Photosynthesis Research 82: 315–325, 2004.
2004 Kluwer Academic Publishers. Printed in the Netherlands.

315

Review

The translational apparatus of Chlamydomonas reinhardtii chloroplast Marı´ a Vero´nica Beligni2,*, Kenichi Yamaguchi1,* & Stephen P. Mayfield2,3 1

Present address: Division of Biochemistry, Faculty of Fisheries, Nagasaki University, Bunkyo-machi, Nagasaki 852-8521, Japan; 2Department of Cell Biology and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Rd, La Jolla, CA 92037, USA; 3Author for correspondence (e-mail: mayfi[email protected]; fax: +1-858-784-9840) Received 1 March 2004; accepted in revised form 17 June 2004

Key words: chloroplast translation, mRNA-binding proteins, ribosomal proteins, trans-acting factors, translation factors, 5¢ UTR

Abstract Genetic and biochemical studies have revealed that chloroplast gene expression in Chlamydomonas is controlled primarily post-transcriptionally, including events that effect mRNA processing and stability, and during the translation of plastid mRNAs into proteins. Many of the proteins required for chloroplast gene expression are encoded in the nuclear genome, and most of these proteins have yet to be identified biochemically. Emergence of the draft sequence of the Chlamydomonas nuclear genome has enabled us to carry out a prediction and comparative analysis of the proteins required for chloroplast mRNA translation. Putative translation factor genes have been identified by homology search, and functional chloroplast ribosomal protein genes have been compiled based on our recent proteomic studies. This bioinformatic and proteomic analysis shows that the translational apparatus of Chlamydomonas is related to that of bacteria, but is more complex. Chlamydomonas chloroplasts contain all of the general translation factors found in bacteria, and a majority of the ribosomal proteins are conserved between plastids and bacteria. However, Chlamydomonas contains a number of additional proteins and protein domains associated with the plastid ribosome, while some ribosomal proteins are either quite divergent or lacking. In addition, Chlamydomonas chloroplasts contain a number of mRNA specific translation factors that are not found in bacteria.

Introduction The Chlamydomonas reinhardtii plastid genome is a 200 kb circular molecule that encodes approximately 100 genes required for gene expression of the key proteins of the photosynthetic complexes and carbon fixing machinery (Maul et al. 2002). Expression of chloroplast genes is primarily controlled by post-transcriptional events, such as mRNA processing and stability, and most notably during translation of plastids mRNAs into proteins (Mayfield et al. 1995; Rochaix 1996; Choquet

*

Both authors contributed equally to this work.

and Wollman 2002). Biochemical and genetic studies in Chlamydomonas have revealed the involvement of numerous nuclear genes in the post-transcriptional steps of chloroplast gene expression (Barkan and Goldschmidt-Clermont 2000; Zerges 2000; Somanchi and Mayfield 2001). Mechanistically, little is known about how Chlamydomonas chloroplast mRNAs are transcribed and translated, or how these processes are regulated. Identification and characterization of all the components involved in chloroplast transcription, post-transcriptional processing, and translation is needed to gain a complete understanding of the complex processes that together control chloroplast gene expression.

316 Release of the C. reinhardtii nuclear genome sequence (C. reinhardtii genome v1.0) from the JGI (Joint Genome Institute; http://genome.jgi-psf.org/ chlre1/chlre1.home.html), coupled with the completion of the chloroplast genome sequence (Maul et al. 2002) enabled us to search for the set of proteins involved in chloroplast gene expression and its control. Using our recent proteomics results on the Chlamydomonas chloroplast ribosome (Yamaguchi et al. 2002, 2003), we were also able to identify the functional set of chloroplast ribosomal proteins and their genes. Here we report the in silico identification and characterization of proteins involved in chloroplast translation, and in the regulation of translation of chloroplast mRNAs, in C. reinhardtii.

tRNA synthetases and other modifying enzymes of the translational apparatus (enzymes that modify rRNAs, tRNAs, mRNAs and ribosomal proteins) were also not included in this paper, since these proteins function prior to the ribosome cycle and they are not directly involved in translation per se.

