Cytometric analysis of an epitope-tagged transit peptide bound to the chloroplast translocation apparatus

Chloroplast transit peptides are necessary and sufficient for the targeting and translocation of precursor proteins across the chloroplast envelope. However, the mechanism by which transit peptides engage the translocation apparatus has not been investigated. To analyse this interaction, we have developed a novel epitope-tagged transit peptide derived from the precursor of the small subunit of pea Rubisco. The recombinant transit peptide, His-S-SStp, contains a removable dual-epitope tag, His-S, at its N-terminus that permits both rapid purification via immobilized metal affinity chromatography and detection by blotting, flow cytometry and laser-scanning confocal microscopy. Unlike other chimeric precursors, which place the passenger protein C-terminal to the transit peptide, His-S-SStp bound to the translocation apparatus yet did not translocate across the chloroplast envelope. This early translocation intermediate allowed non-radioactive detection using fluorescent and chemiluminescent reporters. The physiological relevance of this interaction was confirmed by protein import competitions, sensitivity to pre- and post-import thermolysin treatment, photochemical cross-linking and organelle fractionation. The interaction was specific for the transit peptide since His-S alone did not engage the chloroplast translocation apparatus. Quantitation of the bound transit peptide was determined by flow cytometry, showing saturation of binding yet only slight ATP-dependence. The addition of GTP showed inhibition of the binding of His-S-SStp to the chloroplasts indicating an involvement of GTP in the formation of this early translocation intermediate. In addition, direct visualization of His-S-SStp and Toc75 by confocal microscopy revealed a patch-like labeling, suggesting a co-ordinate localization to discrete regions on the chloroplast envelope. These findings represent the first direct visualization of a transit peptide interacting with the chloroplast translocation apparatus. Furthermore, identification of a chloroplast-binding intermediate may provide a novel tool to dissect interactions between a transit peptide and the chloroplast translocation apparatus.     

Targeting of proteins to the thylakoid lumen by the bipartite transit peptide of the 33 kd oxygen-evolving protein.

Various chimeric precursors and deletions of the 33 kd oxygen-evolving protein (OEE1) were constructed to study the mechanism by which chloroplast proteins are imported and targeted to the thylakoid lumen. The native OEE1 precursor was imported into isolated chloroplasts, processed and localized in the thylakoid lumen. Replacement of the OEE1 transit peptide with the transit peptide of the small subunit of ribulose-1,5-bisphosphate carboxylase, a stromal protein, resulted in redirection of mature OEE1 into the stromal compartment of the chloroplast. Utilizing chimeric transit peptides and block deletions we demonstrated that the 85 residue OEE1 transit peptide contains separate signal domains for importing and targeting the thylakoid lumen. The importing domain, which mediates translocation across the two membranes of the chloroplast envelope, is present in the N-terminal 58 amino acids. The thylakoid lumen targeting domain, which mediates translocation across the thylakoid membrane, is located within the C-terminal 27 residues of the OEE1 transit peptide. Chimeric precursors were constructed and used in in vitro import experiments to demonstrate that the OEE1 transit peptide is capable of importing and targeting foreign proteins to the thylakoid lumen.     

In vivo import experiments in protoplasts reveal the importance of the overall context but not specific amino acid residues of the transit peptide during import into chloroplasts

The N-terminal transit peptide of chloroplast proteins is necessary and sufficient to direct proteins to the chloroplasts. However, the requirement of the transit peptide of chloroplast proteins is not fully understood. In this study we investigated the requirement of a transit peptide at the level of amino acid sequence using an in vivo targeting approach. Targeting experiments with green fluorescent protein (GFP) fusion proteins containing varying lengths of the N-terminal region of the small subunit of rubisco complex (RbcS) revealed that at least 73 amino acid residues of the N-terminal region is required to direct GFP to the chloroplasts without affecting the efficiency. Even a small deletion from the C- or N-termini of the minimal length of the transit peptide results in strong inhibition of targeting. Also, a small internal deletion within the minimal transit peptide strongly affected targeting of GFP fusion proteins. However, when we replaced one or two amino acid residues of the transit peptide with corresponding numbers of alanine residues sequentially, all the mutants were imported into chloroplasts with 80 to 100% efficiency. Together these results suggest that the overall context of amino acid sequence, but not any specific amino acid residue, of the transit peptide is critical for targeting to the chloroplasts.     

