Synthetic analogues of a transit peptide inhibit binding or translocation of chloroplastic precursor proteins

Although amino-terminal transit peptides of chloroplastic precursor proteins are known to be necessary and sufficient for import into chloroplasts, the mechanism by which they mediate this process is not understood. Another important question is whether different precursors share a common transport apparatus. We used 20-residue synthetic peptides corresponding to regions of the transit peptide of the precursor to the small subunit of ribulose bisphosphate carboxylase (prSS) as competitive inhibitors for the binding and translocation of precursor proteins into chloroplasts. Synthetic peptides with sequences corresponding to either end of the transit peptide had little to no effect on binding of prSS to chloroplasts, but significantly inhibited its translocation. Synthetic peptides corresponding to the central region of the transit peptide inhibited binding of prSS to chloroplasts. Each of the peptides inhibited binding or translocation of precursors to light-harvesting chlorophyll a/b protein, ferredoxin, and plastocyanin in the same manner and to a similar extent as prSS transport was inhibited. The results presented in this paper suggest that the central regions of the transit peptide of prSS mediate binding to the chloroplastic surface, whereas the ends of this transit peptide are more important for translocation across the envelope. Furthermore, all of the precursors tested appear to share components in the transport apparatus even though they are sorted to different chloroplastic compartments.     

Domains of a Transit Sequence Required for in Vivo Import in Arabidopsis Chloroplasts

Nuclear-encoded precursors of chloroplast proteins are synthesized with an amino-terminal cleavable transit sequence, which contains the information for chloroplastic targeting. To determine which regions of the transit sequence are most important for its function, the chloroplast uptake and processing of a full-length ferredoxin precursor and four mutants with deletions in adjacent regions of the transit sequence were analyzed. Arabidopsis was used as an experimental system for both in vitro and in vivo import. The full-length wild-type precursor translocated efficiently into isolated Arabidopsis chloroplasts, and upon expression in transgenic Arabidopsis plants only mature-sized protein was detected, which was localized inside the chloroplast. None of the deletion mutants was imported in vitro. By analyzing transgenic plants, more subtle effects on import were observed. The most N-terminal deletion resulted in a fully defective transit sequence. Two deletions in the middle region of the transit sequence allowed translocation into the chloroplast, although with reduced efficiencies. One deletion in this region strongly reduced mature protein accumulation in older plants. The most C-terminal deletion was translocated but resulted in defective processing. These results allow the dissection of the transit sequence into separate functional regions and give an in vivo basis for a domain-like structure of the ferredoxin transit sequence.     

Mutation of a neutral amino acid in the transit peptide of rat mitochondrial malate dehydrogenase abolishes binding and import

We have investigated the function of a leucine residue in the transit peptide of the rat mitochondrial malate dehydrogenase precursor using in vitro mutagenesis. Amino acid replacement of leucine 13 with glutamic acid and asparagine abolished import into mitochondria, while substitutions with proline, histidine, and arginine severely diminished uptake. In contrast, glutamine, tyrosine, valine, and alanine replacement resulted in normal levels of import, suggesting that there is a requirement for an uncharged residue at this position. Mutants involving rearrangements of the native sequence at positions 12-14 were imported as efficiently as the wild-type mitochondrial malate dehydrogenase, indicating that there was not an obligatory order of amino acid residues. However, deletion of leucine 13 resulted in diminished import. Binding studies with isolated mitochondria revealed that several position 13 mutants were deficient in binding to the mitochondrial surface, accounting for the reduced import of these proteins. This impairment could be distinguished from the effects due to decreased positive charge. We conclude that while translocation depends on the net positive charge, binding to the mitochondrial surface is mediated by uncharged residues within the transit peptides of mitochondrial precursor proteins.     

The Arabidopsis thaliana genome encodes at least four thioredoxins m and a new prokaryotic-like thioredoxin

Screening of cDNA libraries at low stringency and complete sequencing of EST clones with homology to thioredoxins allowed us to characterize five new prokaryotic type Arabidopsis thaliana thioredoxins. All present N-terminal extensions with characteristics of transit peptides. Four are clustered in a phylogenetic tree with the chloroplastic thioredoxin m from red and green algae and higher plants, and their transit peptides have typical characteristics of chloroplastic transit peptides. One is clearly divergent and defines a new prokaryotic thioredoxin type that we have named thioredoxin x. Its transit peptide sequence presents characteristics of both chloroplastic and mitochondrial transit peptides. The five corresponding genes are expressed at different levels, but mostly in green tissues and in in-vitro cultivated cells.     

