Photosynthesis Research 19:7-22 (1988) © Kluwer Academic Publishers, Dordrecht - Printed in the Netherlands


Using bacteria to analyze sequences involved in chloroplast gene expression A N T H O N Y A. GATENBY, 1 STEVEN J. ROTHSTEIN 2 & D O U G L A S BRADLEY 3 1Central Research and Development Department, Experimental Station, E.L du Pont de Nemours & Co., Wilmington, D E 19898 USA; 2Department o f Molecular Biology and Genetics, University o f Guelph, Guelph, Ontario, Canada, N1G 2W1; 3Department o f Molecular Genetics, Plant Breeding Institute, Trumpington, Cambridge CB2 2LQ, UK

Received 21 September 1987; accepted 24 March 1988

Keywords: mutagenesis, promoters, secretion, transcription Alrstract. The expression of higher plant chloroplast genes in prokaryotic cells has been used to examine organelle sequences involved in promoter recognition by RNA polymerase, and protein translocation through membranes. The similarity in sequence structure between Escherichia coli promoters and the maize chlo . last atpB promoter has been investigated using deletion and single base pair substitution mutants. The atpB mutants were mainly isolated by a selection system in E. coli, and then used as templates for the analysis of transcription using chloroplast RNA polymerase. It was found that both the bacterial and chloroplast RNA polymerases behaved in a similar fashion with the wild-type and mutant promoters, indicating that the sequences involved in promoter recognition share a considerable degree of homology. Signal peptide recognition of pea cytochrome f has also been examined in E. coll. This signal peptide, which is probably responsible for insertion of the protein into the thylakoid membrane, is efficiently recognized in E. coli leading to the inner membrane insertion o f p e t A : : l a c Z fusion proteins. This process requires the bacterial SecA protein and points to a general similarity in the mechanisms of protein translocation within chloroplasts and bacteria. Gene symbols: atpB - ATPase F~ fl subunit, lacZ - fl-galactosidase, petA - cytochromef, psbA - photosystem II 32K protein; rbcL - large subunit of ribulose bisphosphate carboxylase, trnM2 - tRNA2Met


Chloroplast genes exhibit considerable homology with Escherichia coli genes for sequences involved in recognition by R N A polymerase, and subsequent interaction with ribosomes (Whitfeld and Bottomley 1983; Rochaix 1985). This has provided a special opportunity to use bacterial systems to identify features that are involved in chloroplast gene expression. Clearly, examining

the regulatory features of chloroplast gene expression in bacteria is not a completely faithful elucidation of the events occurring in the plant organelle, but it can provide valuable information on the interaction of bacterial RNA polymerase, 70S ribosomes and membrane targeting mechanisms with the chloroplast sequences. It should therefore be considered as an additional tool to aid in the dissection of the molecular events occurring within the chloroplast. A particularly promising approach using this methodology is the ability to select and screen mutations of chloroplast gene function in bacteria, and then to test the mutations in chloroplast derived transcription systems (Bradley and Gatenby 1985). If faithful chloroplast translation and membrane insertion reactions can also be developed, so that mutants generated and selected in bacteria can be tested, then a wide range of events concerning chloroplast biogenesis can be studied. In this review we shall describe two areas in which chloroplast gene expression has been studied using bacterial systems to gain insight into the events occurring in the organelle viz. promoter recognition, and protein translocation.

Characterization of promoters In early experiments it was demonstrated that isolated chloroplast D N A from spinach could direct the expression of a chloroplast protein in vitro using an E. coli cell-free coupled transcription-translation system (Bottomley and Whi.tfield 1979), suggesting that the bacterial RNA polymerase could bind and initiate transcription at sites on the chloroplast genome. These findings were supported by in vivo expression studies in which synthesis of chloroplast proteins could be detected in E. coli using cloned chloroplast genes, in a manner that was consistent with trancription initiating at sites on the chloroplast DNA, rather than occurring from rcadthrough transcription from vector sequences (Gatenby et al. 1981; Gatenby and Cuellar 1985). Using these in vitro or in vivo methods numerous chloroplast genes from several different plant species have been successfully transcribed by E. coil RNA polymerase (Howe et al. 1982; Willey et al. 1983; Fluhr et al. 1983; Bovenberg et al. 1984; Woessner et al. 1984; Zhu et al. 1984; Gatenby and Rothstein 1986). Chloroplast D N A restriction fragments have also been used to provide a sequence for E. coli RNA polymerase to recognize, resulting in the in vivo expression of a downstream promoter deficient gene such as fl-galactosidase (Gatenby et al. 1981; Fukuzawa et al. 1985) or galactokinase (Kong et al. 1984). Although such an approach may identify a promoter that is active in both the chloroplast and E. coli, a problem arises when sequences fortuitously function as promoters in bacteria, but which