Materials and methods

Identification of the complete set of chloroplast ribosomal proteins We have recently identified 52 ribosomal proteins from the C. reinhardtii chloroplast 70S ribosome by LC-MS/MS analysis. This complete set, or near complete set, contains 22 small subunit proteins (orthologues of E. coli S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S12, S13, S14, S15, S16, S17, S18, S19, S20, S21; 28 large subunit proteins (orthologues of E. coli L1, L2, L3, L4, L5, L6, L9, L10, L11, L12, L13, L14, L15, L16, L17, L18, L19, L20, L21, L22, L23, L24, L27, L28, L31, L32, L35, and homologues of spinach PSRP-6 and PSRP-3. In addition to these 52 proteins we identified a novel S1-domain containing protein, PSRP-7, and 2 proteins unique to the Chlamydomonas chloroplast 70S ribosome, RAP38 and RAP41 (Yamaguchi et al. 2002, 2003). As shown in Table 1, we compiled functional genes that correspond to each of these 55 ribosomal and ribosome associated-proteins. Orthologous proteins for L33, L34, L36, PSRP-1 and PSRP-4 were not identified by LCMS/MS analysis of C. reinhardtii 70S ribosomes, probably because trypsin or Lys-C fragmented ions generated from these proteins were too small to be detected by MS/MS analysis. Although the proteins were not identified proteomically, transcripts of these genes were found within the C. reinhardtii EST databases (Yamaguchi et al. 2002, 2003; also see Table 1). The S11 protein was also not identified in our previous proteomic analysis (Yamaguchi et al. 2002), but has now

Computational analyses A BLAST (tblastn) search was performed to identify predicted ORFs by GreenGenie or Genewise using the assembly of the C. reinhardtii genome sequence (C. reinhardtii genome v1.0) released from the JGI (Joint Genome Institute; http://genome.jgi-psf.org/chlre1/chlre1.home.html). Homology comparison was done using BLAST 2 SEQUENCES (National Center for Biotechnology Information, NCBI). Protein masses were calculated by the ProtParam in the ExPasy proteomic tools (http://www.expasy.ch/tools/protparam.html). Prediction of cleavage sites for chloroplast transit peptides were obtained using the ChloroP program (Emanuelsson et al. 1999). Presence or absence of N-terminal methionine of chloroplast-encoded protein was predicted by the penultimate amino acid residue (Giglione and Meinnel 2001).

Results and discussion Transcriptional and post-transcriptional control While the control of transcription, mRNA processing and stability are critical factors in chloroplast gene expression it is out of the scope of this article to attempt a bio-informatic analysis of the proteins involved in these processes. Aminoacyl

Translational control For translation and translational regulation we considered three separate aspects: (a) the plastid ribosome; (b) general translation factors identified in other systems containing 70S ribosomes; and (c) mRNA or chloroplast specific translation factors identified by previous genetic or biochemical analysis.

317 Table 1. In silico identification and characterization of Chlamydomonas reinhardtii chloroplast ribosomal proteins Protein

30S subunit proteins S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 PSRP-1 PSRP-2 PSRP-3 PSRP-4 PSRP-7 50S subunit proteins L1 L2 L3 L4 L5 L6 L9 L10 L11 L12 L13 L14 L15 L16 L17 L18 L19 L20 L21

Gene

EST

Predicted protein

Protein

Allocation

Name or acc. no.a

JGI version 2.0b

Acc. no.c

Frequency

Precursord (AA)

Maturee (AA)

identifiedg

Nucleus Plastid Plastid Plastid Nucleus Nucleus Plastid Plastid Plastid Nucleus Plastid Plastid Nucleus Plastid Nucleus Nucleus Nucleus Plastid Plastid Nucleus Nucleus Nucleus – Nucleus Nucleus Nucleus

genie.424.2 DAA00936 DAA00947 DAA00921 genie.191.7 genie.165.4 DAA00925 DAA00920 DAA00940 genie.4.16 DAA01543 DAA00960 genie.719.2 DAA00926 genie.36.11 genie.666.3 genie.256.4 DAA00937 DAA00916 genie.809.10 genie.355.5 genie.979.4 N.F.f Genie.69.0 scaffold_444 genewise.76.32.1

