Subcellular location and expression level of a chimeric protein consisting of the maize waxy transit peptide and the beta-glucuronidase of Escherichia coli in transgenic potato plants

Summary The transit peptide of the maize waxy protein (a nuclear-encoded amyloplast protein of the maize endosperm) was studied with respect to its role in subcellular protein targeting in transgenic potato plants. TP30, a chimeric precursor protein consisting of the waxy transit peptide and an additional 34 amino acids of the mature waxy protein fused to the -glucuronidase of Escherichia coli, was expressed in potato plants under the control of the 35S promoter of cauliflower mosaic virus. This fusion protein is imported not only into amyloplasts, the natural target organelles in the maize plant, but also into chloroplasts. In contrast, Gus, the -glucuronidase alone, which was also expressed in parallel experiments in transgenic potato plants is always found in the cytosol of the plant cells. As a consequence of the different subcellular locations of TP30 and Gus, we observed differences in the expression rates of the respective proteins in leaf cells, resulting in higher steady state levels of TP30 compared to Gus. In tuber cells, no correlation between intracellular location and expression of the proteins was found.     

Mitochondrial and chloroplast targeting sequences in tandem modify protein import specificity in plant organelles

Protein targeting to plant mitochondria and chloroplasts is usually very specific and involves targeting sequences located at the amino terminus of the precursor. We challenged the system by using combinations of mitochondrial and chloroplast targeting sequences attached to reporter genes. The sequences coding for the presequence of the mitochondrial F₁-ATPase -subunit and the transit peptide of the chloroplast chlorophyll a/b-binding protein, both from Nicotiana plumbaginifolia, were fused together in both combinations, then linked to the reporter genes, chloramphenicol acetyl transferase (CAT) and -glucuronidase (GUS), and introduced into tobacco. Analysis of CAT and GUS activities and proteins in the subcellular fractions revealed that the chloroplast transit peptide alone was not sufficient to target the reporter proteins to chloroplasts. However, when the mitochondrial -presequence was inserted downstream of the chloroplast sequence, import of CAT and GUS into chloroplasts was observed. Using the reciprocal system, the mitochondrial presequence alone was able to direct transport of CAT and, to a lesser extent, GUS to mitochondria; the GUS targeting to mitochondria was increased when the chloroplast targeting sequence was linked downstream of the mitochondrial presequence. Immuno-detection experiments using subcellular fractions confirmed the results observed by enzymatic assays. These results indicate the importance of the amino-terminal position of the targeting sequence in determining protein import specificity and are considered within the hypothesis of a co-translational protein import.     

Protein import pathways in ‘complex’ chloroplasts derived from secondary endosymbiosis involving a red algal ancestor

Heterokont algae such as diatoms and the raphidophyte Heterosigma akashiwo and peridinin-containing dinoflagellates such as Heterocapsa triquetra originally acquired their chloroplasts via secondary endosymbiosis involving a red algal endosymbiont and a eukaryote host, resulting in complex chloroplasts surrounded by four and three membranes, respectively. The precursors of both heterokont and dinoflagellate chloroplast-targeted proteins are first inserted into the ER with removal of an N-terminal signal peptide, but how they traverse the remaining membranes is unclear. Using a nuclear-encoded thylakoid lumen protein, PsbO, from the heterokont alga Heterosigma akashiwo, the dinoflagellate Heterocapsa triquetra and the red alga Porphyra yezoensis we show that precursors without the ER signal peptide can be imported into pea chloroplasts. In the case of the H. triquetra and Porphyra PsbO, the precursors were processed to their predicted mature size and localized within the thylakoid lumen, using the Sec-dependent pathway. We report for the first time a stromal processing peptidase (SPP) activity from an alga of the red lineage. The enzyme processes the Heterosigma PsbO precursor at a single site and appears to have different substrate and reaction specificities from the plant SPP. In spite of the fact that we could not find convincing homologs of the plant chloroplast import machinery in heterokont (diatom) and red algal genomes, it is clear that these three very different lines of algae use similar mechanisms to import chloroplast precursors.     