9 are not authentic chloroplast gene promoters. For example, it has been observed that a maize rbcL restriction fragment that is known not to contain the authentic rbcL transcription initiation site, nevetheless has a measurable level of promoter activity in E. coli (A.A. Gatenby and R.E. Cuellar unpublished). The clearest evidence for authentic chloroplast promoter recognition by E. coli RNA polymerase has been obtained from experiments using high resolution transcript mapping, sometimes in combination with mutagenesis. Thus, Shinozaki and Sugiura (1982) using tobacco rbcL and atpB genes, Tohdoh et al. (1981) using tobacco rRNA genes, Erion et al. (1983) using spinach rbcL and Boyer and Mullet (1986) using pea psbA, were able to demonstrate that purified E. coli RNA polymerase would initiate transcription in the same position on chloroplast DNA as would the corresponding chloroplast RNA polymerase. Fukuzawa et al. (1985) also observed recognition of a chloroplast promoter from a liverwort with the bacterial RNA polymerase. These studies have shown that the transcription initiation site is located downstream from regions of - 10 and - 35 base pairs that have a high degree of homology to the consensus sequence found for E. coli promoters (Rosenberg and Court 1979). Several authors have combined both mutagenesis and transcript mapping to characterize chloroplast promoter sequences. Hanley2Bowdoin et al. (1985) using the rbcL gene and chloroplast RNA polymerase suggested that chloroplast and prokaryotic promoters share sequence homology. This observation was supported by introducing a mutation to the maize rbcL promoter that increased the spacing between the putative - 1 0 and - 3 5 promoter elements from 18 to 20 base pairs, but without changing the structure of the elements themselves. Transcription of the insertion mutant by homologous RNA polymerase was depressed in vitro relative to wildtype levels, indicating that the spacing of the - 1 0 and - 3 5 region is important for effficient transcription by RNA polymerase. Gruissem and Zurawski (1985b) also obtained data that are consistent with a prokaryotic model for chloroplast promoter function. Using a chloroplast transcription extract and synthetic DNA fragments containing the defined transcriptional start sites of rbcL, atpB and psbA they were able to use an in vitro assay to examine the relative strengths of wild-type and hybrid - 1 0 and - 3 5 canonical sequences. The introduction of single, base pair changes into the - 10 region of the psbA promoter reduced transcription levels in the chloroplast extract and were analogous to similar base pair changes which lower promoter efficiency in E. coli. A chloroplast hybrid promoter that had absolute homology to the canonical - 10 and - 3 5 region, was the most efficient in the in vitro transcription system. A mutational analysis of the