The Arabidopsis thaliana genome encodes at least four thioredoxins m and a new prokaryotic-like thioredoxin

Screening of cDNA libraries at low stringency and complete sequencing of EST clones with homology to thioredoxins allowed us to characterize five new prokaryotic type Arabidopsis thaliana thioredoxins. All present N-terminal extensions with characteristics of transit peptides. Four are clustered in a phylogenetic tree with the chloroplastic thioredoxin m from red and green algae and higher plants, and their transit peptides have typical characteristics of chloroplastic transit peptides. One is clearly divergent and defines a new prokaryotic thioredoxin type that we have named thioredoxin x. Its transit peptide sequence presents characteristics of both chloroplastic and mitochondrial transit peptides. The five corresponding genes are expressed at different levels, but mostly in green tissues and in in-vitro cultivated cells.     

Partitioning of malate dehydrogenase isoenzymes into glyoxysomes, mitochondria, and chloroplasts

Malate dehydrogenase isoenzymes catalyzing the oxidation of malate to oxaloacetate are highly active enzymes in mitochondria, in peroxisomes, in chloroplasts, and in the cytosol. Determination of the primary structure of the isoenzymes has disclosed that they are encoded in different nuclear genes. All three organelle-targeted malate dehydrogenases are synthesized with an amino terminal extension that is cleaved off in connection with the import of the enzyme precursor into the organelle. The sequence of the 27 amino acids of the mitochondrial transit peptide is unrelated to the 37-residue glyoxysomal transit peptide, which in turn is entirely different in sequence from the 57-residue chloroplastic transit peptide. With the exception of malate dehydrogenase and 3-ketoacyl thiolase, peroxisomal enzymes are synthesized without transit peptides and are frequently translocated into the organelle with a peroxisomal targeting signal consisting of a conserved tripeptide at the carboxy terminus of the protein. Based on the observation that this tripeptide (Ala-His-Leu) occurs in the transit peptides of glyoxysomal malate dehydrogenase and peroxisomal 3-ketoacyl thiolase, the possible significance of amino terminal transit peptides for peroxisome import is discussed.     

Characterization of a novel zinc metalloprotease involved in degrading targeting peptides in mitochondria and chloroplasts

We have recently isolated and identified a novel mitochondrial metalloprotease, pre-sequence protease (PreP) from potato and shown that it degrades mitochondrial pre-sequences. PreP belongs to the pitrilysin protease family and contains an inverted zinc-binding motif. To further investigate the degradation of targeting peptides, we have overexpressed the Arabidopsis thaliana homologue of PreP, zinc metalloprotease (Zn-MP), in Escherichia coli . We have characterized the recombinant Zn-MP with respect to its catalytic site, substrate specificity and intracellular localization. Mutagenesis studies of the residues involved in metal binding identified the histidines and the proximal glutamate as essential residues for the proteolytic activity. Substrate specificity studies showed that the Zn-MP has the ability to degrade both mitochondrial pre-sequences and chloroplastic transit peptides, as well as other unstructured peptides. The Zn-MP does not recognize an amino acid sequence per se . Immunological studies and proteolytic activity measurements in isolated mitochondria and chloroplasts revealed the presence of the Zn-MP in both organelles. Furthermore, the Zn-MP was found to be dually imported to both mitochondria and chloroplasts in vitro . In summary, our data show that the Zn-MP is present and serves the same function in chloroplasts as in mitochondria – degradation of targeting peptides.     

Processing, targeting and import into the mitochondria and peroxisomes of proteins coded by nuclear genes]

I have herein discussed two gene-enzyme families in maize whose protein products participate to purge toxic oxidants from cells, and are thus of importance to all aerobic organisms. We have demonstrated that plant mitochondria import precursor proteins (i.e., preSOD-3) in a manner analogous to other eukaryotic cells. The "transit peptide" (TP) of preSOD-3 is 31 amino acid long and has similar properties to other reported TPs for mitochondrial and chloroplastic proteins. Import to peroxisomes is uniquely different from that for mitochondria and chloroplasts in that no consensus presequence seems to be involved. Instead, targeting signals seem to be integral parts of peroxisomal proteins.     