Import of a new chloroplast inner envelope protein is greatly stimulated by potassium phosphate

A cDNA clone encoding a major chloroplast inner envelope membrane protein of 96 kDa (IEP96) was isolated and characterized. The protein is synthesized as a larger-molecular-weight precursor (pIEP96) which contains a cleavable N-terminal transit sequence of 50 amino acids. The transit peptide exhibits typical stromal targeting information. It is cleaved in vitro by the stromal processing peptidase, though the mature protein is clearly localized in the inner envelope membrane. Translocation of pIEP96 into chloroplasts is greatly stimulated in the presence of 80 mM potassium phosphate which results in an import efficiency of about 90%. This effect is specific for potassium and phosphate, but cannot be ascribed to a membrane potential across the inner envelope membrane. Protein sequence analysis reveals five stretches of repeats of 26 amino acids in length. The N-terminal 300 amino acids are 45% identical (76% similarity) to the 35 kDa -subunit of acetyl-CoA carboxyl-transferase from Escherichia coli. The C-terminal 500 amino acids share significant similarity (69%) with USOI, a component of the cytoskeleton in yeast.Abbreviations P_i phosphate - IEP inner envelope membrane protein - pIEP precursor form of IEP - SSU small subunit of ribulose-1,5-bisphosphate carboxylase oxygenase - IEP96_pep peptide specific antiserum to IEP96 - IEP96_pol polyspecific antiserum to IEP96     

Expression of a bacterial lysine decarboxylase gene and transport of the protein into chloroplasts of transgenic tobacco

A possible approach for altering alkaloid biosynthesis in plants is the expression of genes encoding key enzymes of a pathway such as lysine decarboxylase (ldc) in transgenic plants. Two strategies were followed here: one focused on expression of the gene in the cytoplasm, the other on subsequent targeting of the protein to the chloroplasts. Theldcgene fromHafnia alvei was therefore (a) placed under the control of the 1 promoter of the bidirectional Tr promoter fromAgrobacterium tumefaciens Ti- plasmid, and (b) cloned behind therbcS promoter from potato fused to the coding region of therbcS transit peptide. Bothldc constructs, introduced intoNicotiana tabacum with the aid ofA. tumefaciens, were integrated into the plant genome and transcribed as shown by Southern and northern hybridization. However, LDC activity was only detectable in plants expressing mRNA under the control of therbcS promoter directing the LDC fusion protein into chloroplasts with the aid of the transit peptide domain. In plants expressing the processed bacterial enzyme cadaverine levels increased from nearly zero to 0.3–1% of dry mass.     

Expression and import of an active cellulase from a thermophilic bacterium into the chloroplast both in vitro and in vivo

A bacterial thermostable cellulase, the endo-1,4--D-glucanase E1 from Acidothermus cellulolyticus, was imported into chloroplasts, and an active enzyme was recovered both in vitro and in vivo. Precursor fusion proteins were synthesized with E1 or its catalytic domain, CD, fused to the transit peptide of ferredoxin or ribulose-bisphosphate carboxylase activase for stromal targeting. A spacer region of 1, 5 or 15 amino acids was included carboxy to the transit peptide. The efficiency of import and processing by the stromal processing peptidase depended on the nature of the transit peptide and the passenger protein, and increased with the length of the spacer between them. Besides finding E1 or CD in the stroma, protein was arrested in the envelope during import showing that structural features of E1 and CD, along with their proximity to the transit peptide, influence translocation. The cellulose binding domain and/or serine/proline/threoline-rich linker of E1 may impede efficient import. Significantly, most precursors for E1 and CD synthesized by in vitro translation possessed endoglucanse activity that was temperature-dependent, and required the residues AGGGY at the N-terminus of E1 and CD. Furthermore, activity was detected upon import into chloroplasts. Based on the in vitro analyses, five precursor fusion proteins were selected to determine if E1 and CD would be successfully targeted to chloroplasts in vivo. In transgenic tobacco plants, E1 and CD accumulated in both the stromal and membrane fractions and, importantly, chloroplast extracts showed endoglucanase activity.     