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11 spinach trnM2 gene also supported the view that the arrangement of D N A sequences recognized by the chloroplast R N A polymerase resembles the prokaryotic promoter organization (Gruissem and Zurawski 1985a). Given the close similarity in structure between the chloroplast and E. coli promoters, as outlined above, an attempt was made to see if a selection scheme for the isolation of chloroplast promoter mutants in E. coli could be devised (Bradley and Gatenby 1985). To do this the 5' end of the maize atpB gene containing the promoter, ribosome binding site and the first ten codons of the ATPase fl subunit were fused in-frame to lacZ in an M13 phage vector to give M 11 (Fig. 1). A series of Bal31 deletion mutants were also made by treating the D N A with this nuclease prior to cloning into the M 13 vector. Transfectants were examined on plates containing 5-bromo-4-chloro-3indolyl-fl-D-galactopyranoside for differences in phage fl-galactosidase levels. A wide range of blue phenotypes were recovered, indicating that the chloroplast atpB D N A sequences that were operating as a promoter in E. coli were doing so in a complex fashion. The mutant deletion end points and the relative levels of fl-galactosidase produced by each phage were measured (Fig. 1). The deletion of sequences > 120 base pairs upstream of the putative atpB promoter (phage M27) caused the production of three times more fl-galactosidase activity than the parent phage ( M l l ) . The M27 deletion removed two nucleotides from the rbcL - 1 0 promoter region that was adjacent to the atpB gene, but which is transcribed divergently. Further deletion of this rbcL promoter region resulted in even higher levels of fl-galactosidase activity. These results suggest that, at least in E. coli, loss of the rbcL promoter improves the transcriptional efficiency of the adjacent atpB promoter. Possibly the close juxtaposition of the rbcL and atpB genes leads to mutual interference of RNA polymerase binding and that once the rbcL promoter is deleted this occlusion effect is abolished. Deletions can extend into the chloroplast insert as far as the atpB - 35 region (M27, M30, M26, M22) without a negative effect on atpB expression. However, when sequences inclusive of the - 3 5 region are deleted, expression of the atpB::lacZ gene product is reduced to almost background levels (M38, M40, M37), suggesting that a critical promoter region must reside between" the M22 and M38 deletions. To assay directly and compare the atpB promoter activity of each Bal31 mutant, S1 nuclease protection experiments were carried out using RNA from both phage infected E. coli cells and from maize chloroplast in vitro transcription reactions. The size of the S1 protected R N A indicated that transcription is initiated in E. coli at the same site as that used in maize chloroplasts. The level of atpB transcription measured in E. coli correlated well with the fl-galactosidase activities recorded in Fig. 1. When the in vitro

12 transcription reactions were carried out in a maize chloroplast extract, the levels of fl-ATPase specific transcription obtained from each of the deletion templates exhibit variation that is quantitatively similar to that seen in E. coli cells. Again, deletions that remove the rbcL promoter region seem to have an enhancing effect on the transcriptional activity of the atpB promoter. As with transcription in E. coli, when a deletion extends into the 35 region of the atpB promoter (phage M38), atpB transcription was not detected using maize chloroplast RNA polymerase, showing that the region is important in the identification of a chloroplast promoter by its RNA polymerase. To define more accurately those sequences that comprise a chloroplast promoter, single base substitution mutants were sought that would alter atpB promoter function in E. coli. The effects of the mutations on chloroplast RNA polymerase promoter recognition were then assayed using the chloroplast in vitro transcription system. Several different methods were used to isolate point mutations in the maize atpB promoter (Bradley and Gatenby 1985), but with all of these the mutant phenotype was selected in vivo using altered levels of atpB promoter transcribed fl-galactosidase activity following phage infection as a screen. The DNA sequences of the single base change mutants recovered using these techniques is shown in Table 1, together with the relative levels of fl-galactosidase produced. The mutations M50, M51, M54 and M75 were all isolated independently at least twice, suggesting that target site saturation had been achieved with this -

Table 1. The D N A sequence and relative levels of lacZ activity of single base pair substitution mutants of the maize atpB promoter. The LacZ activity of promoter mutant M113 is presented as a percentage of the value obtained for the wild-type promoter phage M114. The LacZ activities of the remaining promoter mutants are all expressed as a percentage of phage M22. Underlined nucleotides designate the sites of mutations. The E. coli consensus sequence is that of Hawley and McClure (1983). Reprinted from Bradley and Gatenby (1985). Phage

