Current Genetics

Curr Genet (1992)22:421-427

9 Springer-Verlag 1992

Identification of a chloroplast-encoded secA gene homologue in a chromophytic alga: possible role in chloroplast protein translocation Carol D. Scaramuzzi, Roger G. Hiller, and Harold W. Stokes

School of Biological Sciences, Macquarie University, Sydney, NSW 2109, Australia Received May 15, 1992 Summary. SecA is one of seven Sec proteins that comprise the prokaryotic protein translocation apparatus. A chloroplast-encoded secA gene has been identified from the unicellular chromophytic alga Pavlova lutherii. The gene predicts a protein that is related to the SecA proteins of Escherichia coli and Bacillus subtilis. The presence of secA, as well as the previously described sec Y and hsp70 genes, on the chloroplast genome of P. lutherii suggests that this eukaryotic organism utilises protein translocation mechanisms similar to those of bacterial cells. Key words: Chloroplast genome - Chromophytic alga secA gene - Protein translocation

Introduction

The protein translocation apparatus of bacterial cells is composed of seven secretory (Sec) proteins, SecY (Suh et al. 1990), SecA (Schmidt et al. 1988; Sadie et al. 1991), SecE (Schatz et al. 1989), SecD (Gardell et al. 1978), SecB (Kumamoto et al. 1989), SecC and SecF, as well as two additional chaperone proteins, GroEL and Trigger factor (TF) (For reviews see Saier et al. 1989; Austen and Westwood 1991). Prokaryotic protein translocation is a co-ordinated series of interactions involving these proteins although the precise details are unknown. The 63 kDa Trigger factor maintains the newly synthesised protein in an open conformation. A second chaperone, SecB, which binds to the protein via a signal sequence, further maintains the nascent polypeptide in a stable and pre-translocation conformation. This complex is recognized by the soluble, cytosolic protein SecA, which is peripheral to the membrane at the exposed cytosolic regions of SecY (Suh etal. 1990). SecA binds ATP and couples ATPase hydrolysis to transmembrane protein translocation (Lill et al. 1989; Matsuyama et al. 1990). Release and ATPase activity are stimulated at the membrane by SecY which also Correspondence to: C. D. Scaramuzzi

potentiates the action of SecA and forms part of a channel which allows translocation of the protein. This model is based on in-vitro studies of prokaryotic protein translocation-utilising precursors for the maltose-binding protein, the outer membrane protein A, and the outer membrane porin. Targeting and transportation of nuclear-encoded chloroplast proteins into higher plant chloroplasts, with the aid of transit peptides, has been extensively researched (For reviews see Smeekens et al. 1990; Keegstra 1989). However, protein transport into and within the chloroplasts of the Chromophyta has not been investigated and is complicated by the existence of four chloroplast membranes. The chromophytic algae are hypothesised to have evolved via a double endosymbiosis, where the first event involved the phagocytosis of a photosynthetic bacterium and the second event involved the phagocytosis of a photosynthetic eukaryote, thus giving rise to four chloroplast membranes. The outer two membranes comprise the chloroplast endoplasmic reticulum (CER). The outermost of these is thought to be derived from the host cell endomembrane system whereas the inner CER is thought to be derived from the plasma membrane of the eukaryotic endosymbiont (Whatley and Whatley 1981; McFadden 1990). The two innermost membranes of the chromophytic chloroplast are homologous to the membranes that surround the chloroplasts of higher plants and the green algae (Whatley and Whatley 1981), and the innermost of these is analogous to the plasma membrane of the original endosymbiont. Saier et al. (1989) suggest that there is a "unified mechanism by which proteins are inserted into and translocated across biological membranes". They conclude that protein targeting and transmembrane transport processes in bacterial systems are remarkably similar to those of the eukaryotic endoplasmic reticulum and that both processes occur by seemingly "homologous mechanisms with conservation of many specific functions". They further extrapolate that similar mechanisms are likely to be found for mitochondrial-targeted proteins. The identification of sec Y (Scaramuzzi et al.

422 1922 b), hsp70 (Scaramuzzi et al. 1992a) and, as reported here, a secA gene homologue, on the chloroplast genome of P. lutherii would support such a hypothesis for the translocation of proteins across internal membranes including those of the chloroplast.

