that there is only one thermolysin-sensitive membrane components [40]. In other reports, Toc159 in C. reinhardtii [7], we have detected an additional one. The its insertion into the outer membrane required both ATP or GTP and two proteins (Cre17.g707500 and Cre17.g734300) have 23% identical proteinaceous components [76,78]. In a different study, targeting of amino acid sequences, with identity in the N-terminal regions, in- Toc34 was not affected by protease treatment of chloroplasts, but GTP cluding the GTPase domains, rising to 31.1% (Fig. 2A). Interestingly, and GDP were required for maximal integration [79]. It was also dem- the best hit of Cre17.g707500 in A. thaliana is AtToc120, while the clos- onstrated that Toc34 can target to protein-free liposomes, and that in- est homolog of Cre17.g734300 in A. thaliana is AtToc90. The N-termini sertion can be stimulated by GTP [80]. In addition to these external of the two C. reinhardtii Toc159 family members are not acidic, with factors, efficient targeting of Toc34 into the chloroplast outer envelope one acidic amino acid out of 22 and 16 residues, respectively. There membrane requires its C-terminus and the GTP binding domain [79].Fi- are four Toc159 paralogs in P. patens. All of them are more similar to nally, a double-positive charge at the cytosolic side of the transmem- AtToc132 than to other Toc159 members in A. thaliana.Inmostge- brane α-helix was shown to be a determinant for its correct topology nomes at Phytozome, there are 3–5 Toc159 receptors. However, in the in the outer envelope membrane [81]. trees Malus domestica and P. trichocarpa, there are up to 8–9Toc159 genes, probably due to recent whole-genome duplications [74,75]. 2.2.3. In vivo functional studies We also found a new member of the Toc159 family in A. thaliana In vivo functional studies have been most often carried out using (At4g15810), which we designate as AtToc100 because its theoretical A. thaliana. There are two Toc34 receptors in A. thaliana, AtToc33 and molecular mass is 99.7 kDa with 918 amino acids in length. The protein AtToc34, with AtToc33 being the ortholog of pea Toc34 [82,83].Both most similar to this new member in A. thaliana is AtToc159, though they of them have intrinsic GTPase activities. L.-X. Shi, S.M. Theg / Biochimica et Biophysica Acta 1833 (2013) 314–331 321

AtToc33 antisense plants display a pale yellowish phenotype, are pH sensitive. These data have been interpreted to indicate that while AtToc34 antisense plants show a weaker phenotype [84]. The the different pH sensitivities of monomeric and dimeric AtToc33 two paralogs also have different organ-expression patterns, which may serve as a regulatory factor in protein import. may reﬂect their precursor speciﬁcity [84]. Kinetic studies on the G-domains of AtToc33, AtToc34, PsToc34 and

The null mutant of AtToc33 (ppi1) is pale only for the first two AtToc159 revealed that Toc34 G-domains have higher Vmax and lower weeks after germination and then turns green and seems comparable Km values than of those Toc159 [93]. These authors also demonstrated to the wild type plants [85]. The mutant displays reduced sizes of that GTP hydrolysis by PsToc34 is stimulated by a chloroplast transit prolamellar bodies in etioplasts and fewer thylakoids in chloroplasts peptide in a manner similar to that of a GTPase activating protein. [85], and the abundance of many proteins of the photosynthetic com- It was demonstrated in vitro that GTP and phosphorylation of plexes is reduced [83]. Import efficiencies of mutant chloroplasts iso- Toc34 regulated the interaction of Toc34 with a transit peptide in lated from young ppi1 leaves are decreased compared with the wild peas [101,102]. Phosphorylation of PsToc34 resulted in a loss of GTP type chloroplasts, indicating a role of Toc34 in plastid protein import binding capability [103].InA. thaliana, AtToc33 can be phosphorylat- in vivo [85]. Studies with this mutant suggest that AtToc33 is specifically ed, while AtToc34 cannot [77,104]. However, in vivo studies demon- involved in import of photosynthetic proteins. Conversely, AtToc34 is strated that an AtToc33 possessing a mutated phosphorylation site presumed to be involved in the import of non-photosynthetic or house- could still fully rescue the ppi1 (AtToc33) mutant albino phenotype keeping proteins [86]. The plastid morphology in the guard cells and [105]. Further analysis of this transgenic plant indicated that phos- roots in the ppi1 mutant is comparable with that in wild type plants phorylation of AtToc33 can affect early development, but is less im- [62], indicating that AtToc33 is not essential in these types of cells and portant for later stages [105,106]. organs. Characterization of the ppi3 (AtToc34-free) mutant suggests PhosphorylationofToc34,aswellasToc159canoccurintheGTPase that AtToc34 is more important during the development of plastids in domains and disrupts homo- and hetero-dimer formation [107].Phos- roots and therefore is more specifically involved in the import of phorylation also interferes with the interaction of Toc34 with other non-photosynthetic proteins [87]. Toc components [107]. Taken together, it appears that phosphorylation Activity of these receptors in specific tissues is also evidenced by the of the two GTPase can result in inhibition of downstream steps of pro- fact that deletion of Toc receptors arrests embryonic development at tein import. different stages [88]. The development of an embryo of an AtToc132/ AtToc159 mutant stops at the heart stage, while the embryo of 2.2.5. Evolutionary considerations AtToc33/AtToc34 mutant arrests slightly earlier, at the globular stage. Similar to Toc159, Toc34 is not found in cyanobacterial genomes and may be of eukaryotic origin [4,7]. C. reinhardtii contains only one Toc34. 