- 35

M 114 M 113 M22 M50 M51 M54 M75 M76 M38


E. coli consensus sequence TTGACA

- l0


Relative LacZ activity 100 % 0.4% 100% 0.9% 0.9% 0.3% 0.5% 7.3% 0.2%

13 mutant selection. The levels of atpB specific transcription from genes with single base changes in their promoters were determined in S1 nuclease protection experiments. R N A purified from E. coli cells infected with each of the mutant genes gave levels of Sl-protected R N A that mirrored the fl-galactosidase activities presented in Table 1. The atpB point mutants M50, M51, M54, M75 and M 113 produced no detectable atpB specific RNA. The results of chloroplast in vitro transcription reactions on each of the atpB promoter mutants showed that transcription is reduced. These data indicated that there are no sequences essential for atpB transcription upstream from the - 35 region. They also demonstrate that not only is the - 35 region important for chloroplast promoter function, but that the sequence is similar in detail to the - 35 region of E. coli promoters. Deletions can extend to within one nucleotide of the atpB - 35 region without loss of promoter activity in either E. coli or in a chloroplast in vitro transcription system. The atpB -- 10 sequence by itself is not sufficient for promoter recognition in the chloroplast transcription system or in E. coli. Similar conclusions have been reached by Link (1984) and Gruissem and Zurawski (1985a). The six different point mutations isolated within the - 35 region of atpB (Table 1) affect five of the six positions that make up the E. coli - 35 consensus sequence. The 3'-most nucleotide of the six-base - 35 consensus sequence, TTGACA, is the least conserved position (Hawley and McClure 1983), thus it is not surprising that chloroplast - 35 down mutations were not found at this site. No mutations were recovered within the - 10 region of the atpB promoter using a strong selection for promoter down mutants. This could be due to the fact that the atpB - 3 5 region is an optimal E. coli - 3 5 sequence and mutations in the - 10 region do not diminish promoter function sufficiently to be picked up by the promoter mutant selection method. Alternatively, the 10 region may be repeated within the atpB promoter, and a second, less optimal - 10 region can be found. The apparent similarity between chloroplast and E. coli gene control signals can be successfully exploited in order to isolate in E. coli chloroplast mutations that are of functional importance. It should be possible to carry out similar genetic studies in E. coli to investigate chloroplast transcription termination sites and chloroplast ribosome binding sequences, thus allowing us to gain important information about chloroplast gene expression. Although the resemblance between chloroplast and bacterial promoters has enabled a selection technique to be devised to obtain mutant chloroplast promoters in E. coli it is important to realise that there are a number of differences between chloroplast and bacterial promoters. These differences have been reviewed by Hanley-Bowdoin and Chua (1987) and they include sensitivity to spacing mutations, level of specificity, and promoter preference -

14 by the RNA polymerases. The chloroplast and E. coli RNA polymerases also have a number of different physical properties. It has also been found that not all chloroplast genes possess the - 10 and - 35 consensus sequences. Gruissem et al. (1986) have described a subpopulation of tRNA genes that do not require these upstream promoter sequences for their transcription.

Protein translocation

The proteins of the chloroplast are, for the most part, encoded and synthesized outside the organelle, and are transported through the chloroplast envelope after translation is completed (reviewed by Schmidt and Mishkind 1986). A few proteins are chloroplast encoded and are synthesized in the stroma. Whether imported, or synthesized within the chloroplast, there are several organelle compartments in which the various proteins will finally reside. These are the outer and inner membranes of the envelope, the envelope intermembrane space, the stroma, thylakoid membrane, or thylakoid lumen. The major multi-subunit complexes of the thylakoid membranes are the cytochromef/b6 complex, ATP synthase and photosystems I and II, and each complex contains both nuclear and chloroplast encoded polypeptides. It has been demonstrated that during import of the precursor to the light-harvesting chlorophyll a/b protein (pre-LHCP) into isolated chloroplasts the precursors were converted to their final size and became integrated into the thylakoid membrane in a chlorophyll-proteiia complex (Schmidt et al. 1981). The existence of a pathway within chloroplasts for incorporating soluble precursor proteins into thylakoid membranes has been suggested by Cline (1986) who identified an activity in chloroplast lysates that incorporates pre-LHCP into thylakoid membranes. Hageman et al. (1986) described a novel protease located in the thylakoids that processes an intermediate form of preplastocyanin to the mature protein. It was suggested that a thylakoid-transfer domain exists in preplastocyanin and that this shared a resemblance to a prokaryotic signal peptide, with the inference that the thylakoid protease would function analogously to the prokaryotic signal peptidase. Kirwin et al. (1987) have partially purified the thylakoid protease involved in plastocyanin biogenesis and have considered the possibility that the protease may process some chloroplast-synthesized proteins, in addition to the imported preplastocyanin. We are also interested in the mechanism which translocates proteins through thylakoid membranes, and in the remainder of this paper will be described experiments in which the expression of pea cytochromefin E. coli has been used to examine the properties of the signal peptide, which is thought to initiate membrane insertion.


