Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase.

We have developed a nuclear transformation system for Chlamydomonas reinhardtii, using micro-projectile bombardment to introduce the gene encoding nitrate reductase into a nit1 mutant strain which lacks nitrate reductase activity. By using either supercoiled or linear plasmid DNA, transformants were recovered consistently at a low efficiency, on the order of 15 transformants per microgram of plasmid DNA. In all cases the transforming DNA was integrated into the nuclear genome, usually in multiple copies. Most of the introduced copies were genetically linked to each other, and they were unlinked to the original nit1 locus. The transforming DNA and nit+ phenotype were stable through mitosis and meiosis, even in the absence of selection. nit1 transcripts of various sizes were expressed at levels equal to or greater than those in wild-type nit+ strains. In most transformants, nitrate reductase enzyme activity was expressed at approximately wild-type levels. In all transformants, nit1 mRNA and nitrate reductase enzyme activity were repressed in cells grown on ammonium medium, showing that expression of the integrated nit1 genes was regulated normally. When a second plasmid with a nonselectable gene was bombarded into the cells along with the nit1 gene, transformants carrying DNA from both plasmids were recovered. In some cases, expression of the unselected gene could be detected. With the advent of nuclear transformation in Chlamydomonas, it becomes the first photosynthetic organism in which both the nuclear and chloroplast compartments can be transformed.

Gene Amplification Can Correct a Photosynthetic Growth Defect Caused by mRNA Instability in Chlamydomonas Chloroplasts.

Chlamydomonas reinhardtii chloroplast transformants that lack an inverted repeat normally found at the 3[prime] end of the chloroplast atpB gene have a slow phototrophic growth phenotype due to reduced accumulation of atpB mRNA and the chloroplast ATPase [beta] subunit. We have recovered transformants exhibiting more robust phototrophic growth at a moderate frequency (~1% relative to slow-growing transformants). Quantitative DNA blot analysis indicated that in one class of these robust photosynthetic transformants, the introduced plasmid DNA is maintained at high copy number-~25 copies per chloroplast genome or 2000 copies per cell. Partial restriction digests resulted in a ladder with at least 15 visible fragments, indicating that most of the transforming DNA is organized as a long head-to-tail tandem repeat. Total atpB transcription and accumulation of atpB mRNA and the ATPase [beta] subunit were increased approximately fivefold relative to transformants that carry a single copy of the truncated atpB gene. The amplified DNA was stably maintained at high copy number under mixotrophic growth conditions. It was inherited uniparentally from the mt+ parent, and its synthesis was sensitive to 5-fluoro-2[prime]-deoxyuridine, an inhibitor of chloroplast DNA synthesis. Therefore, we conclude that the tandem repeat is maintained in the chloroplast. Restriction enzymes that fail to digest the transforming plasmid but have recognition sites in chloroplast DNA did not alter the electrophoretic mobility of the tandem repeat, suggesting that it is not integrated in the chloroplast genome. We conclude that the tandem repeat is probably episomal and hypothesize that its replication is independent of the chloroplast genome.

A Nuclear Mutation That Affects the 3[prime] Processing of Several mRNAs in Chlamydomonas Chloroplasts.

We previously created and analyzed a Chlamydomonas reinhardtii strain, [delta]26, in which an inverted repeat in the 3[prime] untranslated region of the chloroplast atpB gene was deleted. In this strain, atpB transcripts are unstable and heterogeneous in size, and growth is poor under conditions in which photosynthesis is required. Spontaneous suppressor mutations that allow rapid photosynthetic growth have been identified. One strain, [delta]26S, retains the atpB deletion yet accumulates a discrete and stable atpB transcript as a consequence of a recessive nuclear mutation. Unlike previously isolated Chlamydomonas nuclear mutations that affect chloroplast mRNA accumulation, the mutation in [delta]26S affects several chloroplast transcripts. For example, in the atpA gene cluster, the relative abundance of several messages was altered in a manner consistent with inefficient mRNA 3[prime] end processing. Furthermore, [delta]26S cells accumulated novel transcripts with 3[prime] termini in the petD-trnR intergenic region. These transcripts are potential intermediates in 3[prime] end processing. In contrast, no alterations were detected for petD, atpA, or atpB mRNA 5[prime] ends; neither were there gross alterations detected for several other mRNAs, including the wild-type atpB transcript. We suggest that the gene identified by the suppressor mutation encodes a product involved in the processing of monocistronic and polycistronic messages.