Materials and methods Molecular cloning and DNA sequencing. Cultivation, harvesting,

preparation of chloroplast DNA, construction of chloroplast clone banks and hybridisation techniques have all been previously described (Scaramuzzi et al. 1992a, b). Manipulations of DNA were performed according to standard protocols (Maniatis et al. 1982) or, when using DNA-modifying enzymes, to the manufacturer's instructions. DNA fragments for subcloning into MI 3, or for use as hybridisation probes, were gel-purified using GeneClean II (Bio 101, LaJolla, Calif, USA). The bacterial host strains JM101 (Yanisch-Perron et al. 1985) and HB101 (Maniatis et al. 1982) were used for all cloning procedures. DNA sequencing was performed by the chain-termination method (Sanger et al. 1980) using a Sequenase kit (United States Biochemical Corporation). Sequencing of both single- and double-stranded templates was performed according to the manufacturer's protocols. Primers used were either the universal primer supplied or else synthetic oligonucleotides synthesised on a Pharmacia Gene Assembler Mark II. Oligonucleotides were designed to provide extensive overlap between gels and, where necessary, compressions were resolved by substitution of dITP for dGTP. Restriction mapping. Chloroplast DNA (200-400 ng) was restricted with the endonucleases, BssHII; SmaI; NarI and NaeI (individually

and in combination). DNA was fractionated on 0.5% agarose gels and transferred to a Zeta-probe nylon membrane (Bio-Rad) according to the manufacturer's instructions. Probes were made radioactive by oligoqabelling using a kit and protocols provided by Bresatec (Australia). Hybridisations using homologous DNA probes were routinely carried out at 65 ~ as previously described (Scaramuzzi et al. 1992a). For heterologous probes, hybridisation temperatures were lowered to 50 ~ Chloroplast DNA was screened with P. lutherii probes specific for hsp70 (pMAQ801), rpoB/rpoC1 (pMAQS07; Scaramuzzi et al. 1992a), see Y (pMAQS04), atpH/atpI (pMAQS06; Scaramuzzi et al. 1992b), secA (pMAQ805); Scaramuzzi 1991) and E. coli secB (Kumamoto and Nault 1989).

P. tutherii SecA protein shows high sequence similarity with the bacterial SecA proteins. Similarly, the three sequences show very good alignment beginning at position 635 in P. lutherii. Consequently, P. lutherii SecA has an additional 109 amino acids in this region when compared to B. subtilis. E. coli has an additional 30 amino acids at the same point although we can see no similarity between the extra 30 amino acids in E. coli and the additional amino acids in P. lutherii. The P. lutherii SecA protein can be subdivided into four regions of homology (Fig. 2). In regions I (residue 1 to 229) and II (residue 315 to 524) the three proteins are approximately 52% identical and 70% similar (Table 1). The percent identity/similarity in the intervening space of approximately 84 amino acid residues is 12/27 for E. coli and 7/17 for B. subtilis. The percent identity/similarity of Region III (residue 635 to 759) is 51/77 and 48/73 for E. coli and B. subtilis, respectively. The percent identity/ similarity for Region IV (residue 850 to 904) is 40/72 and 34/60 for E. coli and B. subtilis, respectively (Table 1). The additional 109 amino acids in P. lutherii are located in the middle of a highly conserved region of the bacterial SecA proteins and create the two domains which we show as regions II and III on Fig. 2. The additional 109 amino acids (525 to 635) include a significant number of charged residues. Based on the amino-acid groupings o f Dayhoff et al. (1978) - D E Q N , LMVI, TSAPG, K H R , Y W F and C - 40% of the residues are charged and an additional 15% are polar in this portion of SecA. There are four repeats of a doublet composed of residues from the D, E, Q, N grouping with a hydrophobic (LMVI) residue on either side. These four motifs are L D D M , INNL, VEEL, VEEL. Both VEEL motifs are preceded by the amino-acid sequence E K N R L / I which appears as an imperfect tandem repeat from residue 558 to 597 (Fig. 3). The motif I N N L is preceded by a similar amino-acid motif K N E L . Overall, the predicted SecA protein from P. lutherii contained 33.8% charged amino-acid residues and of these 9.3% are glutamic acid (E) and 8.5% are

Results Identification o f P. lutherii secA

During an investigation of the chloroplast genome of P. lutherii, we identified a chloroplast D N A clone (pMAQ805) possessing an open reading frame (ORF) which, when used in database searches, displayed sequence homology to previously identified SecA proteins ofE. coli and B. subtilis. Consequently, a 3.8 kb region of pMAQ805 was sequenced which contained the complete sequence of secA. The seeA gene is composed of 2673 nucleotides (position 775-3448 in Fig. 1) and predicts a polypeptide of 891 amino acids with a molecular weight of 101,911 daltons. Comparison of SecA proteins of E. coli and B. subtills with the P. lutherii SecA is shown in Fig. 2. To optimally align the three sequences it is necessary to introduce a large gap in the sequences of E. eoli and B. subtilis beginning at position 525 (Fig. 1). Up to this position the