Fig. 2. Identification of pea cytochrome f synthesized in E. coil minicells containing the chloroplast petA gene. Minicells were labelled with [35S]methionine and the cytochrome f polypeptides were identified by immunoprecipitation. Lanes: A, total plasmid-encoded products from minicells; B, immunopre¢ipitate obtained with antibodies to charlock (Sinapsis arvensis) cytochrome f and protein A-Sepharose. The 37 kDa polypeptide is the same molecular weight as mature cytochromef. Reprinted from Rothstein et al. (1985).

Cytochrome f is a chloroplast-encoded membrane protein that is synthesized within the organdie on membrane-bound ribosomes. It is a component of the membrane cytochrome b-f complex involved in photosynthetic electron transfer between photosystem II and photosystem I. The polypeptide has a transmembrane arrangement in the thylakoid membrane, with the N-terminal region in the intrathylakoid space, and a 15 amino acid C-terminal sequence in the stroma (Willey et al. 1984). A single membranespanning region near the C-terminus holds the polypeptide in the membrane. Cytochromefis synthesized as a higher molecular weight form in an E. coli transcription-translation system, and it was suggested that a signal peptide may be present to enable the N-terminal part of the protein to be secreted across the thylakoid membrane (Willey et al. 1983). This suggestion was supported by DNA sequence analysis, which demonstrated that the amino acid sequence immediately preceding the mature N-terminus had properties similar to signal peptides used to direct polypeptides to membrane locations (Willey et al. 1984). To determine whether the higher molecular weight form of pea cytochrome fobserved in an E. coli cell free transcription-translation system could also

16 CYtochrome f - l a c Z


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Fig. 3. Construction ofpetA::lacZ gene fusions. Various parts of the 5' end of the cytochrome fgene were fused in the correct reading frame with lacZ (striped line) at the XhoI (X), EcoRI (E), BamHI (Ba), and BclI (Be) sites. The region encoding the signal peptide (stippled) and the cleavage site giving mature cytochromefin the chloroplast are shown. All the plasmids except p454 encode the complete signal peptide and cleavage site. Numbers in parentheses indicate the number of amino acid residues of cytochrome f encoded from the cleavage site to the restriction enzyme site. Reprinted from Rothstein et al. (1985).

be synthesized in vivo in bacteria, an E. coli minicell-producing strain was transformed with a plasmid encoding the chloroplast gene (petA). Two polypeptides were immunoprecipitated from aSS-labelled minicells using an antiserum raised against cytochrome f (Rothstein et al. 1985). The larger polypeptide (39 kDa) was similar in size to the form synthesized in an in vitro transcription-translation reaction, and the smaller polypeptide (37 kDa) is similar in size to mature cytochrome f found in chloroplast thylakoid membranes (Fig. 2). The demonstration of precursor and mature

17 Table 2. Cell fractionation of pea cytochrome f-fl-galactosidase fusion proteins. E. coli cells containing petA::lacZ gene fusions, or induced wild-type lacZ, were disrupted by sonication. The cell-free extract was separated into a washed membrane fraction and a cytoplasmic fraction by ultracentrifugation and then asasayed for fl-galactosidase activity. The figures show the percentage distribution of total fl-galactosidase activity in the original cell extract. Reprinted from Rothstein et al. (1985).

w.t. control p454 p456 13458 p490



2 43 80 85 78

82 36 9 10 20

Figures represent percentage of total fl-galactosidase activity.