Expression of chimeric genes by the light-regulated cabII-1 promoter in Chlamydomonas reinhardtii: a cabII-1/nit1 gene functions as a dominant selectable marker in a nit1- nit2- strain.

In Chlamydomonas reinhardtii, expression of the cabII-1 gene increases dramatically in response to light (cabII-1 encodes one of the light-harvesting chlorophyll a/b-binding proteins of photosystem II). We have used a region upstream of the cabII-1 gene in translational fusions to the bacterial uidA gene (encodes beta-glucuronidase) and transcriptional fusions to the Chlamydomonas nitrate reductase gene (nit1). Chlamydomonas transformants carrying intact copies of the chimeric uidA gene do not express beta-glucuronidase at the level of enzyme activity or mRNA accumulation. Methylation in the cabII-1 promoter region of the introduced gene is extensive in these strains, suggesting that newly introduced foreign genes may be recognized and silenced by a cellular mechanism that is correlated with increased methylation. Transformants that express the chimeric cabII-1/nit1 gene have been recovered. In contrast to the endogenous nit1 gene, the chimeric cabII-1/nit1 gene is expressed in ammonium-containing medium. Moreover, nit1 mRNA accumulation is dramatically stimulated by light, with a time course that is indistinguishable from that of the endogenous cabII-1 gene. The cabII-1/nit1 gene has been used to select transformants in a nit1- nit2- Chlamydomonas strain (CC400G) and should be useful for transformation of the large number of mutants in the Ebersold-Levine lineage, which carry the same mutations.

3'end maturation of the Chlamydomonas reinhardtii chloroplast atpB mRNA is a two-step process.

Inverted repeat (IR) sequences are found at the 3' ends of most chloroplast protein coding regions, and we have previously shown that the 3'IR is important for accumulation of atpB mRNA in Chlamydomonas reinhardtii (D. B. Stern, E.R. Radwanski, and K. L. Kindle, Plant Cell 3:285-297, 1991). In vitro studies indicate that 3' IRs are inefficient transcription termination signals in higher plants and have furthermore defined processing activities that act on the 3' ends of chloroplast transcripts, suggesting that most chloroplast mRNAs are processed at their 3' ends in vivo. To investigate the mechanism of 3' end processing in Chlamydomonas reinhardtii chloroplasts, the maturation of atpB mRNA was examined in vitro and in vivo. In vitro, a synthetic atpB mRNA precursor is rapidly cleaved at a position 10 nucleotides downstream from the mature 3' terminus. This cleavage is followed by exonucleolytic processing to generate the mature 3' end. In vivo run-on transcription experiments indicate that a maximum of 50% of atpB transcripts are transcriptionally terminated at or near the IR, while the remainder are subject to 3' end processing. Analysis of transcripts derived from chimeric atpB genes introduced into Chlamydomonas chloroplasts by biolistic transformation suggests that in vivo processing and in vitro processing occur by similar or identical mechanisms.

petD mRNA maturation in Chlamydomonas reinhardtii chloroplasts: role of 5' endonucleolytic processing.