C_330096 – – – C_60181 C_160040 – – – C_30180 – – C_740047 – C_200006 C_740007 C_660081 – – C_740035 C_280075 C_300023 N.F. C_390052 N.F. C_160057

BI873502 – – – AV396809 BE212144 – – – AV623307 – – AV623187 – BE337315 BE726302 AI625916 – – BE726479 AV633276 AV634980 N.F. BI529066 BE452645 AV626377

17 – – – 17 61 – – – 11 – – 30 – 14 23 12 – – 25 26 43 – 23 3 27

436 570 712 257 673 171 168 153 191 169 130 133 164 100 141 130 105 137 92 166 184 286 – 298 >135 >560

395 570 711 256 622 117 167 152 190 127 129 132 135 99 108 100 80 137 91 128 156 227 – 262 82 560

MS1, IX1 MS1 MS1, IX1 MS1 MS1 MS1 MS1 MS2 MS1 MS1 N.I.h MS1 MS1 MS1 MS1 MS1 MS1 MS1 MS1 MS1 MS1 IX2 N.I. MS1 N.I. MS1

Nucleus Plastid Nucleus Nucleus Plastid Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Plastid Nucleus Plastid Nucleus Nucleus Nucleus Plastid Nucleus

genie.554.3 DAA00915 genie.649.1 genie.1209.1 DAA00915 genie.409.2 genie.39.22 genie.488.12 genie.289.6 genie.872.2 genie.36.10 DAA00918 genie.2626.0 DAA00917 genie.349.7 genie.217.8 genie.484.5 DAA00909 genie.296.1

C_390109 – C_890015 C_480024 – C_710070 C_950057 C_270194 C_260130 C_230149 C_200119 – C_500047 – C_20013 C_180036 C_1110036 – C_190156

BE024867 – BE724898 BU653022 – BE121745 BG844693 AV392867 BI528668 AV635280 AV623161 – BM003119 – BI874648 BE129406 BI999322 – AV390396

20 – 34 23 – 20 15 4 17 46 52 – 19 – 13 33 20 – 16

297 278 259 243 206 207 204 235 176 162 225 122 246 136 173 145 153 112 179

261 277 236 176 205 197 183 197 140 130 195 122 221 136 124 119 122 111 128

MS2 MS2, MS2 MS2 MS2, MS2 MS2, MS2 MS2 MS2, MS2 MS2 MS2 MS2, MS2 MS2 MS2 MS2 MS2

IX1

IX1 IX1

IX1

IX1

318 Table 1. Continued Protein

L22 L23 L24 L25 L27 L28 L29 L30 L31 L32 L33 L34 L35 L36 PSRP-5 PSRP-6 70S–specific proteins RAP38 RAP41

Gene

EST

Predicted protein

Protein

Allocation

Name or acc. no.a

JGI version 2.0b

Acc. no.c

Frequency

Precursord (AA)

Maturee (AA)

identifiedg

Nucleus Plastid Nucleus – Nucleus Nucleus – – Nucleus Nucleus Nucleus Nucleus Nucleus Plastid – Nucleus

genie.188.12 DAA00914 genie.493.4 N.F. genie.1003.1 genie.99.6 N.F. N.F. genie.2142.1 genie.264.1 genie.1735.2 genie.31.27 genie.928.2 DAA00913 N.F. scaffold_241

C_230075 – C_1180006 N.F. C_190003 C_270188 N.F. N.F. C_640061 C_100047 C_830026 C_1390029 C_1000009 – N.F. N.F.