Table 1. Percent identify/percent similarity of the derived P. lutherii SecA over four regions of the derived SecA proteins of E. coli and B. subtilis *

Region

Region I (I -229) Region II (315- 524) Region III (635-759) Region IV (850-904)

% identify/% similartiy E. coil

B. subtilis

52/70

52/69

52/68

51/70

51/77

48/73

40/72

34/60

* The four regions of homology are boxed in Fig. 2. Substitutions were designated conservative if both amino acids fall within one of the following exchange groups - TSAGP, RKH, FWY, DEQN, LIMV, C (Dayhoff et al. 1978)

423 lysine (K). These findings are consistent with the other bacterial SecA proteins and confirm that P. lutherii SecA is also a soluble protein. Two open reading frames were identified in the sequenced region of pMAQ805. The first of these, ORF93, is located on the complementary DNA strand with respect to secA, 193 bases upstream of the start codon of secA (position 303-582 in Fig. 1). The second unidentified O R F (ORF42) is located 53 bases downstream (3') from secA and on the same D N A strand as seeA (position 3502-3628 in Fig. 1). The two ORFs predict polypeptide sequences of 93 and 42 amino-acid residues respectively, and neither displayed any significant similarities to sequences lodged in the databases.

Restriction mapping

Using our available P. lutherii chloroplast and other clones we are currently generating a restriction map of the P. lutherii chloroplast chromosome. For this purpose we have digested D N A with six restriction endonucleases that have recognition sequences containing only [G + C] residues. Such enzymes are rare base cutters in this genome since the [G + C] content is 37% (Scaramuzzi et al. 1992 a) and should generate DNA fragments of average length 25 kb. Probing of Southern blots of the appropriate digests with probes specific for secA and rpoB/C1 revealed that these genes are located within 10 kb of each other. A restriction map of this region is shown in Fig. 4. The P. lutherii chloroplast genes, hsp70, atpI/atpH and secY, are not linked to secA or rpoB/rpoC1 since they hybridised to different D N A fragments. An E. coli secB did not hybridise to any fragments of P. lutherii chloroplast DNA.

Discussion

This is the first report of a chloroplast-encoded SecA, and its presence in the chloroplast of the chromophytic alga P. lutherii raises some interesting questions about protein translocation into and within the organelle. It also raises the unprecendeted possibility of protein export from the chloroplast, since the SecA protein is a component of the prokaryotic protein translocation (export) apparatus. The secA gene of P. lutherii is linked to the R N A polymerase genes; its presence and linkage to rpoB/rpoCl further supports the finding that the gene arrangement of the chloroplast genome in P. lutheriiis novel (Scaramuzzi et al. 1992b). SecA has been previously described from only two bacteria, E. coli (Schmidt et al. 1988) and B. subtilis (Sadaie et al. 1991). In E. coli there is an unidentified ORF located upstream of seeA, called genex, which is co-transcribed with secA (Schmidt et al. 1988). Mutation of genex results in a secA- phenotype (Schmidt et al. 1988). ORF93 found on the complementary strand in P. lutherii upstream ofseeA has no sequence similarity to genex or to the O R F located upstream ofB. subtilis seeA. ORF42, downstream from secA in P. lutherii, is unidenti-