Complex processing of primary transcripts occurs during the expression of higher-plant chloroplast genes. In Chlamydomonas reinhardtii, most chloroplast genes appear to possess their own promoters, rather than being transcribed as part of multicistronic operons. By generating specific deletion mutants, we show that petD, which encodes subunit IV of the cytochrome b6/f complex, has an RNA processing site that is required for accumulation of monocistronic petD mRNA in petD promoter deletion mutants; in such mutants, transcription of petD originates from the upstream petA promoter. The 5' ends of transcripts initiated at the petD promoter are probably also generated by processing, since the 5' end of monocistronic petD mRNA is the same in wild-type strains as it is in the petD promoter mutants. The location and function of the processing site were further examined by inserting petD-uidA fusion genes into the chloroplast genome (uidA is an Escherichia coli gene that encodes beta-glucuronidase). When a promoterless petD-uidA fusion gene was inserted downstream of petA, a monocistronic uidA transcript accumulated, which was apparently initiated at the petA promoter and was processed at a site corresponding precisely to the petD mRNA 5' end. When a construct including only sequences downstream of +25 relative to the mature mRNA 5' end was inserted into the same site, a dicistronic petA-uidA transcript accumulated but no monocistronic uidA transcript could be detected, suggesting that a processing site lies at least partially within the region from -1 to +25. Beta-glucuronidase activity was not detected in transformants that accumulated only the dicistronic petA-uidA transcript, suggesting that the first 25 bp of the 5' untranslated region are required for translation initiation. One explanation for this translational defect is that Chlamydomonas chloroplasts cannot translate the second coding region of some dicistronic messages.

Small cis-acting sequences that specify secondary structures in a chloroplast mRNA are essential for RNA stability and translation.

Nucleus-encoded proteins interact with cis-acting elements in chloroplast transcripts to promote RNA stability and translation. We have analyzed the structure and function of three such elements within the Chlamydomonas petD 5' untranslated region; petD encodes subunit IV of the cytochrome b(6)/f complex. These elements were delineated by linker-scanning mutagenesis, and RNA secondary structures were investigated by mapping nuclease-sensitive sites in vitro and by in vivo dimethyl sulfate RNA modification. Element I spans a maximum of 8 nucleotides (nt) at the 5' end of the mRNA; it is essential for RNA stability and plays a role in translation. This element appears to form a small stem-loop that may interact with a previously described nucleus-encoded factor to block 5'-->3' exoribonucleolytic degradation. Elements II and III, located in the center and near the 3' end of the 5' untranslated region, respectively, are essential for translation, but mutations in these elements do not affect mRNA stability. Element II is a maximum of 16 nt in length, does not form an obvious secondary structure, and appears to bind proteins that protect it from dimethyl sulfate modification. Element III spans a maximum of 14 nt and appears to form a stem-loop in vivo, based on dimethyl sulfate modification and the sequences of intragenic suppressors of element III mutations. Furthermore, mutations in element II result in changes in the RNA structure near element III, consistent with a long-range interaction that may promote translation.

The petD gene is transcribed by functionally redundant promoters in Chlamydomonas reinhardtii chloroplasts.

FUD6, a nonphotosynthetic mutant of Chlamydomonas reinhardtii, was previously found to be deficient in the synthesis of subunit IV of the cytochrome b6/f complex, the chloroplast petD gene product (C. Lemaire, J. Girard-Bascou, F.-A. Wollman, and P. Bennoun, Biochim. Biophys. Acta 851:229-238, 1986). The lesion in FUD6 is a 236-bp deletion between two 11-bp direct repeats in the chloroplast genome. It extends from 82 to 72 bp upstream of the 5' end of wild-type petD mRNA to 156 to 166 bp downstream of the 5' end. Thus, the deletion extends into the putative promoter and 5' untranslated region of petD. No petD mRNA of the normal size can be detected in FUD6 cells, but a low level of a dicistronic message accumulates, which contains the coding regions for subunit IV and cytochrome f, the product of the upstream petA gene. petD transcriptional activity in FUD6 is not significantly altered from the wild-type level. This transcriptional activity was eliminated by petA promoter disruptions, suggesting that it originates at the petA promoter. We conclude that the petD-coding portion of most cotranscripts is rapidly degraded in FUD6, possibly following processing events that generate the 3' end of petA mRNA. A chloroplast transformant was constructed in which only the sequence from -81 to -2 relative to the major 5' end of the petD transcript was deleted. Although this deletion eliminates all detectable petD promoter activity, the transformant grows phototrophically and accumulates high levels of monocistronic petD mRNA. We conclude that the petD gene can be transcribed by functionally redundant promoters. In the absence of a functional petD promoter, a lack of transcription termination allows the downstream petD gene to be cotranscribed with the petA coding region and thereby expressed efficiently.