AV388591 – AV633462 N.F. BG853464 BI722670 N.F. N.F. BG853327 AV639564 BE337448 BE337870 AV624636 – N.F. AW661516

22 – 23 – 48 47 – – 28 26 35 20 76 – – 28

175 95 170 161 195 – – 136 98 101 124 114 37 – 66

132 95 137 – 132 162 – – 105 62 75 67 72 37 – 53

MS2 MS2, IX1 MS2 N.I. ED, IX1 MS2 N.I. N.I. MS2 MS2 N.I. N.I. MS2 N.I. N.I. MS2

Nucleus Nucleus

genie.2124.0 genie.1149.5

C_150008 C_150160

AV620219 AV635725

34 15

401 439

360 404

MS2 MS2

a Gene name (for nuclear-encoded protein) from the JGI database version 1.0 or accession number (for plastid-encoded protein) is indicated. b Gene names of nuclear-encoded proteins according to JGI Chlamydomonas v2.0 are included. c The accession number of the longest EST clone is indicated as the representative. d Cytoplasmic precursor or plastid pro-protein sequence was deduced from nucleic acid sequence. e Predicted by ChloroP program (for nuclear-encoded protein) or by penultimate amino acid residue (for plastid-encoded protein). f N.F., not found. g MS, identified by tandem mass spectrometry (Yamaguchi et al. 2002, 2003); IX, identified by immuno-cross reaction (RandolphAnderson et al. 1989; Bubunenko and Subramanian. 1994); ED, identified by Edman degradation (Liu et al. 1988). h N.I., not identified.

been identified following revision of the chloroplast genome (Table 1). Thus, at least 58 plastid ribosomal proteins are potentially present in the Chlamydomonas chloroplast: 38 encoded in the nuclear genome and 20 encoded in the plastid genome. Homologues of bacterial L25, L30 and L29, and homologues of spinach PSRP-2 and PSRP-5 were not identified in either the Chlamydomonas nuclear genome sequence or the latest EST databases. Genes for plastid ribosomal proteins L25 and L30 are also missing from the Arabidopsis genome, and the proteome of the spinach plastid ribosome lacks L25 and L30 proteins (Yamaguchi and Subramanian 2000). Each of these data suggests that chloroplast ribosomes do not contain orthologs of these ribosomal proteins. L29 protein is dispensable in E. coli (Kramer et al.

2002), while in maize there is about 1.5 times more L29 protein in ribosomes of greening leaves than in ribosomes of dark grown leaves (Zhao et al. 1999). These data suggest that L29 may not be directly required for translation in 70S ribosomes, but rather participate in reorganization of particular ribosomal proteins during translational control or in plastid development in higher plants. Plastid specific ribosomal proteins (PSRPs) are proteins that are present in chloroplast ribosomes but absent from bacterial and mitochondrial ribosomes. PSRPs were originally identified from spinach chloroplast ribosome (Subramanian 1993), and seven PSRPs (PSRP-1 to PSRP-7) have been identified to date. PSRPs have been proposed to be involved in unique aspects of translation control found in chloroplast, like light-activated

319 translation, and PSRPs may have a number of functions within translation (Yamaguchi and Subramanian 2003). Counterparts of the spinach PSRP-1, PSRP-3, PSRP-4, and PSRP-6 have been identified from Chlamydomonas (Yamaguchi et al. 2002, 2003). Orthologs of the spinach PSRP-2 and PSRP-5 proteins were not identified in C. reinhardtii, either by proteomic analysis of database search. A novel PSRP (PSRP-7) and two ribosome associated proteins RAP38 and RAP41 identified from Chlamydomonas chloroplast ribosome are all present in higher plant genomes, although their products may not be tightly associated with plastid ribosomes, as they are in C. reinhardtii (discussed in Yamaguchi et al. 2002, 2003). The frequency of ESTs for ribosomal proteins is variable (Table 1), but the variability is lower than that of the general and mRNA-specific translation factors (Tables 2 and 3). Identification of the general 70S-type translation factors in chloroplasts In bacteria, translation involves at least 11 general translation factors: initiation factors 1, 2 and 3 (IF1, IF2, and IF3), elongation factors Tu, Ts and G (EF-Tu, EF-Ts, EF-G), release factors 1, 2 and 3 (RF1, RF2, and RF3), and ribosome recycling factor (RRF or RF4) (Reviewed in Ramakrishnan 2002). In addition, elongation factor P (EF-P), a protein that stimulates the peptidyltransferase activity, has been recognized as an essential translation factor in E. coli (Aoki et al. 1997). To date, only the chloroplast encoded elongation factor Tu (EF-Tu) has been characterized from Chlamydomonas. The rest of the general factors are nuclear encoded, and have yet to be cloned or characterized. We searched for genes encoding orthologs of bacterial translation factors in the nuclear genome of C. reinhardtii in silico. Average amino acid sequence similarity between ribosomal proteins of Chlamydomonas chloroplast and those of Arabidopsis chloroplast, Synechocystis, and E. coli are 63%, 68% and 60%, respectively (Yamaguchi et al. 2002, 2003). Since the greatest similarity was found between Chlamydomonas and Synechocystis, we started our search using the Synechocystis protein sequences as probes to identify potential chloroplast translation factors from Chlamydomonas. As summarized in Table 2, orthologs of all of the Synechocystis translation factors could be identified in the nuclear genome