The transit peptide of a chloroplast thylakoid membrane protein is functionally equivalent to a stromal-targeting sequence.

The role of transit peptides in intraorganellar targeting has been studied for a chlorophyll a/b binding (CAB) polypeptide of photosystem II (PSII) and the small subunit of ribulose-1,5-bisphosphate carboxylase (RBCS) from Pisum sativum (pea). These studies have involved in vitro import of fusion proteins into isolated pea chloroplasts. Fusion of the CAB transit peptide to RBCS mediates import to the stroma, as evidenced by assembly of RBCS with chloroplast-synthesized large subunit (RBCL) to form holoenzyme. Similarly, fusion of the RBCS transit peptide to the mature CAB polypeptide mediates import and results in integration of the processed CAB protein into the thylakoid membrane. Correct integration was indicated by association with PSII and assembly with chlorophyll to form the light-harvesting chlorophyll a/b protein complex (LHCII). We interpret these results as evidence that the CAB transit peptide is functionally equivalent to a stromal-targeting sequence and that intraorganellar sorting of the CAB protein must be determined by sequences residing within the mature protein. Our results and those of others suggest that import and integration of CAB polypeptides into the thylakoid proceeds via the stroma.     

Peculiar properties of the PsaF photosystem I protein from the green alga Chlamydomonas reinhardtii: presequence independent import of the PsaF protein into both chloroplasts and mitochondria

It has previously been shown that presequences of nuclear-encoded chloroplast proteins from the green alga Chlamydomonas reinhardtii contain a region that may form an amphiphilic -helix, a structure characteristic of mitochondrial presequences. We have tested two precursors of chloroplast proteins (the PsaF and PsaK photosystem I subunits) from C. reinhardtii for the ability to be imported into spinach leaf mitochondria in vitro. Both precursors bound to spinach mitochondria. The PsaF protein was converted into a protease-protected form with high efficiency in a membrane potential-dependent manner, indicating that the protein had been imported, whereas the PsaK protein was not protease protected. The protease protection of PsaF was not inhibited by a synthetic peptide derived from the presequence of the N. plumbaginifolia mitochondrial F₁ subunit. Furthermore, if the presequence of PsaF was truncated or deleted by in vitro mutagenesis, the protein was still protease-protected with approximately the same efficiency as the full-length precursor. These results indicate that PsaF can be imported by spinach mitochondria in a presequence-independent manner. However, even in the absence of the presequence, this process was membrane potential-dependent. Interestingly, the presequence-truncated PsaF proteins were also protease-protected upon incubation with C. reinhardtii chloroplasts. Our results indicate that the C. reinhardtii chloroplast PsaF protein has peculiar properties and may be imported not only into chloroplasts but also into higher-plant mitochondria. This finding indicates that additional control mechanisms in the cytosol that are independent of the presequence are required to achieve sorting between chloroplasts and mitochondria in vivo.Abbreviations cTP chloroplast transit peptide - mTP mitochondrial targeting peptide - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - pF₁(1,25) a synthetic peptide derived from the first 25 residues of the Nicotiana plumbaginifolia mitochondrial ATP synthase F₁ subunit - PsaF(2–30) and PsaF(2–61) mutant proteins lacking regions corresponding to residues 2–30 and 2–61 in the PsaF precursor protein, respectively     

The chlorophyll a/b-binding protein inserts into the thylakoids independent of its cognate transit peptide

In order to determine if the cognate transit peptide of the light-harvesting chlorophyll a/b-binding protein (LHCP) is essential for LHCP import into the chloroplast and proper localization to the thylakoids, it was replaced with the transit peptide of the small subunit (S) of ribulose-1,5-bisphosphate carboxylase/oxygenase, a stromal protein. Wheat LHCP and S genes were fused to make a chimeric gene coding for the hybrid precursor, which was synthesized in vitro and incubated with purified pea chloroplasts. My results show that LHCP is translocated into chloroplasts by the S transit peptide. The hybrid precursor was processed; and most importantly, mature LHCP did not remain in the stroma, but was inserted into thylakoid membranes, where it normally functions. Density gradient centrifugation showed no LHCP in the envelope fraction. Hence, the transit peptide of LHCP is not required for intraorganellar routing, and LHCP itself contains an internal signal for localization to the correct membrane compartment.     