A dominant mutation in the Chlamydomonas reinhardtii nuclear gene SIM30 suppresses translational defects caused by initiation codon mutations in chloroplast genes.

A suppressor of a translation initiation defect caused by an AUG to AUU mutation in the Chlamydomonas reinhardtii chloroplast petD gene was isolated, defining a nuclear locus that we have named SIM30. A dominant mutant allele at this locus, sim30-1d, was found to increase the translation initiation rate of the mutant petD mRNA. sim30-1d was also able to suppress the translational defect caused by an AUG to AUC mutation in the petD gene, and an AUG to AUU mutation in the chloroplast petA gene. We therefore suggest that the SIM30 gene may encode a general chloroplast translation factor. The ability of sim30-1d to suppress the petD AUG to AUU mutation is diminished in the presence of one or more antibiotic resistance markers located within the 16S and 23S rRNAs, suggesting that the activity of the sim30-1d gene product in translation initiation may involve interaction with ribosomal subunits.

Cloning and characterization of the actin-encoding gene of Chlamydomonas reinhardtii.

The genomic and complementary DNA sequences were determined for the unique actin-encoding gene in Chlamydomonas reinhardtii (Cr). The deduced amino acid (aa) sequence of this actin was similar to most known actin sequences, with the highest identity (98.1%) being with that of Volvox carteri actin. The Cr actin-encoding gene has one intron in the 5'-untranslated region and eight introns in the coding region. The latter eight introns occur at the same positions as those in the V. carteri actin-encoding gene. The 5'-upstream region contains four short stretches of sequence similar to the so-called 'tub box', a characteristic sequence proposed to be responsible for the regulation of synthesis of various axonemal proteins upon deflagellation and during the cell cycle. Southern blot analysis indicated that the Cr genome has only a single actin-encoding gene. An antibody specific for the 11-aa peptide corresponding to the N-terminal sequence of this actin was found to react with a 43-kDa protein associated with flagellar inner-arm dynein. These findings indicate that a single actin functions in both the cytoplasm and flagella of this organism.

High-frequency nuclear transformation of Chlamydomonas reinhardtii.

By using a method in which cell-wall-deficient Chlamydomonas reinhardtii cells were agitated in the presence of DNA, glass beads, and polyethylene glycol, nuclear transformation rates of approximately 10(3) transformants per micrograms of plasmid DNA were achieved. The nitrate reductase gene from wild-type Chlamydomonas was used to complement a mutation in the corresponding gene of a strain containing nit1-305. Transformants were selected by growth with nitrate as sole source of nitrogen. The transforming DNA integrated into the genome at a low-copy number in nit+ transformants. When cells carrying nit1-305 were agitated in the presence of two plasmids, one with the gene for nitrate reductase and the second with an unselected gene, the unselected gene was present in 10-50% of nit+ transformants. This high frequency of cotransformation will allow any cloned gene to be introduced into Chlamydomonas. Moreover, the overall efficiency of transformation should be high enough to permit isolation of genes from genomic libraries by complementation of stable nuclear mutants. The availability of efficient nuclear and chloroplast transformation in Chlamydomonas provides specific advantages for the study of chloroplast biogenesis, photosynthesis, and nuclear-chloroplast genome interactions.

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.

Expression of a gene for a light-harvesting chlorophyll a/b-binding protein in Chlamydomonas reinhardtii: effect of light and acetate.