fled, as is the ORF seen downstream from B. subtilis secA and there is no similarity between the two ORFs. The predicted protein of the DNA sequence presented here has sufficient identity to the bacterial SecA proteins to be unequivocally identified as SecA (Table 1). Furthermore, the hydropathy analysis (data not shown) indicates that the predicted SecA protein of P. lutherii is hydrophylic, consistent with the bacterial SecA proteins and their function in these organisms. A striking feature of the predicted P. lutherii protein is the presence of an additional 109 amino acids (which contains an imperfect tandem repeat sequence) at position 525 of the E. coli sequence (Fig. 1). Interestingly, the E. eoli SecA protein has an additional 30 amino acids at the same location when compared with that of B. subtilis but there is no homology between the P. lutherii and E. coli proteins over this region. SecA has been shown to be required for secretion of most of the periptasmic and outer membrane proteins thus far investigated (Strauch et al. 1986; Baker et al. 1987; Saier et al. 1989) and the movement of proteins is coupled to ATP hydrolysis at one or more ATP-binding sites probably associated with the amino-terminal end of the protein (Lill eta1. 1989; Matsuyama etal. 1990). Sadaie et al. (1991) suggest that the amino-acid conservation in the SecA amino-terminal supports this finding and that ATP-binding occurs in the N-terminal region. Although we found no typical, GxxxxGKTxxxxxI/V, ATP-binding consensus sequence (Walker etal. 1982; Gill et al. 1986) there are two adjacent regions (underlined in Fig. 2) which resemble the ATP-binding consensus sequence, incorporating the essential GKT/GKS motif. The first (MxxGxGKTxxxxxP) is more likely as a binding site since the second (LxxxSGKSxxxxxV) is not conserved in the other SecA proteins. However, both are likely candidates for ATP-binding sites since ATP hydrolysis has been demonstrated from two regions of the SecA amino-terminus ofE. coli (Lill et al. 1989) and a survey of 50 ATP-binding proteins by Gill et al. (1986) suggests considerable latitude in the outer elements of the ATPbinding consensus sequence. The distribution of charged and hydrophobic residues within the imperfect tandem repeat of P. lutherii SecA may reflect differences in proteins that require translocation across chloroplast membranes, including the thylakoids, which would not be found in bacterial systems. Alternatively, it may indicate that SecA is multifunctional in P. lutherii, where the additional 109 amino acids form a protein structure of unknown function peculiar to the protein translocation mechanisms of the chromophytic chloroplast with its four surrounding membranes. It has already been suggested that SecA is a multifunctional protein with a central role in protein translocation in E. coli. (Saier et al. 1989; Matsuyama et al. 1990). Possible roles include recognition of signal peptides (Matsuyama et al. 1990), ATP-binding, ATP-hydrolysis and protein translocation (Lill et al. 1989), SecY-SecA interactions (Fandl et al. 1988) and an involvement in the electrochemical force required (Yamada et al. 1989). A diagrammatic representation of prokaryotic transmembrane protein translocation is shown in Fig. 5. This

424 PstI

100

CTGCAGGAAAATAGTATTGAATCAAACAAATTAGATATAGAAATGG~TT~GCTCGATCC~GCTATAAAG~TGCAGTAG~TCAGGCTTGCGCCGCCTTTGTTTC~TATCTGCGAT 2O0 ~GAAAAG~TTA~AGTTAGACACTCTTAGACATCTAAAAGTAAAT~CTT~cTGTTAAAA~C~CTATC~TTATTATAAACAAAGAAA~TATA~TCAAAGGCAGCAGAAATGTT 300 ~TGAAAGAGATACTCTTTTCTACAAAA~TGAG~TGAAATT~T~TT~TAAAAAGTTTACTTTTGT~GTAGGGTAAATTGAC~GTATTTTGTAAATATTTATTTTTAG~TACT * K Q L Y P L N V L I K Y I N I K L T S 400 ATTAGTAAAA~TTTTATAc~GGAcTTTCAT~GTTAAAAGAGGTAGTGTAGCTA~GT~GAAAGAAAAAGTGTTTTAAAGATAGCTAGCGGGTTTAGAG~TCGTCAAATA~cT N T F S K I C P S E H S N F S T T Y S S Y S L F L T K F .

I A L P N L P D D F L S

500

600

TTTTCGTA~GCAAACGC~GTTATGAAATCTATGTTACATGCATATATTGGGCGAAAAAATAAAAAGCGTACATTTAGGCA~TATGGATTGGACGCATCAAAGCATTTGCGAATCA K R V A F A L T I F D I N C A Y I P R F F L F R V N L C I H I P R M < o r f 9 3 70O AAAG~TC~TACAGTAAATTTATGATGAACTTAAAAAAGTCC~TATTGGTCTAAATAGAAAAATGCTAT~CATT~TAGTTTTAGAAGGTTTAG~TTTGTG~CTTTTAAATA 80O AAAATTCTATGTAAAGTTACATAAAATTTTAGTGAAATATGGCAAATATAGTTTTATGTTAAAG~CATACTTAGAAAAAC~CACAAAGTGATTTATACcGGTAC~G~TATCGTT~ s e c A > M L K D I L R K T T Q S D L Y R Y E N I V K 900 AAAAATAAAC~TCTCGAAAGGGTTATGAAACCTTT~CC~TG~G~CTTAGAGCTAAAACATTGGGTTTTAGAAAATC~TAGAG~CGGGCAAAGCATTGAC~TATACTTCCAGA K I N D L E R V M K P L T N E E L R A K T L G F R K S I E D G Q S I D N I L P E EcoRl AGCCTTTG~TTAGTACGGG~GCGTCTTT~GAATTCTAGGGCTTAGACATTATGATGTGC~TTGATTGGTGGTTGTATACTCCAcGATAGTAAAATCGCCGAG~G~GACAGGC~ A F G L V R E A S L R I L G L R H Y D V Q L I G G C I L H D S K I A E M K T G E 1100