Identification of cDNAS encoding plastidtargeted glutathione peroxidase

A cDNA was isolated from pea leaf RNA which encodes a phospholipid hydroperoxide glutathione peroxidase (PHGPX; E.C. 1.1.1.1.9). The N-terminal section of this PHGPX encodes a recognisable chloroplast transit peptide. Efficient import in vitro of the pre-PHGPX protein into the stroma of isolated pea chloroplasts confirmed that the PHGPX is a chloroplast-located enzyme. The pea PHGPX has highly conserved homologues in Arabidopsis, citrus and Nicotiana sylvestris and the authors suggest that these proteins are also localised in the chloroplast and not in the cytosol as previously supposed.     

Tissue-specific and developmental-specific expression of an Arabidopsis thaliana gene encoding the lipoamide dehydrogenase component of the plastid pyruvate dehydrogenase complex

We describe an Arabidopsis thaliana gene, ptlpd2, which codes for a protein with high amino acid similarity to lipoamide dehydrogenases (LPDs) from diverse species. Ptlpd2 codes for a precursor protein possessing an N-terminal extension predicted to be a plastid-targeting signal. Expression of the ptlpd2 cDNA in Escherichia coli showed the encoded protein possessed the predicted LPD activity. PTLPD2 protein, synthesized in vitro, was efficiently imported into isolated chloroplasts of Pisum sativum and shown to be located in the stroma. In addition, fusion proteins containing the predicted transit peptide of PTLPD2 or the entire protein fused at the N-terminus with the green fluorescent protein (GFP), showed accumulation in vivo in chloroplasts but not in mitochondria of A. thaliana. Expression of ptlpd2 was investigated by introducing ptlpd2 promoter--glucuronidase (GUS) gene fusions into Nicotiana tabacum. GUS expression was observed in seeds, flowers, root tips and young leaves. GUS activity was highest in mature seeds, decreased on germination and increased again in young leaves. Expression was also found to be temporally regulated in pollen grains where it was highest in mature grains at dehiscence. Database searches on ptlpd2 sequences identified a second A. thaliana gene encoding a putative plastidial LPD and two genes encoding proteins with high similarity to the mitochondrial LPD of P. sativum.     

Identification and characterization of gene-based SSR markers in date palm (Phoenix dactylifera L.)

Background

Clp/Hsp100 chaperones are involved in protein quality control. They act as independent units or in conjunction with a proteolytic core to degrade irreversibly damaged proteins. Clp chaperones from plant chloroplasts have been also implicated in the process of precursor import, along with Hsp70 chaperones. They are thought to pull the precursors in as the transit peptides enter the organelle. How Clp chaperones identify their substrates and engage in their processing is not known. This information may lie in the position, sequence or structure of the Clp recognition motifs.

Results

We tested the influence of the position of the transit peptide on the interaction with two chloroplastic Clp chaperones, ClpC2 and ClpD from Arabidopsis thaliana (AtClpC2 and AtClpD). The transit peptide of ferredoxin-NADP⁺ reductase was fused to either the N- or C-terminal end of glutathione S-transferase. Another fusion with the transit peptide interleaved between two folded proteins was used to probe if AtClpC2 and AtClpD could recognize tags located in the interior of a polypeptide. We also used a mutated transit peptide that is not targeted by Hsp70 chaperones (TP1234), yet it is imported at a normal rate. The fusions were immobilized on resins and the purified recombinant chaperones were added. After a washing protocol, the amount of bound chaperone was assessed. Both AtClpC2 and AtClpD interacted with the transit peptides when they were located at the N-terminal position of a protein, but not when they were allocated to the C-terminal end or at the interior of a polypeptide.

Conclusions

AtClpC2 and AtClpD have a positional preference for interacting with a transit peptide. In particular, the localization of the signal sequence at the N-terminal end of a protein seems mandatory for interaction to take place. Our results have implications for the understanding of protein quality control and precursor import in chloroplasts.     