of C. reinhardtii. To unambiguously confirm functional genes for all of these general factors, further scaffold sequence or cloning and characterization of the individual genes will need to be undertaken. Identification of mRNA specific trans-acting factors Biochemical and genetic studies in Chlamydomonas have revealed the involvement of numerous nuclear genes in post-transcriptional steps of chloroplast gene expression (Mayfield et al. 1995; Rochaix 1996; Zerges 2000; Somanchi and Mayfield 2001). All of the chloroplast mRNA-specific trans-acting factors identified and characterized so far are encoded in the nuclear genome, and several of these factors have been cloned, sequenced and partially characterized. We searched the Chlamydomonas database for homologs of any cloned genes encoding mRNA-specific trans-acting factors or proteins that affect any aspect of chloroplast translation. We attempted to identify any related genes present in the Chlamydomonas genome and summarized the information on the identified genes and ESTs in terms of protein size and percent similarity between the identified protein and the Chlamydomonas homolog (Table 3). All of the mRNA-specific trans-acting factor genes characterized in Chlamydomonas were identified as single-copy genes in the genome. For the C. reinhardtii Tbc2 protein, the amino-terminal half of the protein is contained within scaffold_2357 (genie.2357.1) and the carboxy-terminal half within scaffold_422 (genie.422.10) with a 276 amino acid stretch from the middle of the protein missing from the JGI version 1.0 database, but both gene models were contained within a single gene in the JGI version 2.0 database (C_80020). The frequencies of ESTs for the psbA mRNAbinding proteins cPABP and cPDI is much greater than for other chloroplast translation factors. This increased frequency may be related to abundance of the encoded proteins, which have been shown to directly bind to the abundant psbA mRNA, or may simply indicate that EST frequency and protein levels for these proteins, like the ribosomal proteins, are not directly correlated. We identified genes and ESTs from Chlamydomonas that had sufficient similarity to trans-acting factors from higher plants to suggest they could be orthologs (Table 3). Whether these proteins are true functional counterparts will need to be

Nucleus Nucleus Nucleus Plastid Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus

genie.594.1 genie.1145.0 genewise.1927.11.1 P17746 genie.76.21 genie.435.12 genie.111.15 genie.90.21 genie.3485.0 genie.368.10 Genie.1005.1 C_160057 C_50177 C_810066 C_3330002 C_2700005 C_900035 C_110077

Predicted protein

5 0 0

161 278 >94 148 1013 776 216 361 80 331 269

94 235 >94 147 962 720 190 327 50 310 240

Frequency Precursorc Matured (AA) (AA)

AV387886 26 AV640405 265 BE238024 17 N.F. 0 N.F. 0 N.F. 0 BE725867 18

C_1780014 AV632253 C_42003 N.F.h N.F. N.F.