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.     

Chloroplast transit peptides: structure, function and evolution

It is thought that two to three thousand different proteins are targeted to the chloroplast, and the 'transit peptides' that act as chloroplast targeting sequences are probably the largest class of targeting sequences in plants. At a primary structural level, transit peptide sequences are highly divergent in length, composition and organization. An emerging concept suggests that transit peptides contain multiple domains that provide either distinct or overlapping functions. These functions include direct interaction with envelope lipids, chloroplast receptors and the stromal processing peptidase. The genomic organization of transit peptides suggests that these domains might have originated from distinct exons, which were shuffled and streamlined throughout evolution to yield a modern, multifunctional transit peptide. Although still poorly characterized, this evolutionary process could yield transit peptides with different domain organizations. The plasticity of transit peptide design is consistent with the diverse biological functions of chloroplast proteins.     

The C terminus of a chloroplast precursor modulates its interaction with the translocation apparatus and PIRAC.

The import of proteins into chloroplasts involves a cleavable, N-terminal targeting sequence known as the transit peptide. Although the transit peptide is both necessary and sufficient to direct precursor import into chloroplasts, the mature domain of some precursors has been shown to modulate targeting and translocation efficiency. To test the influence of the mature domain of the small subunit of Rubisco during import in vitro, the precursor (prSSU), the mature domain (mSSU), the transit peptide (SS-tp), and three C-terminal deletion mutants (Delta52, Delta67, and Delta74) of prSSU were expressed and purified from Escherichia coli. Activity was then evaluated by competitive import of (35)S-prSSU. Both IC(50) and K(i) values consistently suggest that removal of C-terminal prSSU sequences inhibits its interaction with the translocation apparatus. Non-competitive import studies demonstrated that prSSU and Delta52 were properly processed and accumulated within the chloroplast, whereas Delta67 and Delta74 were rapidly degraded via a plastid-localized protease. The ability of prSSU-derived proteins to induce inactivation of the protein-import-related anion channel was also evaluated. Although the C-terminal deletion mutants were less effective at inducing channel closure upon import, they did not effect the mean duration of channel closure. Possible mechanisms by which C-terminal residues of prSSU modulate chloroplast targeting are discussed.     

Chloroplast Import Signals: The Length Requirement for Translocation In Vitro and In Vivo

Protein translocation of cytosolically synthesized proteins requires signals for both targeting of precursor proteins to the surface of the respective compartment and their transfer across its membrane. In contrast to signals for peroxisomal and endoplasmic reticulum translocation, the signals for mitochondrial and chloroplast transport are less well defined with respect to length and amino acid requirements. To study the properties of signals required for translocation into chloroplasts in vitro and in vivo, we used fusion proteins composed of transit peptides and the Ig-like module of the muscle protein titin as passenger. We observed that about 60 amino acids—longer than the transit peptide length of many experimentally confirmed chloroplast proteins—are required for efficient translocation. However, within native chloroplast precursor proteins with transit peptides shorter than 60 amino acids, extension appears to be present as they are efficiently imported into organelles. In addition, the interaction of an unfolded polypeptide stretch of 60 or more amino acids with receptors at the chloroplast surface results in the unidirectionality of protein translocation into chloroplasts even in the presence of a competing C-terminal peroxisomal targeting signal. These findings prove the existing ideas that initial targeting is defined by the N-terminal signal and that the C-terminal signal is sensed only subsequently.     

Arabidopsis nuclear-encoded plastid transit peptides contain multiple sequence subgroups with distinctive chloroplast-targeting sequence motifs

The N-terminal transit peptides of nuclear-encoded plastid proteins are necessary and sufficient for their import into plastids, but the information encoded by these transit peptides remains elusive, as they have a high sequence diversity and lack consensus sequences or common sequence motifs. Here, we investigated the sequence information contained in transit peptides. Hierarchical clustering on transit peptides of 208 plastid proteins showed that the transit peptide sequences are grouped to multiple sequence subgroups. We selected representative proteins from seven of these multiple subgroups and confirmed that their transit peptide sequences are highly dissimilar. Protein import experiments revealed that each protein contained transit peptide-specific sequence motifs critical for protein import into chloroplasts. Bioinformatics analysis identified sequence motifs that were conserved among members of the identified subgroups. The sequence motifs identified by the two independent approaches were nearly identical or significantly overlapped. Furthermore, the accuracy of predicting a chloroplast protein was greatly increased by grouping the transit peptides into multiple sequence subgroups. Based on these data, we propose that the transit peptides are composed of multiple sequence subgroups that contain distinctive sequence motifs for chloroplast targeting.     