The sequence surrounding the translation initiation codon of the pea plastocyanin gene increases translational efficiency of a reporter gene

The 5-upstream region of the pea plastocyanin gene (petE) directed 5–10-fold higher levels of -glucuronidase (GUS) activity than the cauliflower mosaic virus 35S promoter in transgenic tobacco plants, although the levels of GUS mRNA were similar. The sequence (AAAAAUGG) around the translation initiation codon of petE enhanced translation of the GUS mRNA 10-fold compared to translation from the GUS translation initiation codon in transgenic tobacco plants and transfected protoplasts.     

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.     

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.     

Pea Chloroplast DnaJ-J8 and Toc12 Are Encoded by the Same Gene and Localized in the Stroma

Toc12 is a novel J domain-containing protein identified in pea (Pisum sativum) chloroplasts. It was shown to be an integral outer membrane protein localizing in the intermembrane space of the chloroplast envelope. Furthermore, Toc12 was shown to associate with an intermembrane space Hsp70, suggesting that Toc12 is important for protein translocation across the chloroplast envelope. Toc12 shares a high degree of sequence similarity with Arabidopsis (Arabidopsis thaliana) DnaJ-J8, which has been suggested to be a soluble protein of the chloroplast stroma. Here, we isolated genes encoding DnaJ-J8 from pea and found that Toc12 is a truncated clone of one of the pea DnaJ-J8s. Protein import analyses indicate that Toc12 and DnaJ-J8s possess a cleavable transit peptide and are localized in the stroma. Arabidopsis mutants with T-DNA insertions in the DnaJ-J8 gene show no defect in chloroplast protein import. Implications of these results in the energetics and mechanisms of chloroplast protein import are discussed.Most chloroplast proteins are encoded by the nuclear genome and synthesized in the cytosol as higher molecular mass precursors with an N-terminal extension known as the transit peptide. Precursor proteins are imported into chloroplasts through a translocon complex located at the chloroplast envelope. Translocon components associated with the outer membrane are called Toc (for translocon of the outer envelope membrane of chloroplast) proteins, and those associated with the inner membrane are called Tic (for translocon of the inner envelope membrane of chloroplast) proteins. Cleavage of the transit peptide from the precursor by a specific stromal processing peptidase during translocation results in the production of the lower molecular mass mature protein. Various translocon components have been assigned functions in the basic steps of the import process (for review, see Inaba and Schnell, 2008; Jarvis, 2008; Li and Chiu, 2010). For example, Toc159 (the no. indicates the calculated molecular mass of the protein) and Toc34 are receptors for the transit peptides, and Toc75 is the protein-translocating channel across the outer membrane. Toc64, on the other hand, has a dual function: it serves as a docking site for the cytosolic Hsp90 through its cytosolic domain and as a scaffold for translocon components located in the intermembrane space through its intermembrane space domain (Qbadou et al., 2007).Protein import into chloroplasts involves at least two distinct ATP-consuming steps. The first step is called “early import intermediate” or “docking,” in which less than 100 μm ATP is required and precursors are translocated across the outer membrane and come into contact with translocon components in the inner membrane (Olsen et al., 1989; Kouranov and Schnell, 1997; Inaba et al., 2003; Inoue and Akita, 2008). It has been shown that the ATP is used in the intermembrane space (Olsen and Keegstra, 1992), most likely by a yet unidentified intermembrane space Hsp70 called imsHsp70 or Hsp70-IAP (ims for “intermembrane space” and IAP for “import intermediate-associated protein”; Marshall et al., 1990; Schnell et al., 1994; Qbadou et al., 2007). The second ATP-consuming step is the complete translocation of precursors across the two envelope membranes into the stroma. This step requires about 1 mm ATP. The ATP is most likely used by the stromal Hsp93 and chloroplast Hsc70 associated with the translocon to drive protein translocation into the stroma (Nielsen et al., 1997; Shi and Theg, 2010; Su and Li, 2010).Hsp70 family proteins are involved in many cellular processes, including protein folding, protein