1200

~GAAAAA~CT~TT~TACTACCTGCATATTTAAATGCGCT~T~AAAAAGT~TACAcATAGTTACAGTG~TGAGTATTTAGCTAAACGTGATAGTTTATCGGTAGGCA~GT G K T L V A I L P A Y L N A L S G K S V H I V T V N E Y L A K R D S L S V G R V 1300 ACTTAGTTTT~TT~GTCTTAGTGTTG~TT~TCTTAGCT~TATG~TA~GAAGAGAGGC~GAAAAC~ACAAATGTGA~GTTATATATAC~CAAATAGTGAGCTAGGTTTTGATTA L S F L G L S V G L I L A D M N R E E R Q E N Y K C D V I Y T T N S E L G F D Y 1400 TTTGAGGGAT~CCTAGTTGGAAATCcTTCTGAGAAAGTTCAAAATGGTTTTG~TTTGCGAT~TTGATGAGGTTGATTCTGTTTT~TTGACG~GCGCG~CcACT~T~TCTC L R D N L V G N P S E K V Q N G P E F A I I D E V D S V L I D E A K T P L I I S 1500 CC~TCACTAGAAA~TTAAAT~TATTTACCT~CGGCGAAAAATGTAGcGCAAGCATTTGAAATAAATACTCACTACGAAATAGATAAAAGAAACCGT~TGTTTATTTAAATG~TC R S L E T L N N I Y L T A K N V A Q A F E I N T H Y E I D K R N R N V Y L N E S 1600

ECORI

TGGTAGCAAACTA~CTGAAAAATTAcTTGGCGTTAGTA~ATATATAAATTT~CTGG~CTTATATATTA~ATGC~TAAAAGCAAAAG~TTCTATACGAAA~ATAAA~TTATCT G S K L A E K L L G V S S I Y K F E T G T Y I L N A I K A K E F Y T K D K D Y L 1700

1800

TGT~T~GAAATCAAATTAC~TTGTA~TGAGTT~CCGG~AGAATTTTGAAAGGTAGG~GCTGGGGTGATGGTCTCCATC~GCTATAG~GCT~GG~GGCGT~CGGTTGGTAG V M R N Q I T I V D E F T G R I L K G R R W G D G L H Q A I E A K E G V T V G S EcoRl CGAAACCATGACTATGGCCTCGATCACTTACCAG~TTTCTTTTTGTTTTACAAAAAGTTATCCGGTATGACTGGTACAGCATT~CAGAAGCG~GG~TTCAAAAAAATATAT~TCT E T M T M A S I T Y Q N F F L F Y K K L S G M T G T A L T E A K E F K K I Y N L 2000 ATCTGTGGACTGCGTACC~T.~`T~G`~AGTT~TCGTATAGACkAAGAG~TGTTGTTTACAAATCTCTATATGCTA~&ATGG'~AAGCTGTCCTATATG~TCCTT~GT~TACATGA $ V B C V P I N K K V N R I D K E D V V Y K S L Y A K W K A V L Y E S L S I H E 2100 G~GGTA~CCATTACTTATAGGTAC~GT~CGTCAAAAACTCA~ATCGTTTCAGGTTTATTG~GG~TAC~TATAAAGCATAGTTTACTT~TGCGAAACCGGAAAATGCAGC Q G R P L L I G T S N V K N S E I V S G L L K E Y N Z K H S L L N A K P E N A A 2200 T~TGAATCTGAAAT~TTGCCCAAGCCGGTAGAAAAGG~GCGTTAC~TTGC~CAAATATGG~GGGGCGTG~TACT~ATATTTTATTAGGTGGT~TCCAGACTTTTTGA~C~GGG N E S E I I A Q A G R K G S V T I A T N M A G R G T D I L L G G N P D F L T K G 2300

2400

T~GTT~TACATATTTA~TCTATAGTCTTATCCTTA~TGATAT~CAGTGCCTAAAAAC~GTT~TT~T~CCTTAAATATAAATATGTTATTTCTGAG~G~TAGGTTAGA E L R Y I F R S I V L S L D D M T V P K N E L I N N L K Y K Y V I S E K N R L D 2500 TGTCGAAG~TT~TT~TAAACTTAAATC~GCTTATACTGTTCCTGAGAAAAATAGGATAGGTGTCG~G~TT~TTGAAAATATTGACGAGTCTTTTC~CCAGTAGATAAATTCGA v~