sized c y t o c h r o m e f i n E. coli raised the intriguing possibility that processing was occurring. Minicells have been shown to process secretory proteins poorly, which might account for the presence of the precursor-size cytochrome f. However, caution is required in this interpretation because of difficulties encountered in identifying the correct initiation codon for the pea petA gene. The D N A sequence reveals three in-frame A U G codons upstream from the mature N-terminus, although only one is preceded by a recognizable ribosome binding site (Willey et al. 1984). The two forms of cytochrome f synthesized in E. coli could arise by the use of two different translational start sites by the heterologous bacterial system. Due to the very low levels of expression of c y t o c h r o m e f i n E. coli, it was difficult to carry out cell fractionation or pulse-labeling experiments to determine whether signal peptide recognition was occurring. To circumvent this problem, in-frame gene fusions were constructed with lacZ such that four different hybrid fl-galactosidase molecules could be synthesized (Fig. 3). This enabled fl-galactosidase assays to be used in cell fractionation and mutant analysis experiments (Rothstein et al. 1985). The shortest fusion (p454) was within the signal sequence, and the other three fusions all contained an intact signal peptide and contained 51 (p456), 134 (p458) or 224 (p490) N-terminal amino acids of the mature cytochrome f. If the cytochrome f signal peptide is recognized by the E. coli. secretory pathway, it would be expected that the fl-galactosidase fusion proteins would behave in a manner similar to proteins containing a bacterial signal peptide fused to fl-galactosidase. A characteristic feature of these fusions is that they usually become trapped in the cytoplasmic membrane, probably because membrane incompatible amino acid sequences in fl-galactosidase jam export sites (Silhavy et al. 1983). Table 2 shows the result of assaying a washed membrane fraction and a cytoplasmic fraction from cells contain-

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Fig. 4. Analysis of cytochrome f-fl-galactosidase binding to membranes by use of sucrose gradient centrifugation. A crude sonicated cell extract was layered on the gradients and centrifuged. The gradients were fractionated and assayed for enzyme activity. Top, wild-type fl-galactosidase; middle, p490-encoded fusion protein; bottom, the gradient containing the p490 transformant extract assayed for the cytoplasmic membrane marker NADH dehydrogenase, fl-galactosidase units are lamol of o-nitrophenylgalactoside hydrolyzed per min per fraction, and N A D H dehydrogenase units are nmol of NADH oxidized per min per fraction. Reprinted from Rothstein et al. (1985).


ingpetA::lacZ fusions, or wild-type lacZ. Induced wild-type fl-galactosidase activity is located primarily in the cytoplasm. C y t o c h r o m e f f u s i o n proteins that contain the signal peptide (encoded by p456, p458 and p490), however, are located mainly in the membrane fraction. The p454 encoded fusion protein, which has a partially disrupted signal peptide, is distributed between the cytoplasm and membrane fractions. To demonstrate that cosedimentation of the signal peptide fusion proteins with the membrane fraction during differential centrifugation was not due to general insolublity, cell extracts were centrifuged on sucrose gradients (Fig. 4). The wild-type enzyme is located mainly in the soluble fraction at the top of the gradient, but the enzyme fused to cytochrome f is located in the lower part of the gradient. The gradients were also assayed for the inner membrane marker N A D H dehydrogenase and the peak fraction coincided with the p490 encoded fl-galactosidase activity. The location of the fusion proteins in the inner membrane was demonstrated by using the differential solubility properties of the inner and outer membranes in the presence of sarkosyl and EDTA. Most of the membrane bound petA::lacZ fusion proteins are released under conditions that solubilize the inner membranes of E. coli. The results obtained from cell-fractionation experiments indicate that export of cytochrome f through the bacterial cytoplasmic membrane was initiated but then blocked by the structure of fl-galactosidase. This suggests that the chloroplast signal peptide is recognized as such in E. coli, particularly since disruption of the signal peptide (as encoded in p454) leads to inefficient membrane localization (Table 2). If this interpretation is correct, then E. coli mutants known to disrupt secretion of bacterial proteins would be expected to have an effect on petA signal peptide recognition and initiation of export. The secA gene of E. coli is thought to encode a component of the bacterial secretion machinery, and mutations in this gene selectively interfere with the synthesis or export of secreted proteins. The strain MM52 is a temperature sensitive conditional-lethal secA mutant (Oliver and Beckwith 1981). At the non-permissive temperature, the precursors of a number of secreted proteins accumulate in the cytoplasm, although some periplasmic proteins are correctly localized. MM52(secA) or M C4100(wt) cells containing the petA::lacZ gene fusions were grown to early log phase at 30 °C and then were either shifted to 41 °C, or maintained at 30 °C, and were assayed for the cellular location of the /3-galactosidase activity at various times. At 30 °C the bulk of the enzyme activity ws found in the washed membrane fraction in both MM52 and MC4100. However, at 41 °C over a period of 3-4 hr in strain MM52, most of the enzyme was found in the soluble fraction and was not associated with the membranes. In the control strain MC4100 at 41 °C, most of the enzyme