2.2.4. Structure, dimerization and phosphorylation Strikingly, its N-terminus upstream of the GTPase domain is extremely The crystal structure of the cytosolic portion of pea Toc34 in com- acidic, with 1 acidic residue per 2.4 amino acids [7].Asmentioned plex with GDP and Mg2+ reveals that this receptor forms a dimer above, while many Toc159 members have an N-terminal acidic domain, [89]. It was proposed that each monomer served as a GTPase activat- the two CrToc159 do not. It has been proposed that the A-domain may ing protein (GAP) for the other via an arginine finger, R133, located at have exchanged between CrToc159 and CrToc34 [7]. Whether this the interface of the two monomers [89]. However, it was later dem- “A-domain” in CrToc34 has a function similar to that in Toc159 is not onstrated that R133 is important for the formation of dimers as di- known. merization was not observed in a pea R133A mutant [90]. A similar Two Toc34 homologs with over 95% identity to each other were result was also observed in an equivalent AtToc33 mutant. It was found in maize, and named ZmToc34-1 and ZmToc34-2 [108]. In spin- also shown that this residue in the Arabidopsis protein is not involved ach, it was reported that two Toc34 receptors of spinach displayed in activation of the other monomer [91,92]. Consistent with this, the substrate specificities [109]. The authors showed that six more spe- GTPase activities of AtToc33 [91] and pea Toc34 [93] are not influenced cies have two Toc34 genes [109]. A survey at Phytozome suggests by dimerization. The crystal structures of PsToc34 and AtToc33 further that 22 out of 29 land plants have more than one Toc34 gene, indicat- reveal that dimerization is independent of the nucleotide loading ing that the substrate selection via Toc34 isoforms might be common state, and that the PsToc34 dimer is not self-activating, confirming among land plants. that a separate GAP is required [94]. It was hypothesized that either an additional factor activates the homodimer or Toc159 exchanges for an AtToc33 to become an active heterodimer GTPase [92]. Indeed, 2.3. Toc75 using a split-ubiquitin system, it was demonstrated that AtToc159 and AtToc33 formed heterodimers in vivo [95]. In another study, it was 2.3.1. The protein conducting channel of the Toc complex reported that the dimeric AtToc33 and PsToc159 display higher GTPase Toc75 (formerly named IAP75 and OEP75) is localized in the chloro- activities than their respective monomers [90].Recently,itwasob- plast outer envelope membrane [44] and was identified in contact with served that homodimerization of AtToc33 actually reduces nucleotide early import intermediates captured using a Protein A tagged pre- exchange and inhibits GTPase activity. Transit peptide binding triggers cursor protein [39]. The same protein was identified independently as dissociation of monomers from the dimer and stimulates GDP exchange interacting with importing precursors [42,110]. Using a label-transfer for GTP [96,97], thereby enabling Toc33 for another round of GTP hy- cross-linking approach, an interaction between Toc75 and incoming pre- drolysis and precursor binding. cursor was again confirmed [46,111]. This major protein in the chloro- Studies using a Toc34 dimerization defective mutant also indicate plast outer envelope membrane has often been co-purified with that dimerization of receptors has a role in protein import. It is spec- Toc159 and Toc34 in various outer envelope membrane preparations ulated that receptor–receptor interaction participates in the initiation [20,21,39,42,43,112,113]. Topology studies have shown pea Toc75 to of the GTP-dependent protein translocation [98]. Complementation have a β-barrel structure [111]. with various AtToc33 point mutants in a ppi1 (AtToc33 mutant) back- The Toc75 precursor possesses an N-terminal cleavable bipartite ground suggests that GTP-binding and dimerization are not essential, transit peptide that directs it into the stroma first (though perhaps but they can enhance import [99]. Both reports support the regulato- not completely), and then to the outer membrane [110,114–116]. ry roles of dimerization of Toc34 in protein import. The targeting of Toc75 uses the general import pathway before inser- The GTPase activity of dimeric AtToc33 is insensitive to pH, while tion to the outer membrane [26]. A glycine-rich stretch present in the that of monomeric AtToc33 is pH-dependent [100]. Moreover, dimer- transit peptide is crucial for directing the protein to the chloroplast ization of AtToc33 and translocation of precursors into chloroplasts outer envelope membrane [116,117]. A type I signal peptidase (SPase I) 322 L.-X. Shi, S.M. Theg / Biochimica et Biophysica Acta 1833 (2013) 314–331 located in the envelope and thylakoid membranes [118] is responsible for 2.4. Toc64/OEP64 the full maturation of Toc75 [119,120]. Toc75 belongs to the Omp85/D15 family of β barrel proteins. Bacte- 2.4.1. Characterization rial Omp85 is essential for viability and plays a role in outer membrane Toc64/OEP64 was suggested to be a Toc component in pea in 2000 protein assembly [121,122]. Toc75 from Synechocystis PCC 6803 is also [147].Itcouldbeco-purified with Toc159, Toc34 and Toc75, and could essential and is located in the lipopolysaccharide layer (outer mem- be recovered in complexes containing precursor and Toc/Tic components brane of the cyanobacteria) [123,124]. This cyanobacterial protein can [147]. Moreover, Toc64/OEP64 antibody could co-immunoprecipitate form a high conductance channel in reconstituted liposomes [123]. other Toc components [148]. The pea Toc64/OEP64 is an integral The pea and Synechocystis Toc75 are structural homologs, which led to membrane protein possessing three transmembrane domains (TMDs) the suggestion that this protein constitutes the Toc protein conducting [28,149], an inactive amidase region and three tetratricopeptide repeats channel. The channel properties of pea Toc75 have been confirmed (TPR). The TPR domain of Toc64/OEP64 is located at the C-terminus and [125]. is exposed to the cytosol [147]. This TPR domain possesses ~70% Three repeats of POTRA (for Polypeptide-transport-associated) do- α-helical content [150] and belongs to the bicarboxylate clamp type mains in Toc75 have been identified at the protein's N-terminus, and in- [151]. It has been reported to provide a docking site for cytoplasmic formation about its overall structure has come from the crystal structures Hsp90 to deliver precursors to the receptor Toc34 of the core Toc com- of Omp85 from the thermophilic cyanobacterium Thermosynechococcus plex in a nucleotide-depended manner [149,151]. The second and third elongatus [126] and from the mesophilic cyanobacterium Anabaena sp. transmembrane domains lead to a loop in the intermembrane space PCC 7120 [127]. Interestingly, the cyanobacterial Omp85 is more similar [149]. This loop is suggested to be a part of the translocon because of to plant Toc75 than to proteobacterial Omp85 [126]. The POTRA domains its recognition of precursor proteins and its interaction with other Toc are speculated to have a chaperone-like function [128]. Consistent with and Tic components, e.g. Tic22 [149]. this suggestion, it was demonstrated that the N-terminal region is in- A Toc64/OEP64 double mutant (ΔPpToc64-1 ΔPpToc64-2) generat- volved in the recognition of precursor proteins and formation of Toc ed in the moss P. patens does not display any growth phenotype or complex [129]. Its topology in the outer envelope membrane has been defects in protein import [152]. Interestingly, chloroplasts in the dou- studied. It is shown that the POTRA domains of Toc75 face the cytoplasm ble mutant appeared thinner and longer than in wild type plants. [130]. Thus, all three core Toc components, Toc75, Toc159 and Toc34, These findings suggested that Toc64/OEP64 may not be a core compo- have cytosolic portions that serve as receptors for precursor proteins nent in chloroplast protein import, leading to the suggestion that it be and/or assembly factors for forming the Toc complex. Toc75 is required re-named OEP64 to reflect the uncertainty in its role in protein import for the import of nearly all proteins that enter the chloroplast. It is also [152]. A mutant involving all three Toc64/OEP64 genes in Arabidopsis involved in targeting outer envelope membrane proteins, such as (Toc64-III/V/I) also failed to reveal an import-related phenotype, again Toc75 itself, Toc159, OEP14 and Toc64/OEP64 [26,54,110,131,132]. suggesting that it may not be crucial for protein import [153]. The chlo- Arabidopsis Toc75 (AtToc75-III, At3g46740) mutants display an roplast shape phenotype noted for the Toc64/OEP64 mutants in moss embryo lethal phenotype, with embryo development arrested at the was not observed in the Arabidopsis mutants. two-cell stage [88,133]. Three Toc75 genes have been identified in A. thaliana [82]. However, expression of Toc75-I cannot be detected 2.4.2. Targeting and localization [133,134], which may suggest that this is a pseudogene. Toc75-IV ex- Toc64/OEP64 does not have a cleavable transit peptide [154].In presses at a much lower level than at Toc75-III and its knockout plants vivo targeting of Arabidopsis Toc64/OEP64 and analysis of GFP fusion display a mild phenotype [133]. Therefore, it is most likely that only a constructs suggests that the signals for Toc64/OEP64 targeting to the single functional Toc75 in A. thaliana (At3g46740) [135]. outer envelope membrane are the lysine-rich flanking region at the C-terminus and in the hydrophobic amino acid residues in the TMDs 2.3.2. OEP80 [155]. In vitro targeting of Toc64/OEP64s from P. patens requires nucle- In addition to Toc75, another OMP85 member, OEP80 (Outer otides, proteinaceous factors and its N-terminal region [26,131].Inad- Envelope Protein, 80 kDa), is present in pea and A. thaliana (also dition, insertion of OEP14 and PrSSU competes with the insertion of referred to as AtToc75-V, At5g19620) [136]. This protein is also predict- the two moss Toc64/OEP64s, indicating that some components of Toc/ ed to have a β-barrel structure [137]. OEP80 is targeted by a different Tic are involved in targeting of Toc64/OEP64 into the chloroplast outer mechanism from that used by Toc75, and is independent of the gen- envelope membrane [131]. eral pathway. OEP80 does not have an N-terminal cleavable transit Three genes in A. thaliana encode Toc64/OEP64 and its related pro- peptide; instead, the targeting signal is present in its mature form teins [82]. Toc64-III (At3g17960) is localized in chloroplasts. AtmtOM64 [26,138]. (At5g09420) resides in mitochondria and is proposed to have a function Like Toc75, an OEP80 deletion mutant is embryo lethal, with arrest similar to the mitochondrial preprotein receptor Tom70 in mammalian of embryo development occurring at the globular stage [139,140].By and fungal cells [156,157]. AtAMI1 (At1g08980) is present in the cyto- using an inducible RNAi approach, studies on plants silenced for plasm [158,159], and contains the amidase region, but lacks the TPR do- AtToc75-III or AtOEP80 suggest that both proteins are important for main [159]. Three Toc64/OEP64s are found in P. patens. Unlike those in chloroplast biogenesis at postembryonic stages of plant development vascular plants, the moss Toc64/OEP64 proteins possess the catalytic [141]. These studies also confirmed the involvement of Toc75 in pro- residues in the amidase region [152]. Two of the moss Toc64/OEP64 tein import in vivo [141]. (PpToc64-1 and -2) are more similar to the Arabidopsis and pea chloroplast Toc64/OEP64 and are indeed targeted to chloroplasts [152],while 2.3.3. Evolutionary considerations the third one is more similar to the mitochondrial AtmtOM64 as judged Toc 75 is suggested to be of cyanobacterial origin. It has apparently by the percentage of identical amino acids shared between their overall evolved by partial gene duplication in the N-terminal region of an an- sequences. Phylogenetic analysis of Toc64/OEP64 proteins and their cient prokaryotic channel protein [3,142]. Another gene duplication TPR domains from various species indicates that the proteins and TPR most likely occurred in an ancient eukaryotic organism ~1.2 billion domains of Toc64/OEP64 are divided into two branches; one located years ago, resulting in the formation of Toc75 and OEP80 sister genes in plastids and the other in mitochondria [160]. [143,144]. This channel protein is conserved among monocot and dicot species and is present in various plastid and tissue types [145,146].Sur- 2.4.3. Evolutionary considerations vey at Phytozome indicates that 1–5 copies of Toc75 gene are present in Toc64/OEP64 is absent in cyanobacteria and the green alga all genomes of Viridiplantae linage. C. reinhardtii, but, is present in two other green algae from the L.