Acc. no.b

Allocation Name or acc. no.a

v 2.0a

EST

Gene

10068.67 24748.4 N.A.i 45612.1 103395 79889.5 21080.2 36662.4 5413.8 33889.4 25784

MWe

10.03 4.28 N.A. 5.9 4.24 5.14 5.43 4.86 8.63 5.27 9.37

pIe

N.Ij N.I. N.I. EP N.I. IX N.I. N.I. N.I. N.I. N.I. ssl3441 slr0744 slr0974 sll1099 sll1261 sll1098 slr0434 sll1110 P74476 slr1228 sll0145

67 1001 177 399 218 691 187 365 372 547 182

81 68 63 82 73 73 74 79 89 84 72

(51/62) (53/77) (56/89) (349/418) (118/159) (511/694) (137/185) (285/358) (44/49) (75/88) (126/172)

Protein f Synechocystis orthologue identified Acc. no. Product Similarityg (AA)

a Gene name (for nuclear-encoded protein) from JGI Chlamydomonas version 1.0 or accession number (for plastid-encoded protein) is indicated; v 2.0 indicates the gene names according to JGI Chlamydomonas v 2.0. b The accession number of the longest EST clone is indicated as the representative. c Cytoplasmic precursor or plastid pro-protein sequence was deduced from nucleic acid sequence d Predicted by ChloroP program (for nuclear-encoded protein) or by penultimate amino acid residue (for plastid-encoded protein). e Calculated from predicted mature protein sequence. f EP, identified in the electrophoretic product pattern of chloroplast-made proteins (Breidenbach et al. 1990); IX, identified by immuno-cross reaction (Breitenberger and Spremulli 1980). g Parenthesis stands for (conserved residues)/(compared residues). h Corresponding gene or EST was not found. i Not available. j Not identified yet.

IF-1 IF-2 IF-3 EF-Tu EF-Ts EF-G EF-P RF-1 RF-2 RF-3 RRF

Protein

Table 2. In silico identification and characterization of Chlamydomonas reinhardtii orthologues of Synechocystis PCC6803 translation factors

320

Chlamydomonas

Chlamydomonas

maize

Tab2

Tbc2

CRP1

Arabidopsis

DIM1

Plastid ribosomes

Plastid ribosomes

psbD polypeptide

petD & petA mRNAs

psbC 5¢ UTR

psaB 5¢ UTR

psbA 5¢ UTR

Plastid rRNA methylase. Assembly of ribosomes at low temperature

Co-translational protein folding and/or stabilization Associated with 50S subunit Mutants lack plastid ribosomes

Specific binding and translation activation of psbA mRNA Redox-regulated binding of RB47 to psbA 5¢ UTR Specific binding and translation activation of psaB mRNA Specific binding and translation activation of psbC mRNA Translation activation and/or RNA processing

Experimental evidence and/or proposed role

Tokuhisa et al. (1998)

Rattanachaikunsopon et al.(1999) Han et al. (1992)

Fisk et al. (1999)

Auchincloss et al. (2002)

Kim and Mayfield (1997) Dauvillee et al. (2003)

Yohn et al. (1998)

Reference

v 2.0 C_90199

C_1330016

genewise.8.41.1

C_120210

scaffold_970f (28688-29289)

Gene v 1.0 genewise.1990.7.1

scaffold_64 (415623-415962)

genewise.1051.7.1

C_760013

C_80020

genie.2537.1 genewise.910.25.1

C_80020

C_170003

C_390061

C_250113

v 2.0

genie.422.10

genie.551.2

genie.436.8

genie.33.20

Gene v 1.0

Corresponding gene and EST

1

0

N.F.g

Frequency

13

1

2

0

EST Acc. no.c BQ825671

20021010.3311.1

20021010.4061.1

BQ814102

N.F.

0

N.F.g g

4

35

106

Frequency

>189

Mature (AA) >208

Precursord (AA) N.A.h

N.A.h

219

N.A.h

1011

668

668

308

516

N.A.h

Mature (AA)

N.A.h

N.A.h

1068

758

758

358

532

657

Precursord (AA)

Predicted protein

AV631790

AV630751

AV629679

EST acc. no.c

Bioinformatic analysis in Chlamydomonasb

N.A.h

N.A.h

MW

16012.77

N.A.h

103850.84

70670.23

70670.23

34469.9

56508.34

N.A.h

MW

55 (104/189)

Similaritye (%) 60(125/208)

57 (70/120)

100

100 (668/668) 100 (668/668) 45(284/631)

100

100

100

Similaritye (%)

Not available: full length protein sequence or processing site could not be predicted. For Acc115, predicted protein was 113 aminoacids according to JGI v1.0, but two in frame stop codons downstream of the first Met were found in the

Corresponding EST was not found.