activity remained in the enzyme fraction. At this higher temperature the temperature-sensitive SecA lesion is known to lead to a defect in export. It would appear that membrane insertion of the petA::lacZ fusion protein is also affected by this defect. These results show that the signal peptide of pea cytochrome f is recognized by the E. coli secretory pathway, leading to initiation of export through the bacterial cytoplasmic membrane. This view is reinforced by the observation that deletion of the processing site and part of the signal peptide (p454) leads to inefficient membrane localization. There is, therefore, a considerable degree of functional homology between a chloroplast signal peptide and the secretory mechanisms of bacteria. Possibly a protein with a function analogous to the SecA protein may be involved in insertion of cytochrome f into the chloroplast thylakoid membrane.

References Bottomley W and Whitfeld PR (1979) Cell-free transcription and translation of total spinach chloroplast DNA. Eur J Biochem 93:31-39 Bovenberg WA, Howe CJ, Kool AJ and Nijkamp HJJ (1984) Physical mapping of genes for chloroplast DNA encoded subunit polypeptides of the ATP synthase complex from Petunia hybrida. Curr Gen 8:283-290 Boyer SK and Mullet J E (1986) Characterization of P. sativum chloroplast psbA transcripts produced in vivo, in vitro and in E. coli. Plant Mol Biol 6:229-243 Bradley D and Gatenby AA (1985) Mutational analysis of the maize chloroplast ATPase-fl subunit gene promoter: the isolation of promoter mutants in E. coli and their characterization in a chloroplast in vitro transcription system. EMBO J 4:3641-3648 Cline K (1986) Import of proteins into chloroplasts. Membrane integration of a thylakoid precursor protein reconstituted in chloroplast lysates. J Biol Chem 261:14804-14810 Erion JL, Tarnowski J, Peacock P, Caldwell P, Redfield B, Brot N and Weissbach H (1983) Synthesis of the large subunit of ribulose-l,5-bisphosphate carboxylase in an in vitro partially defined E. coli system. Plant Mol Biol 2:279-290 Fluhr R, Fromm H and Edelman M (1983) Clone bank of Nicotiana tabacum chloroplast DNA: mapping of the alpha, beta and epsilon subunits of the ATPase coupling factor, the large subunit of ribulose bisphosphate carboxylase, and the 32-kDal membrane protein. Gene 25:271-280 Fukuzawa H, Uchida Y, Yamano Y, Ohyama K and Komano T (1985) Molecular cloning of promoters functional in Escherichia coli from chloroplast DNA of a liverwort, Marchantia polyrnorpha. Agric Biol Chem 49:2725-2731 Gatenby AA, Castleton JA and Saul MW (1981) Expression in E. coli of maize and wheat chloroplast genes for large subunit of ribulose bisphosphate carboxylase. Nature 291: 117-121 Gatenby AA and Cuellar RE (1985) Antiterminationis required for readthrough transcription of the maize rbcL gene by a bacteriophage promoter in Escherichia coli. Eur J Biochem 153: 355-359 Gatenby AA and Rothstein SJ (1986) Synthesis of maize chloroplast ATP-synthase fl-subunit fusion proteins in Eseheriehia coli and binding to the inner membrane. Gene 41:241-247