-X. Shi, S.M. Theg / Biochimica et Biophysica Acta 1833 (2013) 314–331 323 genus, Ostreococcus [7]. It is also missing in the genomes of red algae involved in protein import. The route for importing Tic22 into this [161] and the glaucophyte alga Cyanophora paradoxa [162].Searching sub compartment was initially thought to be different from the gen- for Toc64/OEP64 in the Phytozome platform indicates that there are eral pathway [168], but later, it was shown that the Tic22 precursor one or two Toc64/OEP64 genes in the genomes of most green plants, is imported through Toc complex. It is likely to be processed into its with as many as five in some. Taken together, Toc64/OEP64 appears mature form in the intermembrane space [169]. to be of eukaryotic origin and is only present in some green algae and plants. The lack of Toc64/OEP64 in certain genomes may again signify 3.1.2. Evolutionary considerations its non-essential, perhaps regulatory, role in chloroplast protein import. Tic22 is a ubiquitous protein existing in almost all photosynthetic organisms including cyanobacteria and higher plants [7].Itisalsopres- 2.5. Substrate specificity of the import pathway ent in the non-photosynthetic apicoplast of Plasmodium falciparum,the causative agent of malaria [170]. A search for Tic22 using Phytozome re- The general import pathway was initially worked out largely in sults in identification of two families across 31 spices of Viridiplantae. In chloroplasts isolated from peas. The availability of numerous plant the first family (#31850722), there are two members of Tic22 in most genomes now makes available more information about Toc and Tic species, including the moss P. patens and A. thaliana (At3g23710/ components from many plants. It is commonly found that there is Tic22-III and At4g33350/Tic22-IV). The tree P. trichocarpa has three more than one gene encoding a translocon subunit in any given Tic22 genes and green alga, C. reinhardtii and V. carteri each has only species, as exemplified by the five genes related to Toc159 present one gene. The pea Tic22 (Q9ZST9) that has been intensively studied is in A. thaliana. Consistent with this multiplication of homologous subunits, a member of this first family, which contains homologs with lengths more than one Toc (and maybe Tic as well) subcomplexes possessing vary ranging from ~200 to ~400 amino acid residues. In the second different substrate preferences have been identified. For example, Tic22 family (#31808761), there is only one copy in the majority of spe- AtToc159, AtToc90 and AtToc33 are apparently involved in the trans- cies. The sizes of these family members are much larger (~400 to ~600 aa) location of precursors of proteins involved in photosynthesis. In con- than those in the first family. The Arabidopsis At5g62650/Tic22-like be- trast, AtToc132/AtToc120 and AtToc34 show preference for precursors longs to this family. It shares only 13% and 15% identity with the entire se- of non-photosynthetic house-keeping proteins. In line with their func- quences of Tic22-IV and Tic22-III, respectively, as analyzed with ClustalW tional specialties, AtToc159 and AtToc33 have higher expression levels (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Notably, local alignment in leaves, while AtToc132/AtToc120 and AtToc34 have relative higher analysis using Lalign shows regions of higher identity. Some members expression in roots [134]. in this family possess a partial Tic22-like domain (PF4278) near the The recent isolation of a 1 MDa Tic complex containing previously middle of their sequences. There are no representatives of this second unidentified components opens the door to the possibility that multi- family in the green alga C. reinhardtii and V. carteri. It is noteworthy ple Tic complexes exist as well [163]. Whether the new Tic complex that no Tic22 homologs are present in the Prasinophyceae green displays (or mediates) substrate specificity is not known, but it would algae, Ostreococcus lucimarinus and Ostreococcus tauri, the smallest certainly be in keeping with the functions assigned to the various known eukaryotes [7]. sub-complexes of the Toc complex. No in vivo studies on the function of Tic22 have yet been published, although many questions could be addressed at least in the model plants 2.6. Stoichiometry of Toc components A. thaliana and P. patens: Do the two similar Tic22, e.g. AtTic22-IV and Tic22-III, have redundant or specific functions? Where is the third Apurified Toc core complex from pea is ~500 kDa and contains Tic22 located within chloroplasts? Can it complement the functions of Toc159, Toc75 and Toc34 in a 1:4:4–5 molecular stoichiometry [113]. AtTic22-IV and Tic22-III? From an evolutionary point of view, why do More recently, Toc complexes containing the same components with most land plants need two or even three Tic22s while the single celled an apparent molecular weight of ~700 kDa have been detected by and multicellular green alga need only one? histidine- and deoxycholate-based native (HDN-) PAGE analyses [164]. In another report, slightly larger Toc complexes (800–1000 kDa) were 3.2. Tic20 isolated from pea chloroplasts using Blue Native PAGE and size exclusion chromatography [165]. This larger Toc complex also consists of Toc159, 3.2.1. A potential protein channel in the inner membrane Toc75 and Toc34, but in a 1:3:3 stoichiometry [165]. This stoichiometry Tic20 was originally named IAP21 [43] or Tic(21) [46] when it was of Toc complexes appears to be constant in chloroplasts, etioplasts and found to be covalently cross-linked to importing precursor proteins in root plastids [165]. These data suggest that there are 3–4 channels, one pea chloroplasts. It is also associated with other translocon compo- larger GTPase and multiple smaller GTPase per Toc core complex. The nents forming the Toc–Tic supercomplex [166]. Besides being present presence of multiple translocation channels within a single Toc com- in chloroplasts, it was shown that Tic20 is essential for protein import plex is in agreement with the appearance of these complexes in EM into apicoplasts, the plastids in the parasite apicomplexan Toxoplasma images [113]. gondii [171]. Tic20 is an integral protein in the inner envelope membrane 3. The Tic complex [166,171],80%ofwhichisinanα-helical configuration [172]. It is predicted to span the membrane four times [173], and both of its N- and 3.1. Tic22 C-termini are exposed to the stroma [171,172]. A. thaliana contains four Tic20s, AtTic20-I, -II, -IV and -V [163,170,174]. All four AtTic20s 3.1.1. Initial characterization and targeting possess predicted transit peptides and, in one report [162], were exper- Tic22 is an extrinsic membrane protein bound