In Chlamydomonas reinhardtii, the chlorophyll a/b-binding proteins of photosystem II are encoded in the nucleus by a small family of genes. We have studied the expression of one gene, which we call cabII-1, in a green-in-the-dark strain, which can synthesize chlorophyll in the dark or light, and in a yellow-in-the-dark mutant strain, which is able to make chlorophyll only in the light. In light/dark synchronized cultures of both strains, cabII-1 mRNA abundance increases during the first 6 h of a 12-h light phase, remains high for several hours, then declines. A variety of illumination conditions have been used to analyze the cabII-1 mRNA increase: continuous or intermittent red, blue, or white light, with or without 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosystem II. Our results suggest that light induces increased cabII-1 transcript abundance in two ways: 1) by virtue of its role in the light reactions of photosynthesis and 2) by a blue lightstimulated mechanism which is independent of photosynthesis.We have also examined the role of acetate in regulating cabII-1 mRNA levels in the dark. In both green- and yellow-in-the-dark strains, 15 mM Na-acetate, added to synchronized cells in the dark, induces an increase in cabII-1 mRNA abundance with a temporal accumulation pattern very similar to that induced by continuous white light. We suggest that by providing an energy source, acetate stimulates cellular growth, cell cycle progression, and increased cabII-1 mRNA abundance. Interestingly, in cells exposed to light, acetate inhibits the light-induced increase in cabII-1 mRNA abundance by a mechanism which is not yet understood.

Alterations in the Chlamydomonas plastocyanin transit peptide have distinct effects on in vitro import and in vivo protein accumulation.

Nucleus-encoded chloroplast proteins that reside in the thylakoid lumen are synthesized as precursors with bipartite transit peptides that contain information for uptake and intra-chloroplast localization. We have begun to apply the superb molecular and genetic attributes of Chlamydomonas to study chloroplast protein import by creating a series of deletions in the transit peptide of plastocyanin and determining their effects on translocation into isolated Chlamydomonas chloroplasts. Most N-terminal mutations dramatically inhibited in vitro import, whereas replacement with a transit peptide from the gamma-subunit of chloroplast ATPase restored uptake. Thus, the N-terminal region has stroma-targeting function. Deletions within the C-terminal portion of the transit peptide resulted in the appearance of import intermediates, suggesting that this region is required for lumen translocation and processing. Thus, despite its short length and predicted structural differences, the Chlamydomonas plastocyanin transit peptide has functional domains similar to those of vascular plants. Similar mutations have been analyzed in vivo by transforming altered genes into a mutant defective at the plastocyanin locus (K. L. Kindle, manuscript in preparation). Most mutations affected in vitro import more severely than plastocyanin accumulation in vivo. One exception was a deletion that removed residues 2-8, which nearly eliminated in vivo accumulation but had a modest effect in vitro. We suggest that this mutant precursor may not compete successfully with other proteins for the translocation pathway in vivo. Apparently, in vivo and in vitro analyses reveal different aspects of chloroplast protein biogenesis.

Identification and analysis of Dictyostelium actin genes, a family of moderately repeated genes.

Plasmid M6 has been shown to contain sequences complementary to two related abundant mRNA species which differ in length by 100 nucleotides and code for Dictyostelium actin. M6 complementary RNA was isolated by hybridization to immobilized M6 DNA and translated in vitro. The product is identical to major forms of in vivo labeled actin in both mobility on two-dimensional gels and two-dimensional fingerprints of tryptic peptides. Both plasmid M6 and a second plasmid complementary to the actin mRNA complementary region in M6, pDd actin 2 (McKeown et al., 1978), direct the synthesis in minicells of a number of similar polypeptides that are not seen in minicells containing other recombinant plasmids. Three of these polypeptides are similar in two-dimensional gel mobility to Dictyostelium actin and bind to DNAse I agarose. The repetition frequency of isolated restriction fragments from actin mRNA complementary plasmid M6 has been examined. The data from two different experimental approaches (DNA excess hybridizations using plasmid DNA as probe, and hybridization of plasmid probe to DNA blot filters of restriction enzyme-digested Dictyostelium DNA) indicate that the mRNA complementary region is reiterated 15--20 times. When an actin cDNA probe is used in the same experiments, the results suggest that the entire coding region is reiterated. When the two major actin mRNA species are separated and independently translated, each appears to code for one of the two major actin species. The results suggest that there are at least two different functional genes, and possibly more, for Dictyostelium actin.