translocation across membranes, and regulation of protein degradation. Hsp70 proteins are often recruited to perform a certain function by specifically localized J domain-containing proteins. The J domain-containing proteins interact with Hsp70 when Hsp70 is bound to ATP and stimulate ATP hydrolysis by Hsp70. The specific J domain-containing cochaperone that recruits the stromal chloroplast Hsc70 to the inner envelope membrane to assist in protein translocation has not been identified. The specific J domain-containing cochaperone for imsHsp70 for its function in protein import into chloroplasts is proposed to be a protein named Toc12 (Becker et al., 2004).Toc12 was identified as a novel J domain-containing protein from pea (Pisum sativum) chloroplasts. It belongs to the type III J domain proteins containing only the J domain without the Gly- and Phe-rich domain (G/F domain) and the zinc-finger domain originally found in Escherichia coli DnaJ. It has been shown that the protein is synthesized at its mature size of 103 amino acids without a cleavable transit peptide. After import, the protein has been shown to anchor in the outer membrane by its N-terminal part, which has been suggested to form a β-barrel-type domain. Its C-terminal part, composed of the J domain, has been shown to localize in the intermembrane space. Toc12 has been shown to associate with imsHsp70. Toc12 and imsHsp70 interact with the intermembrane space domain of Toc64, which in turn associates with another intermembrane space translocon component, Tic22. It is proposed that the Toc12-imsHsp70-Toc64-Tic22 complex mediates protein translocation across the intermembrane space through specific precursor binding and ATP hydrolysis (Becker et al., 2004; Qbadou et al., 2007). However, the existence of imsHsp70 has only been shown on immunoblots by its reactivity to the monoclonal antibody SPA820 raised against human Hsp70. Its encoding gene has never been identified. The Arabidopsis (Arabidopsis thaliana) Hsp70 gene family has 14 members. Only two of them are localized in chloroplasts, and both have been shown to locate in the stroma (Ratnayake et al., 2008; Su and Li, 2008). A recent study has further shown that the major protein recognized by the SPA820 antibody in pea chloroplasts is located in the stroma, indicating that imsHsp70 is most likely a stromal protein (Ratnayake et al., 2008).Most translocon components were originally identified from pea chloroplasts. While all translocon components identified from pea have easily recognizable Arabidopsis homologs, Toc12 seems to be an exception. The Arabidopsis gene suggested to be the pea TOC12 homolog, At1g80920 (Inoue, 2007; Jarvis, 2008), encodes a protein that is much larger than pea Toc12 and is annotated as J8 (referred to as AtJ8 herein). The entire pea Toc12 has a high sequence similarity to the N-terminal two-thirds of AtJ8. AtJ8 contains an extra C-terminal domain of 60 amino acids that is highly conserved among J8 proteins from other higher plants. However, in contrast to pea Toc12, AtJ8 is predicted to locate in the stroma (Miernyk, 2001; www.arabidopsis.org). Indeed, a fusion protein consisting of the first 80 amino acids of AtJ8 fused at the N terminus of GFP was imported into the chloroplast stroma, and approximately 46 amino acids from the N terminus were processed after import (Lee et al., 2008), indicating that the first 46 amino acids of AtJ8 function as a cleavable stroma-targeting transit peptide. A T-DNA insertion in the AtJ8 gene that causes the truncation of the last three amino acids results in no visible phenotype. However, detailed analyses indicate that the mutant has lower CO₂ assimilation and Rubisco activity than the wild type (Chen et al., 2010).We are interested in identifying J domain-containing proteins interacting with stromal Hsp70. As part of the initial effort, we investigated the suborganellar location of J8 and examined the relationship between Toc12 and J8. We found that, in pea, there are at least two genes encoding J8, which we named PsJ8a and PsJ8b. TOC12 represents part of PsJ8b. Toc12, AtJ8, and the two PsJ8 proteins could be imported into chloroplasts and processed to stromally localized soluble mature proteins. Four alleles of AtJ8 mutants were analyzed, but none of them showed any defect in the import of various chloroplast precursor proteins.     

Behavior of chloroplasts and chloroplast nuclei during spermatogenesis in the fern,Pteris vittata L.