to the outer face of imentally demonstrated to be localized in the chloroplast envelope, the inner membrane of chloroplasts [166]. It was initially named most likely in the inner envelope membrane. Surprisingly, while a dif- IAP25 [43] because this 25 kDa (apparent molecular weight) protein ferent report placed both AtTic20-I and -II in the inner envelope mem- can be covalently cross-linked to precursor proteins at intermediate brane, AtTic20-V was detected in the thylakoid fraction and AtTic20-IV and late stages of import [43,46,166]. Because it is also associated was dually localized in both chloroplasts and mitochondria [173]. with other Toc and Tic subunits [166,167], it has been considered to The function of Tic20 has been studied in vivo in A. thaliana. be a Tic component in the general import pathway serving as a AtTic20 (At1g04940) antisense plants display pale leaves, reduced scaffold between Toc and Tic complexes [166]. To date, Tic22 is the chloroplast protein levels and defects in protein import [175]. Based only protein known to be exposed to the intermembrane space and on the in vivo and biochemical data, Tic20 is proposed to function 324 L.-X. Shi, S.M. Theg / Biochimica et Biophysica Acta 1833 (2013) 314–331 as a protein-conducting channel in the inner membrane of chloro- Tic21 may play a role in proper assembly of the larger complex in the plasts [175]. Tic20 has also been found in a 1-megadalton transloca- inner membrane [163].Thetic21/pic1 mutants display defects in the tion complex detected by Blue-native PAGE (BN-PAGE). In addition import of photosynthetic precursors, but not of house-keeping proteins to Tic20, this complex contains Tic21 and other unidentified compo- [163]. It appears that Tic21, together with Tic20-I, has a preference for nents, but does not contain Toc159, Tic110, Tic40, Hsp93 or Tic22 the import of photosynthetic proteins [174]. [163]. This large complex is suggested to be a translocation intermedi- In a screen for metal transporters in plastids, the Philippar labora- ate functioning between Toc and Tic110–Tic40-chaperone complexes tory isolated an Arabidopsis mutant with a similar phenotype to that [163]. An independent study also found Tic20 in a complex larger mentioned above; they called the corresponding protein PIC1 [179]. than 700 kDa [172]. Strikingly, reconstituted liposomes containing pu- These authors observed impaired development of plastids in the mu- rified Tic20 display features of cation selective channel activity [172], tant. In addition, ferritin clusters increased and protein levels related again suggesting that Tic20 is a protein channel (parallel to Tic110 in to iron stress or transport, photosynthesis, and Fe–S cluster biogene- the inner envelope membrane, see below). sis were regulated differentially in the pic1/tic21 mutants. Based on these observations and studies on PIC1 overexpressing Arabidopsis 3.2.2. Evolutionary considerations plants, it was suggested that PIC1/Tic21 plays a role in iron transport Tic20 is one of the Tic proteins with a prokaryotic origin [4,170].A across the chloroplast inner envelope membrane [179,180]. However, survey using Phytozome shows that the Tic20 gene is represented be- Nakai and his coworkers argue against the iron transporter role of PIC1 tween 2 and 9 times in each of the 31 listed organisms, including by demonstrating that up-regulation of iron homeostasis-related gene green algae and flowering plants. Phylogenetic analyses suggest that expression is not unique to the tic21/pic1 mutant. Indeed, similar regu- Tic20 proteins are divided into two subfamilies/groups [7,176,177], lation occurs in other albino mutants, e.g. tic20 and albino3 [163]. Nev- and it is estimated that the first Tic20 gene duplication occurred ertheless, it might be possible that Tic21 possesses dual functions, ~1.2 billion years ago, after chloroplasts evolved [174]. Since then, participating in the import of proteins and in iron transport. two evolutionary branches have developed. Interestingly, the genes represented by group 1, which are essential for protein import, have 3.3.2. Evolutionary considerations changed during evolution at a faster pace than those in group 2. Wheth- Tic21 is of prokaryotic origin [7] and it appears that Tic21 is structur- er the higher mutation rates of the group 1 genes are due to positive se- ally and functionally conserved from cyanobacteria through higher lection or relaxation of purifying selection is not clear at the present plants [181]. With the exception of Medicago truncatula,inwhichTic21 [177]. appears to be absent, each species of the 31 organisms of Viridiplantae There are four pea Tic20 orthologs in A. thaliana, with two in each of surveyed using Phytozome showed usually one, but as many as four group 1 or group 2. Those aligning most closely with pea Tic20 (AtTic20-I Tic21 genes. and AtToc20-IV) belong to group 1 and play essential roles in chloroplasts; those falling into group 2 (AtTic20-II and AtTic20-V) have 3.4. Tic110 functions that are as yet unknown [7,176,177]. AtTic20-I is mostly expressed in shoots, while Tic20-IV is expressed mainly in roots 3.4.1. A second potential protein channel in the Tic complex [174,176]. Consistent with their expression preferences, Tic20-I T-DNA Tic110 was originally named IEP110 [182,183] and IAP100 [184], insertion lines exhibit albino and seedling lethal phenotypes [174,176], and then changed to Tic110 soon after it was found to be a compo- lack thylakoid membranes [176], fail to accumulate photosynthetic pro- nent of pea chloroplast translocon in the inner envelope membrane teins [174,176] and display impaired protein import [163].Although [182,184,185]. Tic110 was the first discovered Tic protein. It is an Tic20-IV is not essential for A. thaliana viability, it partially complements integral membrane protein anchored via its two transmembrane he- Tic20-I function since the double mutant tic20-I tic20-IV is embryo le- lices at the N-terminus [182,184,186], with the bulk of the soluble thal [174,176]. These data indicate that Tic20-I and -IV have some portion in the stroma [185]. It has been suggested, however, that overlapping functions and substrate/organ specificity. Tic110 may have six transmembrane helices [187], and this point is The group 2 members, Tic20-II and -V, are expressed at