Amino-terminal and hydrophobic regions of the Chlamydomonas reinhardtii plastocyanin transit peptide are required for efficient protein accumulation in vivo

Nucleus-encoded chloroplast proteins of vascular plants are synthesized as precursors and targeted to the chloroplast by stroma-targeting domains in N-terminal transit peptides. Transit peptides in Chlamydomonas reinhardtii are considerably shorter than those in vascular plants, and their stroma-targeting domains have similarities to both mitochondrial and chloroplast targeting sequences. To examine Chlamydomonas transit peptide function in vivo, deletions were introduced into the transit peptide coding region of the petE gene, which encodes the thylakoid lumen protein plastocyanin (PC). The mutant petE genes were introduced into a plastocyanin-deficient Chlamydomonas strain, and transformants that accumulated petE mRNA were analyzed for PC accumulation. The most profound defects were observed with deletions at the N-terminus and those that extended into the hydrophobic region in the C-terminal half of the transit peptide. PC precursors were detected among pulse-labeled proteins in transformants with N-terminal deletions, suggesting that these precursors cannot be imported and are degraded in the cytosol. Intermediate PC species were observed in a transformant deleted for part of the hydrophobic region, suggesting that this protein is defective in lumen translocation and/or processing. Thus, despite its shorter length, the bipartite nature of the Chlamydomonas PC transit peptide appears similar to that of lumen-targeted proteins in vascular plants. Analysis of the synthesis, stability, and accumulation of PC species in transformants bearing deletions in the stroma-targeting domain suggests that specific regions probably have distinct roles in vivo. Abbreviations: cyt, cytochrome; ECL, enhanced chemiluminescence; LSU, large subunit; PC, plastocyanin; TP, transit peptide     

Analysis of chloroplast transit peptide function using mutations in the carboxyl-terminal region

Protein import into chloroplasts requires a transit peptide, which interacts with the chloroplast transport apparatus and leads to translocation of the protein across the chloroplast envelope. While the amino acid sequences of many transit peptides are known, functional domains have been difficult to identify. Previous studies suggest that the carboxyl terminus of the transit peptide for ribulose bisphosphate carboxylase small subunit is important for both translocation across the chloroplast envelope and proper processing of the precursor protein. We dissected this region using in vitro mutagenesis, creating a set of mutants with small changes in primary structure predicted to cause alterations in secondary structure. The import behavior of the mutant proteins was assessed using isolated chloroplasts. Our results show that removal of a conserved arginine residue in this region results in impaired processing, but does not necessarily affect import rates. In contrast, substituting amino acids with low reverse turn or amphiphilic potential for other original residues affected import rate but not processing.     

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.     

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.     

Mechanism of protein import across the chloroplast envelope

The development and maintenance of chloroplasts relies on the contribution of protein subunits from both plastid and nuclear genomes. Most chloroplast proteins are encoded by nuclear genes and are post-translationally imported into the organelle across the double membrane of the chloroplast envelope. Protein import into the chloroplast consists of two essential elements: the specific recognition of the targeting signals (transit sequences) of cytoplasmic preproteins by receptors at the outer envelope membrane and the subsequent translocation of preproteins simultaneously across the double membrane of the envelope. These processes are mediated via the co-ordinate action of protein translocon complexes in the outer (Toc apparatus) and inner (Tic apparatus) envelope membranes.     

Carboxyl-terminal sequences can influence the in vitro import and intraorganellar targeting of chloroplast protein precursors.