Summary The fate of the chloroplasts and chloroplast nuclei (cp-nuclei) was followed during spermatogenesis in the fernPteris vittata L. by epifluorescence microscopy after staining with 4-6-diamidino-2-phenylindole (DAPI) and by quantitation of chloroplast DNA (cp-DNA) by fluorimetry using a video intensified microscope photon counting system (VIMPICS). The spores were grown on solid medium that contained antheridiogen (An_ptd), and formed an antheridium initial on the protonema cell. The antheridium initial divided and produced 16 spermatocytes and 3 surrounding cells. The chloroplasts in the spermatocytes decreased in volume as cell division was repeated, until finally the volume of each chloroplast was 1/15 of that of the primary chloroplasts. The DNA content of the chloroplasts was also reduced to 1/5 of the original value and when the sperm matured, the fluorescence of cp-DNA disappeared. In the 16-cell spermatocyte, the recognition of the fluorescence of chlorophyll in the chloroplasts with a green excitation filter became difficult. But, the plastids could be observed until the final stage of the sperm. From these observations, it appears that there are two steps in the metamorphosis of chloroplasts during spermatogenesis in the fern. The first step involves the decrease in the volume of chloroplasts, accompanied by reduction of the DNA content, and the second step involves the change of the physical state of chloroplasts to amyloplasts and the disappearance of the cp-DNA from the amyloplasts.     

Isolation and immobilization of various plastid subtypes by magnetic immunoabsorption

Antibodies have been prepared which immuno-localize to the outer membrane of the pea chloroplast envelope and cause agglutination of isolated chloroplasts. This antisera is immunoreactive with a variety of plastid forms from both monocotyledonous and dicotyledonous plants. Whether such antibodies might be effectively used for isolation and immobilization of plastids from whole cell lysates has been tested. A system has been developed for immunolabeling various forms of higher plant plastids with biotinylated antibody and streptavidin magnetic nano-particles followed by separation of the plastids in a 0.6 Tesla high gradient magnetic field. Using this magnetic immunoabsorption procedure it has been possible to achieve a high degree of positive enrichment for chromoplasts, amyloplasts, and chloroplasts from whole cell lysates of several plant species. The integrity of these plastids has been examined by in organellar protein synthesis, ¹⁴C-ADP-glucose uptake, flow cytometry, in vitro synthesized precursor import and FITC-cationized ferritin staining of the plastid envelope. Western blot analysis showed significant enrichment for amyloplasts from cytosolic sucrose synthase in maize endosperm. Magnetic immunoabsorption of subcellular structures from whole cell lysates is a new method that may be useful in the in vitro analysis of many different cellular compartments from a wide range of organisms.     

Plastid targeting of E. coli β-glucuronidase and ADP-glucose pyrophosphorylase in maize (Zea mays L.) cells

Summary Dicot and monocot chloroplast targeting peptides (CTPs) were evaluated for their effect on targeting, processing, and expression of two reporter proteins in maize cells. When tested transiently in maize leaf protoplasts, the maize ribulose bisphosphate carboxylase small subunit CTP required the inclusion of the amino terminus of mature small subunit protein to target -glucuronidase (GUS) to the plastid. To remove this amino terminal extension from GUS after import and processing, a repeat of the native processing site was inserted between the native mature protein and the reporter protein. This repeat processing site was used with less efficiency than the native site. Parallel constructs using the Arabidopsis thaliana small subunit and maize granule-bound starch synthase CTPs also localized GUS, but varied in repeat site use and GUS expression levels. Data from the CTP fusions with GUS were generally confirmed with fusions to an allosteric variant of E. coli ADP-glucose pyrophosphorylase. Plastid targeting of this enzyme was required for starch enhancement of transgenic BMS cells.Abbreviations BMS maize Black Mexican Sweet suspension culture cells - CTP chloroplast targeting peptide - glgC16 an allosteric variant of E. coli ADP-glucose pyrophosphorylase - GUS -glucuronidase - LUX luciferase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - SSU small subunit of ribulose bisphosphate carboxylase