relatively still not completely settled. The Tic110 precursor protein is likely high levels through developmental stages with patterns more similar imported first into the stroma and then re-inserted into the inner en- to that of Tic20-I [176]. The functional inter-relationship between velope membrane [188,189] in an ATP-dependent manner [189]. group 1 and 2 proteins was investigated by using double and triple Based on the location of Tic110, it is suggested that the stromal por- mutants [176]. Although the group 2 Tic20s are conserved during evo- tion (>90 kDa) recruits stromal chaperones for the import of precursor lution, they are not essential for chloroplast development and do not proteins [184,185]. The soluble domain of Arabidopsis Tic110 is suggested have redundant roles in protein import, as do the group 1 Tic20s [176]. to form a docking site in stroma for the importing preproteins emerging from the Tic complex at a late stage of translocation [186].Thecentral 3.3. Tic21 role of Tic110 in the chloroplast translocon complex is suggested by its interactions with many Tic, Toc and stromal proteins [190],suchas 3.3.1. Characterization Tic55 [191],Cpn60[184],Toc75[21],ClpC/Hsp93[20,21],Tic40[192], Tic21 was initially designated as CIA5, and alternatively as PIC1 for Tic62 [193],Tic32[167] and Hsp70 [19]. Permease in Chloroplast 1. Tic21/PIC1 is localized in the inner enve- An anion channel of the chloroplast envelope appears to be involved lope membrane and is predicted to have four transmembrane helices, in protein import and can be inactivated by Tic110 antibody [194], with a secondary structure and topology similar to that of Tic20 suggesting that Tic110 may be a candidate for a protein-conducting [7,178,179]. Tic21 is associated with other translocon components, channel. In another report, recombinant Tic110 in planar lipid bilayers e.g. Tic110 and Toc75 [178]. was seen to form a cation-selective high-conductance channel which A tic21 null mutant is albino, accumulates precursor proteins and is sensitive to the addition of transit peptides [195]. A similar channel has impaired ability to import proteins in vitro. The phenotype of exists in the chloroplast inner envelope membrane [195]. In a recent the tic21/tic20 double and the tic20 single mutant is the same [178]. study it was shown that Tic110 forms a Ca2+ and redox-regulated chan- It is proposed that Tic21 functions as part of the inner envelope mem- nel in vitro [187]. These data lead to the conclusion that Tic110 forms brane protein conducting channel [178]. Recently, Tic21 was detected a protein-conducting channel in the inner envelope membrane by NB-PAGE in the 1-megadalton translocation complex in which [187,195]. Notably, Tic110 is absent from the 1-megadalton Tic com- Tic20 is a core component [163]. The majority of Tic21 was found in a plex studied by the Nakai laboratory [163]. This 1 MDa protein complex different 100 kDa complex, which led the authors to suggest that is a translocation intermediate across the inner envelope membrane in L.-X. Shi, S.M. Theg / Biochimica et Biophysica Acta 1833 (2013) 314–331 325 which Tic20 is a core component, suggesting that Tic110 and Tic20 may and external signals to regulate protein import [208]. Interestingly, it be independent channels. was also reported that loss of the BnMs3 gene, a Tic40 member from It has been reported that Arabidopsis Tic110 is present in all Brassica napus, causes the mutant to be male-sterile. It was further dem- three types (Toc159-, Toc132- and Toc120-containing) of translocon onstrated that Tic40s, BnMs3 and BnaC (another Tic40 protein in this subcomplexes [196], and that the protein is essential for plastid biogen- organism) are essential for tapetal development and function [209,210]. esis and plant viability [196,197]. The down-regulation of atTic110 expression affects plastid protein levels, and the translocation of precur- 3.5.2. Evolutionary considerations sors across the inner envelope membrane and the assembly of Tic com- Tic40 is of eukaryotic origin [7]. This component is unique to plexes are impaired in a dominant negative atTic110 mutant [196]. Viridiplantae species, and is lacking in red algae [7,161] and glaucophytes Homozygous Tic110 mutants are embryo lethal, while a heterozygous [162]. There is only one Tic40 gene in most organisms surveyed at Tic110 mutant displays a chlorotic phenotype and defective protein im- Photozome. It is noteworthy that Tic40 is not found in M. truncatula, port [197]. which may suggest that Tic40 gene is not being sequenced in this genome because ~6% of the genes are uncovered [211] or that Tic40 is 3.4.2. Evolutionary considerations not essential in chloroplast protein import. Tic110 appears to have originated in eukaryotes [3,4] and is conserved among various organisms, including red algae [161] and 3.6. Tic55 glaucophytes [162], and in different types of plastids [146].Mostor- ganisms surveyed in Phytozome possess only one Tic110 gene. Some 3.6.1. A potential redox regulator of protein import of them, for example, P. patens in which the entire genome was re- Tic55 was identified as a Tic component in 1997, when it was cently duplicated, have two to four. Two conserved motifs were co-purified with an incoming precursor as well as with Toc159, found in Tic110 in the Viridiplantae species; a leucine-zipper-like Toc34, Toc75 and Tic110 [191]. It is an intrinsic protein anchored in motif (L-x-x-L-x-x-x-L-G) and the sequence F-L-L-P-W-K/R-R [7]. the inner envelope membrane via two transmembrane spans located The functions of these motifs are unclear. The presence of Tic110 in at its C-terminus [7,212]. Tic55 has a Rieske-type iron–sulfur cluster and every genome once it has been evolved may reflect its essential a mononuclear iron-binding site, and could be affinity purified via a CxxC role in chloroplast import. motif by passing it through a thioredoxin column, suggesting that Tic55 is a target of this small redox active protein [213]. Tic55 belongs to the 3.5. Tic40 mono- and di-oxygenase superfamily in which all members contain a Rieske-type 2Fe–2S center as well as a non-heme mononuclear iron 3.5.1. Characterization binding site [214,215]. Besides Tic55, there are four more subfamilies in Tic40 (previous named as Cim/Com44 [198,199] and Toc36 [200]) this superfamily from