Protein oxidation in the leaves and roots of cucumber plants (Cucumis sativus L.), mutant MSC16 and wild type

Reactive oxygen species (ROS) may cause irreversible carbonylation of proteins, resulting in structural and/or functional modifications. Carbonylated proteins were analyzed and compared in tissue extracts or purified mitochondria isolated from the leaves and roots of wild-type (WT) or MSC16 mutant cucumber plants. For analysis of the oxidized protein formation and degradation, several techniques were applied: Western blotting, quantitative, spectrophotometric assay of carbonyl concentration and protease activity measurements. Oxidized proteins were tagged with 2,4-dinitrophenylhydrazine (DNPH) and detected with anti-DNP antibodies. Western blots of 1D gels indicated that, in the leaves of both WT and MSC16 plants, certain oxidized proteins have chloroplastic origin. In MSC16 plants, protein oxidation is probably higher in chloroplasts than in mitochondria. Carbonyl concentration is similar in MSC16 and WT leaf extracts, but this may be the result of twice as high protease activity observed in MSC16 leaf extracts and indicates that chloroplastic proteases may effectively remove the oxidized proteins from chloroplasts. In mitochondria of both WT and MSC16 leaves, the levels of oxidized proteins and protease activity are similar. In MSC16 root extracts, the carbonyl concentration is lower and protease activity is similar as compared to WT plants. Nevertheless, in MSC16 root mitochondria, the 30% lower carbonyl concentration, lower band abundance for oxidized proteins and over 50% higher protease activity indicate that mitochondrial proteases are involved in degradation of the oxidatively damaged proteins. In matrix and membrane subfractions, the levels of oxidized proteins are similar in leaf mitochondria or lower in root mitochondria from MSC16 as compared to WT plants. The results show that the oxidized protein degradation network in MSC16 cucumber mutants is well developed, thus becoming a survival factor for plants with mitochondrial dysfunctions.     

Current views on chloroplast protein import and hypotheses on the origin of the transport mechanism

Most chloroplastic proteins are synthesized as precursors in the cytosol prior to their transport into chloroplasts. These precursors are generally synthesized in a form that is larger than the mature form found inside chloroplasts. The extra amino acids, called transit peptides, are present at the amino terminus. The transit peptide is necessary and sufficient to recognize the chloroplast and induce movement of the attached protein across the envelope membranes. In this review, we discuss the primary and secondary structure of transit peptides, describe what is known about the import process, and present some hypotheses on the evolutionary origin of the import mechanism.Abbreviations DHFR dihydrofolate reductase - EPSP synthase 5-enolpyrovylshikimate-3-phosphate synthase; hsp heat-shock protein - LHCP II light-harvesting chlorophylla/b binding protein - OEE 16, 23, and 33 the 16-, 23-, and 33-kDa proteins of the oxygen-evolving complex - pr precursor - rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - SS rubisco small subunit     

Import into chloroplasts of a yeast mitochondrial protein directed by ferredoxin and plastocyanin transit peptides

Many chloroplast proteins are synthesized in the cytoplasm as precursors which contain an amino terminal transit peptide. These precursors are subsequently imported into chloroplast and targeted to one of several organellar locations. This import is mediated by the transit peptide, which is cleaved off during import. We have used the transit peptides of ferredoxin (chloroplast stroma) and plastocyanin (thylakoid lumen) to study chloroplast protein import and intra-organellar routing toward different compartments. Chimeric genes were constructed that encode precursor proteins in which the transit peptides are linked to yeast mitochondrial manganese superoxide dismutase. Chloroplast protein import and localization experiments show that both chimeric proteins are imported into the chloroplast stroma and processed. The plastocyanin transit sequence did not direct superoxide dismutase to the thylakoids; this protein was found in the stroma as an intermediate that still contains part of the plastocyanin transit peptide. The organelle specificity of these chimeric precursors reflected the transit peptide parts of the molecules, because neither the ferredoxin and plastocyanin precursors nor the chimeric proteins were imported into isolated yeast mitochondria.     