The transit peptide of the lumenal 33-kDa oxygen-evolving polypeptide (OEE1) is capable of directing the import and targeting of the foreign protein dihydrofolate reductase (DHFR) to the thylakoid lumen. The import results from the first part of this study indicate that methotrexate cannot block the import or intraorganellar targeting of OEE1-DHFR in chloroplasts in contrast to that reported for the import of cytochrome oxidase subunit IV (COXIV)-DHFR in mitochondria. These results suggest that the fusion of the OEE1 transit sequence to DHFR affected the protein's methotrexate binding properties. We further examined and compared the transport characteristics of a number of carboxyl-terminal truncated native chloroplast precursors to determine whether carboxyl domains contribute to the import and intraorganellar targeting mechanism of these proteins. The plastid precursors chosen for this study are targeted to one of the following chloroplast compartments: the stroma, the thylakoid membrane, and the lumen. In most cases, removal of carboxyl domains had a dramatic effect on one or more stages of the translocation pathway, such as import, processing, and intraorganellar targeting. The effects of carboxyl deletions varied from precursor to precursor and were dependent on the extent of the deletion. These combined results suggest that carboxyl domains in the mature part of the proteins can influence the function of the transit peptide, and as a result play an important role in determining the import and targeting competence of chloroplast precursors.     

Protein targeting towards the thylakoid lumen of chloroplasts: proper localization of fusion proteins is only observed in vivo.

Routeing of fusion proteins to the thylakoid lumen of the chloroplast was compared in vitro and in vivo. The Escherichia coli protein beta-lactamase was used as a passenger to study this intraorganellar sorting process. The first step, translocation of beta-lactamase into the chloroplast stroma, occurs properly both in vitro and in vivo and is dependent on the presence of a transit peptide in the protein construct. The second step, targeting towards the thylakoid lumen, is more complicated as was also observed previously when other passenger proteins were used. In vitro, the presence of a thylakoid transfer domain is not enough for routeing and proper processing. Only when the complete thylakoid lumen precursor plastocyanin was fused to beta-lactamase was the fusion protein processed adequately, but routeing was still incomplete. However, in vivo, the information present in the thylakoid transfer domain was the only requirement for proper transport towards the thylakoid lumen. These data show that in vivo, the only requirement for targeting of passenger proteins towards the thylakoid lumen is the presence of a transit peptide and a thylakoid transfer domain. Furthermore, we demonstrate that the in vitro import system does not necessarily reflect the in vivo situation with respect to intraorganellar sorting.     

Protein trafficking to the plastid of Plasmodium falciparum is via the secretory pathway

The plastid of Plasmodium falciparum (or 'apicoplast') is the evolutionary homolog of the plant chloroplast and represents a vestige of a photosynthetic past. Apicoplast indispensability indicates that it still provides essential functions to parasites. Similar to plant chloroplasts, the apicoplast is dependent on many nucleus-encoded genes to provide these functions. The apicoplast is surrounded by four membranes, two more than plant chloroplasts. Thus, protein targeting to the apicoplast must overcome additional membrane barriers. In P.falciparum we have analyzed apicoplast targeting using green fluorescent protein (GFP). We demonstrate that protein targeting is at least a two-step process mediated by bipartite N-terminal pre-sequences that consist of a signal peptide for entry into the secretory pathway and a plant-like transit peptide for subsequent import into the apicoplast. The P.falciparum transit peptide is exceptional compared with other known plastid transit peptides in not requiring serine or threonine residues. The pre-sequence components are removed stepwise during apicoplast targeting. Targeting GFP to the apicoplast has also provided the first opportunity to examine apicoplast morphology in live P. falciparum.     

Sorting signals, N-terminal modifications and abundance of the chloroplast proteome

Characterization of the chloroplast proteome is needed to understand the essential contribution of the chloroplast to plant growth and development. Here we present a large scale analysis by nanoLC-Q-TOF and nanoLC-LTQ-Orbitrap mass spectrometry (MS) of ten independent chloroplast preparations from Arabidopsis thaliana which unambiguously identified 1325 proteins. Novel proteins include various kinases and putative nucleotide binding proteins. Based on repeated and independent MS based protein identifications requiring multiple matched peptide sequences, as well as literature, 916 nuclear-encoded proteins were assigned with high confidence to the plastid, of which 86% had a predicted chloroplast transit peptide (cTP). The protein abundance of soluble stromal proteins was calculated from normalized spectral counts from LTQ-Obitrap analysis and was found to cover four orders of magnitude. Comparison to gel-based quantification demonstrates that 'spectral counting' can provide large scale protein quantification for Arabidopsis. This quantitative information was used to determine possible biases for protein targeting prediction by TargetP and also to understand the significance of protein contaminants. The abundance data for 550 stromal proteins was used to understand abundance of metabolic pathways and chloroplast processes. We highlight the abundance of 48 stromal proteins involved in post-translational proteome homeostasis (including aminopeptidases, proteases, deformylases, chaperones, protein sorting components) and discuss the biological implications. N-terminal modifications were identified for a subset of nuclear- and chloroplast-encoded proteins and a novel N-terminal acetylation motif was discovered. Analysis of cTPs and their cleavage sites of Arabidopsis chloroplast proteins, as well as their predicted rice homologues, identified new species-dependent features, which will facilitate improved subcellular localization prediction. No evidence was found for suggested targeting via the secretory system. This study provides the most comprehensive chloroplast proteome analysis to date and an expanded Plant Proteome Database (PPDB) in which all MS data are projected on identified gene models.     