EL

~

D

K ~ K S A ~

~ V ~

E

KN

R~

GV

E

~

~

EN

~D

9

S

~

Q

P V ~

KF

E

Fig. 1. Nucleotide sequence of the P. lutherii secA gene (EMBL database accessmn number X65961) and the surrounding open reading ~ames (ORF93 and ORF42). ORF93 is on the complementa~ DNA strand upstream of secA while ORF42 is downstream from the termination of secA

425 HindXII

.

2600

~TTTT~TACAAAAGCTTTATGAAAAGACAAAA~GCGGTATGTCAGAG~TGTTTAGCCGAAAAGG~TTATTCA~CTTG~TTTA~T~TAGGTACGGAAAAACAC~ I

L

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Q

K

L

Y

E

K

T

K

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R

Y

V

R

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C

L

A

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K

E

E

V

I

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2700

G

G

L

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I

G

T

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K

H

D

HindIII

CTcTAG~GAATAGAc~CCAGTT~GG~GCTGGTAGAC~GGAGACCCTGGCTCTTCAAAATTTTTCTT~GCTTTGAAGACCGCTTGATAGAAATTTTTACTACG~TG~TT S

R

R

I

D

N

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G

R

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F

F

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2800

GAAA~ATATGATAAAAGAACTG~TTTA~GGAT~CC~CCGGTTGAAGGGAAAATAGT~GCCT~GCATAGAGTCAGCT~GAG~TAGAA~CAAAAACTATCAAGT~GAAA K

N

M

I

K

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L

D

L

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D

D

Q

P

V

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G

K

I

V

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L

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2900

3000

~C~TTATTT~CTAC~T~CGTTTTG~TCTA~GCGAAAGGT~TTTAT~CGAGAGAGATAGATTTTT~GTCTTA~TTTTAAAGGTCT~TTCTCCAGTATTTAGAAAAGTT Q

L

F

N

Y

D

N

V

L

N

L

Q

R

K

V

I

Y

D

E

R

D

R

F

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L

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K

G

L

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Q

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L

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3100 AGTA~TGATGT~GTAGCAGAAATGGAAAGGTCTGAAAACC~G~CAAAAGTA~GGTATTGTACTTTTTTGTAAAAAATTTATATGTCTTCCGTACTC~TA~TC~TGCT V

D

D

V

V

A

E

M

E

R

S

E

N

J

Q

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D

K

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I

V

L

F

C

K

K

F

I

C

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Y

S

I

D

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L

3200 TAGC~TTTGTCAAAAGAAGAGATAAAAATATTTTTG~TGATC~GTAAAAAT~GCTATG~TTGAAAGAAATTGAACTAGAAT~TT~GTGTTG~TTGT~T~GTCTCTTGAGTA S

N

L

S

K

E

E

I

K

I

F

L

N

D

Q

V

K

I

S

Y

E

L

K

E

I

E

L

E

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L

R

V

G

L

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S

L

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3300 TGCTTTTTTACTGc~TCTATA~cC~GTTTGGAAAG~GCTTACTAG~TGG~CTGCTAAAAGAAAGTATTGGTTGGAGAGCTTATG~CAAA~TcCTTTACTCGAATAC~ A

F

L

L

Q

S

I

D

Q

V

W

K

E

HindIII

Q

L

T

R

M

E

L

L

K

E

S

I

G

W

R

A

Y

G

Q

R

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P

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Y

Q

3400

AAAGG~GCTTATAGAATATTCGC~TACAAAC~GAAAAAT~TAGT~TTCT~TTT~TTATGTGTTCTACCTCTTTTGCTT~GAAGGGGCTGTTTAGGTTTC~TC K

E

A

Y

R

I

F

A

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T

R

K

I

R

H

S

A

S

H

L

I

M

C

S

T

S

F

3500

A

* 3600

CTAAAAAAAACT~T~GTGTGATGC~GTAGAGGAAACC~TCTTAAAAAAAAAGCAAAC~TAAATGCAAACAATATTTTATCTTTT~TC~GTAGcTGTAGC~CGTATAGTTATAT o r s

AG~TT~CTCCTTATGGTCAAAAAATCTAGAAAGTTT~TCGTAAAGCAAACCTTAGCTAGAAGCACAC~GAAT~ A L T P Y G Q K I *

Fig. 1. (continued)