21 Gruissem W, Elsner-Menzel C, Latshaw S, Narita JO, Schaffer MA and Zurawski G (1986) A subpopulation of spinach tRNA genes does not require upstream promoter elements for transcription. Nucl Acids Res 14:7541-7556 Gruissem W and Zurawski G (1985a) Identification and mutational analysis of the promoter for a spinach chloroplast transfer RNA gene. EMBO J 4:1637-1644 Gruissem W and Zurawski G (1985b) Analysis of promoter regions for the spinach chloroplast rbcL, atpB and psbA genes. EMBO J 4:3375-3383 Hageman J, Robinson C, Smeekens S and Weisbeek P (1986) A thylakoid processing protease is required for complete maturation of the lumen protein plastocyanin. Nature 324:567-569 Hanley-Bowdoin L and Chua N-H (1987) Chloroplast promoters. Trends Biochem Sci 12: 67-70 Hanley-Bowdoin L, Orozco EM and Chua N-H (1985) In vitro synthesis and processing of a maize chloroplast transcript encoded by the ribulose 1,5-bisphosphate carboxylase large subunit gene. Mol Cell Biol 5:2733-2745 Hawley DK and McClure WR (1983) Compilation and analysis of Escherichia coli promoter DNA sequences. Nucl Acids Res 11:2237-2255 Howe CJ, Bowman CM, Dyer TA and Gray JC (1982) Localization of wheat chloroplast genes for the beta and epsilon subunits of ATP synthase. Mol Gen Genet 186" 525-530 Kirwin PM, Elderfield PD and Robinson C (1987) Transport of proteins into chloroplasts. Partial purification of a thylakoidal processing peptidase involved in plastocyanin biogenesis. J Biol Chem 262:16386-16390 Kong XF, Lovett PS and Kung SD (1984) The Nicotiana chloroplast genome IX. Identification of regions active as prokaryotic promoters in Escherichia coli. Gene 31 : 23-30 Link G (1984) DNA sequence requirements for the accurate transcription of a protein-coding plastid gene in a plastid in vitro system from mustard (Sinapis alba L.). EMBO J 3: 1697-1704 Oliver DB and Beckwith J (1981) E. coli mutant pleiotropically defective in the export of secreted proteins. Cell 25:765-772 Rochaix JD (1985) Genetic organization of the chloroplast. Int Rev Cytol 93:57-91 Rosenberg M and Court D (1979) Regulatory sequences involved in the promotion and termination of RNA transcription. Ann Rev Gen 13:319-353 Rothstein SJ, Gatenby AA, Willey DL and Gray JC (1985) Binding of pea cytochromefto the inner membrane of Escherichia coli requires the bacterial secA gene product. Proc Natl Acad Sci USA 82:7955-7959 Schmidt GW, Bartlett SG, Grossman AR, Cashmore AR and Chua N-H (198 l) Biosynthetic pathways of two polypeptide subunits of the light-harvesting chlorophyll a/b protein complex. J Cell Biol 91:468-478 Schmidt GW and Mishkind M L (1986) The transport of proteins into chloroplasts. Ann Rev Biochem 55:879-912 Shinozaki K and Sugiura M (1982) Sequence of the intercistronic region between the ribulose1,5-bisphosphate carboxylase/oxygenase large subunit and the coupling factor fl subunit gene. Nucl Acid Res 10:4923-4934 Silhavy TJ, Benson SA and Emr SD (1983) Mechanisms of protein localization. Micro Biol Rev 47:313-344 Tohdoh N, Shinozaki K and Sugiura M (1981) Sequence of a putative promoter region for the rRNA genes of tobacco chloroplast DNA. Nucl Acids Res 9:5399-5406 Whitfeld PR and Bottomley W (1983) Organization and structure of chloroplast genes. Ann Rev Plant Physiol 34:279-310 Willey DL, Huttly AK, Phillips AL and Gray JC (1983) Localization of the gene for cytochrome f i n pea chloroplast DNA. Mol Gen Genet 189:85-89

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Using bacteria to analyze sequences involved in chloroplast gene expression.

The expression of higher plant chloroplast genes in prokaryotic cells has been used to examine organelle sequences involved in promoter recognition by...
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