plants: CMO (choline monooxygenase), CAO has a single transmembrane span anchoring it in the inner envelope (chlorophyllide a oxygenase), PAO (pheophorbide a oxygenase 1 (also membrane [192]. Similar to Tic110, Tic40 appears to be first imported called ACD 1 for Accelerated-Cell-Death 1 and Lls1 for Lethal-Leaf Spot into the stroma and then targeted back into the inner membrane 1)), and PTC52 (Protochlorophyllide (pchlide)-dependent Translocon [188]. A serine/proline domain and the transmembrane domain of Component of 52 kDa (also named ACD1-like)) [214,215]. Tic40 are required for reinsertion of the protein into the inner mem- There is only one AtTic55 (at2g24820) protein in the Tic55 sub- brane [201]. Interestingly, Tic40 itself is involved in reinsertion of a family in A. thaliana. The closest proteins to AtTic55 in A. thaliana subset of inner membrane proteins [202]. among the mono- and di-oxygenase superfamily members are AtPTC52 Tic40 is associated with importing precursor proteins [199] and can (At4g25650) (26% identical) [216] and PAO (At3g44880). In vivo studies be covalently cross-linked to Tic110 [192,203]. In addition, Tic40 can in- demonstrated that Arabidopsis Tic55 and PTC52 null single mutants have teract with two stromal chaperones, Hsp70 [18,19], and Hsp93 [203].It wild type appearance and do not have defects in protein import, indicat- is predicted to contain two protein–protein interaction regions, a TPR ing that Tic55 may be a regulatory subunit rather than a core Tic compo- domain and a Hip/Hop/Sti1domain which is typically the binding site nent [216]. for Hsp70/Hsp90 interacting co-chaperones [203]. The redox sensing components, Tic32, Tic55, and Tic62, were not The mechanism of Tic40 involvement in protein import has been detected in the proplastid proteome [205], roots or in dark-grown tis- studied in vitro [190]. Based on the interaction between Tic components sues [216]. In contrast, the expression of Tic55 is induced by light and and precursor proteins and on the hydrolysis of ATP it is proposed that photosynthetic development [216]. These data are consistent with the TPR domain of Tic40 binds Tic110, an action stimulated by the the hypothesis that this Tic redox regulon (Tic62, Tic55 and Tic32) importing precursor. This in turn causes release of the transit peptide may sense photosynthetically derived signals and regulate protein from Tic110 for cleavage by stromal processing peptidase. Hsp93, one import [217]. of the two import motors (the other one is Hsp70) then binds the importing protein, accompanied by ATP hydrolysis, which is stimulated 3.6.2. Evolutionary considerations by the Sti1 domain of Tic40 [190].Thesetwodomains,aswellasthe Tic55 has its origins in prokaryotes, with a homolog found in transmembrane span, have been proven to be essential in vivo [204]. Synechocystis sp. PCC 6803 [3,4]. A survey of Tic55 using Phytozome An Arabidopsis Tic40 null mutant is pale green and has decreased im- shows that most organisms contain one or two genes of Tic55 sub- port rates of photosynthetic and non-photosynthetic precursor proteins family. Neither C. reinhardtii nor Selaginella moellendorffii genomes into chloroplasts compared with wild type [197,203]. The chloroplasts contains any Tic55 subfamily members (also see [7]), although they of the tic40 null mutant have a spherical/swollen appearance with are found in P. patens (moss). fewer thylakoids and thinner granal stacks. Tic40 is expressed in leaves, roots, and flowers, at different devel- 3.7. Tic62 opmental stages [197], and in various plastids, such as proplastids [205], etioplasts [206] and chloroplasts. Remarkably, overexpression 3.7.1. A second potential redox regulator of protein import of His-tagged AtTic40 in tobacco causes proliferation of the inner en- Tic62 was detected in a complex containing Tic110 and Tic55 on velope membrane up to 19 layers [207]. It was shown that Tic40 ex- Blue-Native gels [193]. It is a peripheral membrane protein associating hibits natural fluctuations when Tic110 was used as a reference, and with the inner envelope membrane via a hydrophobic patch located Tic40 transgenic plants can tolerate a wide range of Tic40 levels [208]. near its N-terminal region. It contains three additional structural features: This led to the speculation that Tic40 may respond to both internal an N-terminal NADP(H) binding domain, a C-terminal ferredoxin 326 L.-X. Shi, S.M. Theg / Biochimica et Biophysica Acta 1833 (2013) 314–331

NADP+-oxidoreductase (FNR) binding motif, and a central Tic complex- have a wide range of functions and are located in multiple cellular binding region [218]. compartments. Finally, the pea [225] and Arabidopsis Tic32 Tic62 is a member of an extended family of short-chain (At4g23430) lack N-terminal cleavable transit peptides (predicted dehydrogenases/reductases (SDR) and has been shown to possess by TargetP), making it difficult to identify which orthologs are local- the signature active enzyme characteristics [219]. Its location in ized to the chloroplast. For these reasons it cannot be determined chloroplasts and its association with the Tic complex, as well as how many Tic32 genes, if any, are present in the different species with FNR [193], are affected by the ratio of NADPH/NADP+ in the indexed in Phytozome. This situationmayapplytootherorganisms. stroma [219]. Under reducing conditions (high NADPH/NADP+), Tic62 tends to dissociate from the inner envelope membrane and 4. A working model of protein import Tic complex, becoming soluble in the stroma where it has an increased affinity to FNR. In an oxidizing environment, Tic62 migrates back to the Based on our current knowledge of chloroplast protein import, a inner membrane and binds to the Tic complex. The shuttling between working model for the import of a typical stromal protein via the the two chloroplast subcompartments, and binding to NADPH/NADP+ Toc and Tic machineries is as follows. A precursor protein synthesized and FNR, suggest a model in which Tic62 is a sensor and a regulator in the cytosol with an N-terminal transit peptide is directed to the connecting photosynthetic/redox status and protein import in chloro- chloroplast surface, a process which can be facilitated by the cytosol plasts [193,219]. More recently, Tic62 was detected also in the thylakoid