No gene was predicted. Position within the scaffold is indicated between parentheses.

predicted mRNA sequence in v 2.0.

h

g

f

Cytoplasmic precursor length was deduced from available nucleic acid and protein information.

Similarity between the cloned protein an the predicted Chlamydomonas protein is indicated. Some alignments are partial. The number of similar divided the total aminoacids compared are indicated within parentheses.

e

The accession number of the longest EST clone is indicated as the representative. When available, full length Unigene ESTs are shown (Shrager et al. 2003).

For those proteins cloned from species other than Chlamydomonas, the gene showing the highest similarity in the JGI database is included. Version1.0 (v1.0) and version 2.0 (v2.0) of that database were used.

Cloned proteins having a role in different aspects of chloroplast translation are listed.

maize

Iojap

Other translational functions Acc115 Chlamydomonas

Chlamydomonas

cPDI (RB60)

psbA 5¢ UTR

Target

d

c

b

a

Species

Translation activators cPABP Chlamydomonas (RB47)

Protein

Table 3. Nuclear-encoded proteins that affect chloroplast translationa

321

322 determined by genetic and biochemical analysis. Interestingly, when searching for homologs of maize CRP1, we found an ORF (genewise.910.25.1) that showed significant similarity (Table 3). This ORF encodes MCA1, a mitochondrial protein induced at low CO2 levels and proposed to act as an RNA stability factor (Van et al. 2001). When input into ChloroP, MCA1 was predicted to be a chloroplast protein, containing a 57 amino acid transit peptide, suggesting that MCA1 could be dually targeted to both organelles. In maize, crp1 mutants fail to accumulate both petA and petD proteins. For petD, the failure to accumulate protein has been attributed to an inability to cleave the monocistronic petD mRNA from its polycistronic precursor. In contrast, the defect in petA accumulation has been attributed to a loss of translation, raising the possibility that CRP1 is involved in both RNA processing and translation depending upon the mRNA it interacts with (Fisk et al. 1999). However, there is tight correlation between translation and mRNA processing for the psbA mRNA in Chlamydomonas (Bruick and Mayfield 1997), and the same may be true in maize, suggesting that crp1 may be a translation factor that indirectly effects mRNA processing. A weak homology between CRP1 and several other Chlamydomonas proteins (genewise.418.41.1, genewise.147.9.1, genewise.34.100.1 and genie.28.22) in addition to MCA1 and Tbc2, suggests there may be a family of related proteins, some of which may also participate in chloroplast translation. Conclusions on chloroplast translation and translational control in Chlamydomonas reinhardtii The bioinformatic and proteomic analysis discussed above shows that the translational apparatus of the Chlamydomonas chloroplast is related to that of photosynthetic bacteria, but has diverged to incorporate additional domains and factors not found in bacteria. Chlamydomonas chloroplasts are more closely related to chloroplasts of higher plants, but again not all of the proteins identified in C. reinhardtii are shared with land plants. The transcriptional machinery of Chlamydomonas chloroplasts appears to be simpler than those of either higher plant chloroplasts or bacteria. C. reinhardtii contains only a single confirmed chloroplast RNA polymerase, and has