Evidence that populations of Dictyostelium single-copy mRNA transcripts carry common repeat sequences.

Two recombinant plasmids, M4 and KH10, carrying Dictyostelium DNA inserted into the Eco RI restriction endonuclease site of pMB9 by poly(dA)-poly(dT) tailing, were selected for study because they are complementary to abundant mRNA populations from Dictyostelium. Both plasmids have been shown to hybridize a heterogeneous size class of mRNAs which, in the case of KH10, comprise 5-10% of the pulse-labeled poly(A)+ RNA from vegetative cells. Analysis of the sequence organization of the two pieces of Dictyostelium DNA shows that they consist mostly of single-copy sequences with a short DNA sequence which is repeated in the genome and interspersed with single-copy DNA. These and other results suggest that the majority of the hybridization of pulse-labeled mRNA to M4 and KH10 is to the short "repeated" DNA sequences. In the genome, members of these repeat families appear to be transcribed onto a population of different single-copy mRNAs. Additional results show that M4 DNA contains a sequence which is entirely complementary to a discrete mRNA.

Homologous recombination in the nuclear genome of Chlamydomonas reinhardtii.

Nuclear transformation of the unicellular green alga Chlamydomonas reinhardtii has thus far been characterized by integration of the introduced DNA into nonhomologous sites. In this study, the occurrence of homologous recombination events during transformation was investigated with the intent of developing strategies for gene targeting and gene disruption. Homologous recombination was monitored by using nonfunctional 5' and 3' deletion derivatives of the wild-type C. reinhardtii nit1 gene (encodes nitrate reductase) as selectable markers (p5' delta and p3' delta respectively) and the low reverting nit1-305 strain as the transformation recipient. After introduction of the DNA into the cell, intermolecular recombination between p5' delta and p3' delta occurs at a high frequency to restore a functional nit1 gene, indicating the presence of homologous recombination machinery in mitotic cells. Gene-targeting events at the nit1 locus were selected by restoring nit1-305 cells to prototrophy after transformation with only p5' delta and were confirmed by analysis of genomic DNA. By comparing the number of transformants obtained after transformation with p5' delta to the number obtained after transformation with a functional nit1 gene, the frequency of homologous-to-random integration events ranged between 1:1000 after glass bead-mediated transformation and 1:24 after bombardment with DNA-coated tungsten microprojectiles.

Stable nuclear transformation of Chlamydomonas reinhardtii by using a C. reinhardtii gene as the selectable marker.

We have developed a stable nuclear transformation system for the unicellular green alga Chlamydomonas reinhardtii. Transformation was accomplished by introducing the cloned C. reinhardtii oxygen-evolving enhancer protein 1 (OEE1) gene into C. reinhardtii cells by bombardment with DNA-coated tungsten particles. The recipient strain was an OEE1-deficient, nonphotosynthetic, acetate-requiring mutant, which recovered photosynthetic competence after transformation, and was therefore able to grow in the absence of acetate. Analysis of several transformants indicates that transformation has proceeded via second-site integration of the cloned gene, leaving the endogenous mutant gene intact. In genetic crosses of transformants with wild type, both mutant and wild-type phenotypes were recovered, showing that the photosynthetic competence of transformants was due not to reversion of the original locus but rather to expression of the introduced gene. We suggest that the success of the present system is largely due to using a homologous C. reinhardtii gene, leading to stable maintenance and expression of the gene. Transformation with heterologous genes may be problematic because of poor expression due to an unusual codon bias in C. reinhardtii.

Transit peptide mutations that impair in vitro and in vivo chloroplast protein import do not affect accumulation of the gamma-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 gamma-subunit of chloroplast ATPase-coupling factor 1 (CF1-gamma, 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-gamma accumulation in vivo. The CF1-gamma 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-gamma.