Identification of a Hsp70 recognition domain within the rubisco small subunit transit peptide

The interaction between SStp, the transit peptide of the precursor protein to the small subunit of Rubisco (prSSU) and two Hsp70 molecular chaperones, Escherichia coli DnaK and pea (Pisum sativum) CSS1, was investigated in detail. Two statistical analyses were developed and used to investigate and predict regions of SStp recognized by DnaK. Both algorithms suggested that DnaK would have high affinity for the N terminus of SStp, moderate affinity for the central region, and low affinity for the C terminus. Furthermore, both algorithms predicted this affinity pattern for >75% of the transit peptides analyzed in the chloroplast transit peptide (CHLPEP) database. In vitro association between SStp and these Hsp70s was confirmed by three independent assays: limited trypsin resistance, ATPase stimulation, and native gel shift. Finally, synthetic peptides scanning the length of SStp and C-terminal deletion mutants of SStp were used to experimentally map the region of greatest DnaK affinity to the N terminus. CSS1 displayed a similar affinity for the N terminus of SStp. The major stromal Hsp70s affinity for the N terminus of SStp and other transit peptides supports a molecular motor model in which the chaperone functions as an ATP-dependent translocase, committing chloroplast precursor proteins to unidirectional movement across the envelope.     

A novel, bipartite transit peptide targets OEP75 to the outer membrane of the chloroplastic envelope.

OEP75 is an outer envelope membrane component of the chloroplastic protein import apparatus and is synthesized in the cytoplasm as a higher molecular weight precursor (prOEP75). During its own import, prOEP75 is processed first to an intermediate (iOEP75) and subsequently to the mature form (mOEP75). Experiments conducted with stromal extracts indicated that iOEP75 was generated from prOEP75 by the activity of the stromal processing peptidase. The specific processing site was determined and used to divide the prOEP75 transit peptide into N- and C-terminal domains. To determine the targeting functions of the two domains of the transit peptide and of the mature region of prOEP75, we created a deletion mutant construct from prOEP75 and chimeric constructs between domains of prOEP75 and the precursor to a small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Analysis of these constructs by in vitro chloroplastic protein import assays revealed that the transit peptide of prOEP75 is bipartite in that the N- and C-terminal portions contain chloroplastic and intraorganellar targeting information, respectively.     

Functional characterization of sequence motifs in the transit peptide of Arabidopsis small subunit of rubisco

The transit peptides of nuclear-encoded chloroplast proteins are necessary and sufficient for targeting and import of proteins into chloroplasts. However, the sequence information encoded by transit peptides is not fully understood. In this study, we investigated sequence motifs in the transit peptide of the small subunit of the Rubisco complex by examining the ability of various mutant transit peptides to target green fluorescent protein reporter proteins to chloroplasts in Arabidopsis (Arabidopsis thaliana) leaf protoplasts. We divided the transit peptide into eight blocks (T1 through T8), each consisting of eight or 10 amino acids, and generated mutants that had alanine (Ala) substitutions or deletions, of one or two T blocks in the transit peptide. In addition, we generated mutants that had the original sequence partially restored in single- or double-T-block Ala (A) substitution mutants. Analysis of chloroplast import of these mutants revealed several interesting observations. Single-T-block mutations did not noticeably affect targeting efficiency, except in T1 and T4 mutations. However, double-T mutants, T2A/T4A, T3A/T6A, T3A/T7A, T4A/T6A, and T4A/T7A, caused a 50% to 100% loss in targeting ability. T3A/T6A and T4A/T6A mutants produced only precursor proteins, whereas T2A/T4A and T4A/T7A mutants produced only a 37-kD protein. Detailed analyses revealed that sequence motifs ML in T1, LKSSA in T3, FP and RK in T4, CMQVW in T6, and KKFET in T7 play important roles in chloroplast targeting. In T1, the hydrophobicity of ML is important for targeting. LKSSA in T3 is functionally equivalent to CMQVW in T6 and KKFET in T7. Furthermore, subcellular fractionation revealed that Ala substitution in T1, T3, and T6 produced soluble precursors, whereas Ala substitution in T4 and T7 produced intermediates that were tightly associated with membranes. These results demonstrate that the transit peptide contains multiple motifs and that some of them act in concert or synergistically.     