Hsp70/14-3-3 complex or Hsp90/Toc64. On the chloroplast outer membranes where it forms a complex with FNR that is regulated by envelope membrane the precursor protein interacts with receptors light and stromal pH [220].Thefinding that Tic62 is recruited to tether Toc159 and/or Toc34. Receptor sub-complexes, composed for in- FNR to the thylakoid membrane has been proposed to reflect a fine stance by atToc159/Toc90/Toc33 or atToc132/Toc120/Toc34, provide tuning mechanism for FNR availability for photosynthetic electron specificity for different groups of precursors. Upon GTP hydrolysis transport [221]. by Toc34, the precursor is transferred to the protein-conducting Arabidopsis knockout mutants of Tic62 (At3g18890) are viable. channel, Toc75. Translocation across the outer envelope membrane However, loss of Tic62 results in a decrease in the total FNR level, esis driven by low ATP concentrations, b100 μM [226], perhaps after pecially in the portions binding to the envelope and thylakoid mem- conversion of the ATP to GTP by a plastid nucleoside diphosphate ki- branes [220]. This may again indicate a role of Tic62 in maintaining nase. During translocation the precursor contacts with a complex lo- active FNR levels in the thylakoid membranes. cated in the intermembrane space which may include Toc64/OEP64 and/or Tic22. In the next step, the precursor becomes engaged in 3.7.2. Evolutionary considerations the protein-conducting channel formed by Tic20 and Tic21 in the The NADP(H)-binding domain in Tic62 is highly conserved among inner envelope membrane; the transmembrane domain of Tic110 green sulfur bacteria, and in cyanobacteria through higher plants may participate in this channel activity as well. On the trans side of [193,222]. Therefore, Tic62 is considered to be of prokaryotic origin. In the inner membrane, the emerging N-terminal region of the precursor contrast, the FNR-binding domain is a unique feature in vascular plants interacts with the stromal domain of Tic110. The latter component [7,222]. There are multiple (2–8) Tic62 genes in the 31 Viridiplantae ge- also interacts with Tic40 and recruits stromal chaperones Hsp93 and nomes accessed by the Phytozome program. Hsp70 to the Tic complex. Upon ATP hydrolysis by the two motor chaperones, the precursor is translocated into the stroma. The three regula- 3.8. Tic32 tory componentsTic62, Tic55 and Tic32 provide fine tuning of import through redox and Ca2+ signaling. During or shortly after arriving in 3.8.1. A potential Ca2+-dependent regulator of protein import the stroma, the transit peptide is cleaved off by the stromal processing Tic32 is the newest member of the Tic complex. This protein could be peptidase. The mature protein then folds in the stroma, either sponta- purified through a Tic110-containing affinity matrix [167].Itisalsoasso- neously or under the influence of stroma-resident chaperones, such as ciated with importing precursors and mature proteins, as well with Hsp60 [37]. Tic40, Tic22 and Tic62 [167]. Tic32 behaves as an integral membrane pro- Additionally, the survey of the Toc and Tic components from algae tein. Similar to Tic62, Tic32 belongs to the short-chain dehydrogenase/ through vascular plants, along with their phylogenetic analysis, pro- reductase family [167]. It has an NADP(H)-binding domain at the vides insight into the evolutionary significance and the roles of these N-terminus with active dehydrogenase activity and a calmodulin- components. Accordingly, the components can be grouped into four binding domain at the C-terminus [223].Itwasdemonstratedin categories. (1) Essential components include Toc75 and Tic20. They vitro that Tic32 binds to calmodulin in a calcium-dependent manner are of cyanobacterial origin and are present in all organisms containing [223]. In addition, calmodulin affects the dehydrogenase activity. A primary plastids. Interestingly, they are each suggested to function as calmodulin inhibitor and calcium ionophores can inhibit import of the protein-conducting channels in the outer or inner envelope mem- some precursor proteins in a concentration-dependent manner branes, respectively. It can be postulated that the endosymbiont ances- [224]. These data suggest that protein import into chloroplasts is tors of these two channel proteins assumed crucial roles in importing regulated by calcium-signaling, presumably mediated by calmodu- previously host-encoded proteins back into the nascent plastids in the lin, and by redox-signaling [223,224]. The two pathways converge initial period of transition from an endosymbiont to an organelle. The at the Tic complex through Tic32. emerging Toc and Tic complexes were then built around these channels Arabidopsis Tic32 knockout plants are viable and do not display a as they became fixed in place during plant evolution. It is noteworthy noticeable chloroplast protein import phenotype. Thus, Tic32 is not that the two stromal chaperones Hsp70 and Hsp93, which provide driv- essential for chloroplast biogenesis [167,218]. ing force for the import of chloroplast precursors, are both of prokaryotic origin. (2) Other essential core components of import machineries 3.8.2. Evolutionary considerations include Toc159, Toc34, Tic110, and they are of eukaryotic origin. Once Tic32 originated in prokaryotes [4,7]. While there are three Tic32- they assumed a role in protein import they have been maintained in like genes in C. reinhardtii [7],itisdifficult to determine which one is the plant genomes during the course of evolution. These components the ortholog of the pea Tic32 using computational methods. First, align- provide the receptor or docking sites on the cytoplasmic or stromal ment of Tic32 protein sequences from several species shows that the sides of the plastids, respectively. Presumably dispensable at first, they calmodulin-binding domain is highly diverse, even though the would confer higher efficiency and specificity for different types of pre- NADP(H)-binding domain is conserved. Second, proteins with cursors to the Toc and Tic machineries. (3) Facilitating, non-essential NADP(H)-binding domains make up a large family of SDR that components of import machineries include Toc64/OEP64, Tic22 and L.-X. Shi, S.M. Theg / Biochimica et Biophysica Acta 1833 (2013) 314–331 327

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