model incorporates SecB as a chaperonine and SecYSecA interactions at the membrane. Both sec Y (Scaramuzzi et al. 1992 b) and now secA have been found on the chloroplast genome of P. lutherii but we have so far been unable to detect secB. This may indicate that the probe used was insufficiently homologous for detection because of the difference in the [G + C] content between P. lutherii chloroplast DNA and E. coli DNA. The P. lutherii chloroplast genome also contains an hsp70 gene (Scaramuzzi et al. 1992 a) which predicts a protein that is 70% identical to the cyanobacterial DnaK. Until localisation of the SecY, SecA and Hsp70 proteins in the various compartments of the chloroplast can be demonstrated, the involvement of these proteins in specific translocation processes cannot be specified. Nonetheless, it can be extrapolated from their presence on the chloroplast genome that prokaryotic protein translocation mechanisms are inherent within this organelle and may include protein export from, as well as import into, the chloroplast. It has already been suggested that the chromophytic alga P. tricornutum has a different mechanism from that of higher plants for the import of nuclear-encoded lightharvesting proteins into the chloroplast (Grossman et al. 1990; Bhaya and Grossman 1991). This suggestion was based on the amino-terminal regions of several light-harvesting proteins which appeared to resemble signal sequences rather than transit peptides. It has also been proposed that light-harvesting proteins enter the chloroplast by a co-translational process in which polypeptides are inserted and translocated through the ER while still being synthesised on the ribosome (Grossman et al. 1990; Bhaya and Grossman 1991). Subsequently they are incor-

porated into vesicles that fuse with the chloroplast membrane. The presence of vesicles between the CER and the chloroplast membrane of Euglena (Gibbs 1978) and other eukaryotic algae (Gibbs 1981) supports this hypothesis. Vesicular movement of proteins is not uncommon and it is one method proposed for the movement of proteins across the periplasm from the inner to the outer membrane in bacteria (Austen and Westwood 1991). Similarly, eukaryotic protein translocation beyond the ER involves vesicular traffic between membranes. Proteins are contained within vesicles which detach from a donor membrane and then fuse with the target membrane (Austen and Westwood 1991). It is possible that the chromophytic algae, containing four chloroplast membranes, have several coordinated processes for the importation of nuclear-encoded proteins and for the targeting and positioning of these and others proteins within the chloroplast. These processes might include a vesicular mode of protein transport, as well as mechanisms involving the prokaryotic protein translocation apparatus and hsp70 proteins. The presence of hsp70 and see Y on the chloroplast genome of P. lutherii, as well as the linkage of sec Y to the atpI/H genes (Scaramuzzi et al. i 992 a, b), clearly demonstrate that the chloroplast genome organisation of P. lutherii differs from other chromophytic algae, cyanelles, the Chlorophyta and higher plants. The discovery of secA and its linkage to the rpoB/C1 genes, provides further evidence that the higher plant chloroplast genome is an inadequate model for that of the evolution of the chromophytic alga P. lutherii.

426 i

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J l" . 250 . . . . 300 i NGFEFAI~DEVDSVLIDEi~RTPLIISP~LETLNNIYLTAKNVA~FEINTHYE~DKI~!RN-VYLNESGSK . . . . . . . . . . I~EKLLGVSSIYKk"ETGTYIIaN RKLHY*LV***~*I************(~A*DSSEM*KRVNKIIPHLIRQE~EDSETFQGEGHFSVDEK~RQvNLTERGLV*I*E**VKE~*MDE~ESL*SPA RPLH**V******I*******'****~.~KSTKL*VQ/~/~VR . . . . . . . . . . . TLKAEKD*TYI~INT*NVQLTEE~NTK**K~---G* . . . . DNLFDVK 9

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* T K A * A N ** R K V * S R * F D I *** * L E * * D , A * D ** R A * * SQ * N E L * D V $ * V S E T - * N S I R * D V F K A T I D * Y I P P Q S L E * M W D I P G L Q E R L * N D F - - D L D L P I A **RAV**S***V*GN*FDS****LQ**D**RQ**E***KQ*FEVEDSE~

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.... QPQEGDDNKKAKKAPVRKVVDIGRNAPCHCGSGKKYKNCCGRTE

Fig. 2. Comparison of the derived amino-acid sequences of P. lutherii SecA (A) with SecA proteins of E. coli (B) and B. subtilis (C). The predicted protein sequence is shown for all three proteins and identical residues are indicated with an asterisk (*). The four conserved regions (L II, III and IV) are boxed and potential ATP-