How can organellar protein N-terminal sequences be dual targeting signals? In silico analysis and mutagenesis approach

Organellar nuclear-encoded proteins can be mitochondrial, chloroplastic or localized in both mitochondria and chloroplasts. Most of the determinants for organellar targeting are localized in the N-terminal part of the proteins, which were therefore analyzed in Arabidopsis thaliana. The mitochondrial, chloroplastic and dual N-terminal sequences have an overall similar composition. However, Arg is rare in the first 20 residues of chloroplastic and dual sequences, and Ala is more frequent at position 2 of these two types of sequence as compared to mitochondrial sequences. According to these observations, mutations were performed in three dual targeted proteins and analyzed by in vitro import into isolated mitochondria and chloroplasts. First, experiments performed with wild-type proteins suggest that the binding of precursor proteins to mitochondria is highly efficient, whereas the import and processing steps are more efficient in chloroplasts. Moreover, different processing sites are recognized by the mitochondrial and chloroplastic processing peptidases. Second, the mutagenesis approach shows the positive role of Arg residues for enhancing mitochondrial import or processing, as expected by the in silico analysis. By contrast, mutations at position 2 have dramatic and unpredicted effects, either enhancing or completely abolishing import. This suggests that the nature of the second amino acid residue of the N-terminal sequence is essential for the import of dual targeted sequences.     

The chloroplast protein import channel Toc75: pore properties and interaction with transit peptides

The channel properties of Toc75 (the protein import pore of the outer chloroplastic membrane) were further characterized by electrophysiological measurements in planar lipid bilayers. After improvement of the Toc75 reconstitution procedure the voltage dependence of the channel open probability resembled those observed for other beta-barrel pores. Studies concerning the pore size of the reconstituted Toc75 indicate the presence of a narrow restriction zone corresponding to the selectivity filter and a wider pore vestibule with diameters of approximately 14 A and 26 A, respectively. Interactions between Toc75 and different peptides (a genuine chloroplastic transit peptide, a synthetic peptide resembling a transit peptide, and a mitochondrial presequence) show that Toc75 itself is able to differentiate between these peptides and the recognition is based on both conformational and electrostatic interactions.     

Plant aldolase: cDNA and deduced amino-acid sequences of the chloroplast and cytosol enzyme from spinach

We report the sequences of full-length cDNAs for the nuclear genes encoding the chloroplastic and cytosolic fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) from spinach. A comparison of the deduced amino-acid sequences with one another and with published cytosolic aldolase sequences of other plants revealed that the two enzymes from spinach share only 54% homology on their amino acid level whereas the homology of the cytosolic enzyme of spinach with the known sequences of cytosolic aldolases of maize, rice and Arabidopsis range from 67 to 92%. The sequence of the chloroplastic enzyme includes a stroma-targeting N-terminal transit peptide of 46 amino acid residues for import into the chloroplast. The transit peptide exhibits essential features similar to other chloroplast transit peptides. Southern blot analysis implies that both spinach enzymes are encoded by single genes.     

A truncated analog of a pre-light-harvesting chlorophyll a/b protein II transit peptide inhibits protein import into chloroplasts

It is unclear how transit peptides target nuclear-encoded precursor proteins to the chloroplast. This study establishes the feasibility of using synthetic peptides as competitive inhibitors of chloroplast protein import and as probes for the function of domains within transit peptides. We show that peptide pL(1-20), MAASTMALSSPAFAGKAVNY, an analog of the NH2 terminus of a pre-light harvesting chlorophyll a/b protein II from Arabidopsis, inhibits the import of several Arabidopsis and pea precursor proteins into pea chloroplasts. Inhibition occurs at a step between the initial binding of precursors to the chloroplast and the first proteolytic cleavage event and is not due to interference with ATP availability or chloroplast integrity. Presumably this reflects specific binding of the peptide to the import machinery in the chloroplast envelope. Our data are consistent with the suggestion (Karlin-Neumann, G. A., and Tobin, E. M. (1986) EMBO J. 5, 9-13) that two conserved blocks of amino acids near the NH2-terminus of transit peptides (spanned by peptide pL(1-20] participate in protein targeting. Computer analysis also shows peptide pL(1-20) lacks the amphiphilic properties characteristic of pre-sequences of many nuclear-encoded mitochondrial proteins. This shows a difference in the mechanisms for targeting proteins to chloroplasts and mitochondria.