Transport of proteins into chloroplasts

The import of cytoplasmically synthesized proteins into chloroplasts involves an interaction between at least two components; the precursor protein, and the import apparatus in the chloroplast envelope membrane. This review summarizes the information available about each of these components. Precursor proteins consist of an amino terminal transit peptide attached to a passenger protein. Transit peptides from various precurosrs are diverse with respect to length and amino acid sequence; analysis of their sequences has not revealed insight into their mode of action. A variety of foreign passenger proteins can be imported into chloroplasts when a transit peptide is present at the amino terminus. However, foreign passenger proteins are not imported as efficiently as natural passenger proteins, and some chimeric precursor proteins are not imported into chloroplasts at all. Therefore, the passenger protein, as well as the transit peptide, influences the import process. Import begins by binding of the precursor to the chloroplast surface. It has been suggested that this binding is mediated by a receptor, but evidence to support this hypothesis remains incomplete and a receptor protein has not yet been characterized. Protein translocation requires energy derived from ATP hydrolysis, although there are conflicting reports as to where hydrolysis occurs and it is unclear how this energy is utilized. The mechanism(s) whereby proteins are translocated across either the two envelope membranes or the thylakoid membrane is not known.Abbreviations EPSP 5-enolpyruvyulshikimate-3-phosphate - LHCP Chlorophyll a/b binding protein of the light-harvesting complex - NPT-II Neomycin phosphotransferase II - PC Plastocyanin - Pr Precursor - Rubisco Ribulose-1,5,-bisphosphate carboxylase/oxygenase - SS Small subunit of Rubisco     

Expression, isolation, and characterization of a chloroplast targeting peptide

For the first time a method is described in which an N-terminal targeting peptide is isolated from Escherichia coli. After overexpression, purification, and cleavage of a fusion protein the protease-sensitive transit peptide from the chloroplast precursor protein preferredoxin could be isolated by HPLC. It was characterized by N-terminal amino acid sequencing and electrospray mass spectrometry. Its functionality was suggested by in vitro import competition experiments with isolated pea chloroplasts, in which the isolated peptide inhibited the import of radioactively labeled preferredoxin. Results from import competition experiments performed with a transit peptide deletion mutant suggested that the four extreme C-terminal amino acids lack information to interact with the chloroplast import machinery.     

Transit Peptide Mutations That Impair in Vitro and in Vivo Chloroplast Protein Import Do Not Affect Accumulation of the γ-Subunit of Chloroplast ATPase

We have begun to take a genetic approach to study chloroplast protein import in Chlamydomonas reinhardtii by creating deletions in the transit peptide of the γ-subunit of chloroplast ATPase-coupling factor 1 (CF₁-γ, encoded by AtpC) and testing their effects in vivo by transforming the altered genes into an atpC mutant, and in vitro by importing mutant precursors into isolated C. reinhardtii chloroplasts. Deletions that removed 20 or 23 amino acid residues from the center of the transit peptide reduced in vitro import to an undetectable level but did not affect CF₁-γ accumulation in vivo. The CF₁-γ transit peptide does have an in vivo stroma-targeting function, since chimeric genes in which the stroma-targeting domain of the plastocyanin transit peptide was replaced by the AtpC transit peptide-coding region allowed plastocyanin to accumulate in vivo. To determine whether the transit peptide deletions were impaired in in vivo stroma targeting, mutant and wild-type AtpC transit peptide-coding regions were fused to the bacterial ble gene, which confers bleomycin resistance. Although 25% of the wild-type fusion protein was associated with chloroplasts, proteins with transit peptide deletions remained almost entirely cytosolic. These results suggest that even severely impaired in vivo chloroplast protein import probably does not limit the accumulation of CF₁-γ.