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Fig. 3. Amino-acid sequence of the P. tutherii SecA protein (558 to 597) showing the imperfect tandem repeat sequences (boxed). Positive amino-acid residues are denoted as (+), negatively charged residues as ( - ) , and hydrophobic residues as (o)

rpoB/CI

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Fig. 4. Restriction map of a 36 kb fragment of chloroplast DNA showing the linkage between secA and the rpoB/Cl genes. The seeA gene probe hybridised to the 5.0 kb NarI fragment, the rpoB/C1 gene probe hybridised to a 6.0 kb NaeI/SmaI fragment. The DNA fragment separating these is a 6.0 kb NarI/NaeI fragment

427

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Acknowledgements. This research was supported by Macquarie

University Grants to H.W. Stokes and R. G. Hiller. We thank Carol Kumamoto for the E. coli seeB gene.

References Austen BM, Westwood OMR (eds) (1991) Protein targetting and secretion. IRL Press, Oxford Baker K, Mackman N, Jackson M, Holland IB (1987) J Mol Biol 198:693 -703 Bhaya D, Grossman A (1991) Mol Gen Genet 229:400-404 Dayhoff MO, Schwartz RM, Orcott BL (1978) In: Dayhoff MO (ed) Atlas of protein sequence and structure, vol 15, suppl 3. National Biomedical Research Foundation, Washington DC, pp 345-352 Fandl JP, Cabelli R, Oliver D, Tai PC (1986) Proc Natl Acad Sci USA 85:8953-8957 Gardell C, Benson S, Hunt J, Michaelis S, Beckwith J (1987) J Bacteriol 169:1286-1290 Gibbs SP (1978) Can J Bot 56:2883-2889 Gibbs SP (1981) Ann NY Acad Sci 361:193-207 Gill DR, Hatfull GF, Salmond GPC (1986) Mol Gen Genet 205:134-145 Grossman AR, Manodori A, Snyder D (1990) Mol Gen Genet 224:91 - 100 Keegstra K (1989) Cell 56:247-253 Kumamoto CA, Chen L, Fandl J, Tai PC (1989) J Biol Chem 264: 2242- 2249 Kumamoto CA, Nault AK (1989) Gene 75:167-175 Lill R, Cunningham K, Brundage LA, Ito K, Oliver D, Wickner W (1989) EMBO J 8:961-966 McFadden GI (1990)J Cell Sci 95:303-308

Fig. 5. A model for prokaryotic membrane translocation based on the translocation of pro-OmpA (proOuter membrane protein A) across the inner bacterial membrane. Modified from and by permission of Prof. W. Wickner, UCLA

Maniatis T, Fritsch EF, Sambrook J (1982) Molecular Cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York Matsuyama S, Kimura E, Mizushima S (1990) J Biol Chem 265: 8760- 8765 Sadaie Y, Takamatsu H, Nakamura K, Yamane K (1991) Gene 98:101-105 Saier Jr. MH, Werner PK, Muller M (1989) Microbiol Rev 53: 333366 Sanger F, Coulson AR, Barell BG, Smith AJH, Roe BA (1980) J Mol Biol 143:161-178 Scaramuzzi CD (1991) PhD thesis, Macquarie University, Australia Scaramuzzi CD, Stokes HW, Hiller RG (1992a) Plant Mol Biol 18:467-476 Scaramuzzi CD, Stokes HW, Hiller RG (1992b) FEBS Lett 304:119-123 Schatz PJ, Riggs PD, Jacq A, Fath MJ, Beckwith J (1989) Genes Dev 3:1035-1044 Schmidt MG, Rollo EE, Grodberg J, Oliver DB (1988) J Bacteriol 170:3404-3414 Smeekens S, Weisbeek P, Robinson C (1990) Trends Biochem Sci 15-February: 73-76 Strauch KL, Kumamoto CA, Beckwith J (1986) J Bacteriol 166:505-512 Sub JW, Boylan SA, Thomas SM, Dolan KM, Oliver DB, Price CW (1990) Mol Microbiol 4:305-314 Walker JE, Saraste M, Runswick MJ, Gay NJ (1982) EMBO J 8:945-951 Whatley JM, Whatley FR (1981) New Phytol 87:233-247 Yamada H, Matsuyama S, Tokuda H, Mizushima S (1989) J Biol Chem 264:18577-18581 Yanisch-Perron C, Viera J, Messing J (1985) Gene 33:103-119 C o m m u n i c a t e d b y J.-D. R. R o c h a i x