fewer sigma factors than either higher plant chloroplasts or bacteria. The reasons for the reduced number of sigma factors in C. reinhardtii may lie in the fewer developmental stages of algal plastids compared to their higher plant counterparts, and because translational regulation has become more dominant in plastids than in bacterial systems. From this analysis we predict that a number of novel proteins, with functions in translation unique to chloroplasts, will be identified in the future. Some of these factors will be involved in light regulated translation, and some may be involved in responses to other stimuli or stress. In addition, we can predict that some of the unique proteins identified to date, like the PSRPs, may turn out to have biochemical functions related to bacterial translation factors, but these proteins are not presently recognized as orthologs due to low sequence homology. Models for chloroplast translation were initially based upon genetic and biochemical data showing that plastids and bacteria were closely related, and hence these models assumed that translation was mechanistically similar in both systems. More recent studies have incorporated the nuclear factors that affect translation of specific mRNAs, but again the basic machinery was assumed to be the same between bacteria and chloroplast. With the sequencing of the Chlamydomonas nuclear and chloroplast genomes, one can begin to fill in the blanks in these models of translation, and clearly a number of differences between plastid and bacterial translational components predict that the two systems have diverged, with plastids adapting to their eukaryotic environment by adopting a number of new proteins and specialized functions. For Chlamydomonas chloroplast translation, as for land plants, similarities to both prokaryotes and eukaryotes translation can be found. Certainly the majority of the plastid ribosome, both protein and RNA, is conserved between plastids and bacteria. A ribosomal protein S1 has been identified in chloroplasts as have homologues of bacterial IF1, IF2 and IF3, suggesting that chloroplast translation initiation complexes are likely to have many similarities with bacterial initiation complexes. The presence of homologues of bacteriatype elongation factors (EF-Tu, EF-Ts, EF-G and EF-P), and that inhibitors of bacterial elongation also block plastid elongation, suggests that these steps are accomplished, at least partly, in a bac-

323 teria-like fashion. When translation is terminated in prokaryotes, release factors RF1 and RF2 recognize the stop codon, while RF3 releases the completed polypeptide and RF4 facilitates the dissociation of the 70S ribosome from the mRNA and tRNAs (Zerges 2000). The presence of close homologues of release factors in C. reinhardtii suggests that similar events could be used for termination of translation in chloroplasts. Obviously, both elongation and termination in chloroplasts could have special features not yet discovered. In Arabidopsis for example, mutations of the nuclearencoded RF2 gene affects the stability of UGAcontaining plastid mRNAs, suggesting that RF2 is involved in both mRNA stability and protein synthesis (Meurer et al. 2002). There are a significant number of mRNAs containing TGA codons in higher plant chloroplasts and mitochondria, while Chlamydomonas organelles appear not to use TGA stop codons (Meurer et al. 2002). If translation initiation factors are similar, and elongation and termination appears to operate in a bacterial fashion, then where do the additional proteins identified in plastid translation function, and how might these proteins influence chloroplast translation? In eukaryotic translational regulation, the identification of mRNAs prior to initiation complex formation is a key step. Although plastid mRNAs may use bacterial like Shine-Dalgarno interactions for translation initiation, the spacing from the initiation codon to the Shine-Dalgarno element is different for plastid mRNAs compared to bacteria, suggesting that additional factors (proteins) are required to bring the initiation codon into the correct register with the plastid ribosome. Three large extensions were identified in Chlamydomonas plastid ribosomal proteins (S2, S3 and S5). These extra domains are predicted to have RNA-binding activity and to reside on the solvent side of the 30S subunit, in close proximity to the binding site for mRNA entering the ribosome. This is exactly the location on the ribosome one would predict proteins involved in mRNA discrimination would be located. In addition to these ribosomal proteins, the existence of cPABP and the other mRNA specific factors suggests that interactions with 5¢ UTRs of plastid mRNAs may be a prerequisite to initiation complex formation, similar to that observed in eukaryotes. Together these data suggest that regulation of translation initiation, and perhaps pre-selection of mRNAs

for translation, is carried out by the additional proteins identified in chloroplast translation, and that these additional factors are key regulators of plastid translation. Another aspect of this study that has yet to be resolved is that both chloroplast and mitochondrial translation requires a similar set of prokaryotic-like components. The fact that some of the translation factors identified in this study are encoded by single copy genes in Chlamydomonas, suggests that some of these proteins may be targeted to both the chloroplast and mitochondria. Analysis of the predicted transit peptides of general translation factor proteins suggests that some of these factors may be targeted to both the chloroplast and mitochondria, suggesting that regulation of translation within organelles of eukaryotic cell may be more complex than the mere sum of prokaryotic and eukaryotic systems. This study also shows the importance of completing the Chlamydomonas genome sequencing, so that complete databases are available for the forthcoming proteomics and structural characterization of the translational apparatus involved in chloroplast gene expression.

Acknowledgements We thank Dr T. Oda for helpful advice and for enabling us to complete this work in his lab, and Ms M. Tuji for technical assistance.

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