Acyl carrier protein (ACP) import into chloroplasts does not require the phosphopantetheine: evidence for a chloroplast holo-ACP synthase.

Import of the acyl carrier protein (ACP) precursor into the chloroplast resulted in two products of about 14 kilodalton (kD) and 18 kD when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Time course experiments indicate that the latter is a modification derivative of the 14-kD peptide after the removal of the transit peptide. Substitution of serine 38 by alanine, eliminating the phosphopantetheine prosthetic group attachment site of ACP, produced a precursor mutant that gave rise to only the 14-kD peptide during import, showing that the modified form depends on the presence of serine 38. Furthermore, these results demonstrate that the prosthetic group is not essential for ACP translocation across the envelope or proteolytic processing. Analysis of the products of import by nondenaturing, conformationally sensitive gels showed reversal of the relative mobility of the 14-kD peptide and the modified form, raising the possibility that the modification is the addition of the phosphopantetheine. Proteolytic processing and the modification reaction were reconstituted in an organelle-free assay. The addition of coenzyme A to the organelle-free assay completely converted the 14-kD peptide to the modified form at 10 micromolar, and this only occurred with the wild-type substrate. Reciprocally, treatment of the products of a modification reaction with Escherichia coli phosphodiesterase converted the modified ACP from back to the 14-kD peptide. These results strongly support the conclusion that there is a holo-ACP synthase in the soluble compartment of the chloroplast capable of transferring the phosphopantetheine of coenzyme A to ACP.     

Both ATP and an energized inner membrane are required to import a purified precursor protein into mitochondria.

We have investigated the energy requirement of mitochondrial protein import with a simplified system containing only isolated yeast mitochondria, energy sources and a purified precursor protein. This precursor was a fusion protein composed of 22 residues of the cytochrome oxidase subunit IV pre-sequence fused to mouse dihydrofolate reductase. Import of this protein required not only an energized inner membrane, but also ATP. ATP could be replaced by GTP, but not by CTP, TTP or non-hydrolyzable ATP analogs. Added ATP did not increase the membrane potential of respiring mitochondria; it supported import even if the proton-translocating mitochondrial ATPase and the entry of ATP into the matrix were blocked. We conclude that ATP exerts its effect on mitochondrial protein import outside the inner membrane.     

A soluble protein is imported into Euglena chloroplasts as a membrane-bound precursor.

The Euglena precursor to the small subunit of ribulose-15-bisphosphate carboxylase/oxygenase (pSSU) is a polyprotein. To determine the transport route from cytoplasm to chloroplast, Euglena was pulse labeled with 35S-sulfate and the organelles were separated on sucrose gradients. After a pulse, pSSU was found in the endoplasmic reticulum (ER) and Golgi apparatus. During a chase, ER-and Golgi-localized pSSU decreased concomitant with the appearance of SSU in chloroplasts. SSU was not found in pSSU-containing ER and Golgi fractions. Na2CO3 did not remove pSSU from ER or Golgi membranes, indicating that it was an integral membrane protein. pSSU was inserted in vitro into canine microsomes, and Na2CO3 did not remove pSSU from the microsomal membrane. The in vivo and in vitro experiments show that Euglena pSSU is inserted into the ER membrane and transported as an integral membrane protein to the Golgi apparatus before chloroplast import and polyprotein processing.     

Effect of precursor protein phosphorylation on import into isolated chloroplasts from Chlamydomonas.

In higher plants, chloroplast-destined precursor proteins are thought to be phosphorylated. Mediated by a specific 14-3-3 protein, these phosphorylated proteins bind to the chloroplast surface and are subsequently imported into the chloroplast. We demonstrate that also in the green alga Chlamydomonas reinhardtii the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase becomes phosphorylated by a plant protein kinase and that the phosphorylation site is located in the transit peptide. The phosphorylation status of the precursor protein regulates its import into chloroplasts especially at an early step during this process. The possible physiological function is discussed.     

The import of ferredoxin-NADP+ reductase precursor into chloroplasts is modulated by the region between the transit peptide and the mature core of the protein.

Protein transport across organelles' membranes requires that precursor proteins adopt an unfolded structure in order to be translocated by the import machinery. Ferredoxin-NADP+ reductase precursor, as well as many others, acquires a tightly folded structure that needs to be unfolded before or during its import. Several steps of chloroplast protein import are not fully understood. In particular, the role of different regions of the precursor protein has not been completely elucidated. In this work, we have studied the import into chloroplasts of precursor proteins with inclusions of amino acid spacers between the transit peptide and the mature protein, and with deletions in the N-terminal region of the mature enzyme. We measured the import rate constants for these precursors and the results indicate that the distance between the transit peptide and the core of the mature protein determines the import kinetics. The longer precursors were imported into the organelle faster than the wild type form. Precursors with deletions in the N-terminal region of the mature protein also showed increased import rates compared to the wild type. Homology studies amongst all family members reveal that only chloroplastic proteins possess this region. We suggest that even if the first amino acids of the mature protein do not contribute to its overall structural stability, they condition the kinetic parameters of the import reaction. Besides, the distance between the transit peptide and the mature protein core may be modulating the import rate at which the chloroplast incorporates this protein from the cytosol.     

Import of proteins into chloroplasts. Membrane integration of a thylakoid precursor protein reconstituted in chloroplast lysates

Many of the thylakoid membrane proteins of plant and algal chloroplasts are synthesized in the cytosol as soluble, higher molecular weight precursors. These precursors are post-translationally imported into chloroplasts, incorporated into the thylakoids, and proteolytically processed to mature size. In the present study, the process by which precursors are incorporated into thylakoids was reconstituted in chloroplast lysates using the precursor to the light-harvesting chlorophyll a/b protein (preLHCP) as a model. PreLHCP inserted into thylakoid membranes, but not envelope membranes, if ATP was present in the reaction mixture. Correct integration into the bilayer was verified by previously documented criteria. Integration could also be reconstituted with purified thylakoid membranes if reaction mixtures were supplemented with a soluble extract of chloroplasts. Several other thylakoid precursor proteins in addition to preLHCP, but no stromal precursor proteins, were incorporated into thylakoids under the described assay conditions. These results suggest that the observed in vitro activity represents in vivo events during the biogenesis of thylakoid proteins.     

Isolated plant mitochondria import chloroplast precursor proteins in vitro with the same efficiency as chloroplasts.

Most chloroplast and mitochondrial proteins are synthesized with N-terminal presequences that direct their import into the appropriate organelle. In this report we have analyzed the specificity of standard in vitro assays for import into isolated pea chloroplasts and mitochondria. We find that chloroplast protein import is highly specific because mitochondrial proteins are not imported to any detectable levels. Surprisingly, however, pea mitochondria import a range of chloroplast protein precursors with the same efficiency as chloroplasts, including those of plastocyanin, the 33-kDa photosystem II protein, Hcf136, and coproporphyrinogen III oxidase. These import reactions are dependent on the Deltaphi across the inner mitochondrial membrane, and furthermore, marker enzyme assays and Western blotting studies exclude any import by contaminating chloroplasts in the preparation. The pea mitochondria specifically recognize information in the chloroplast-targeting presequences, because they also import a fusion comprising the presequence of coproporphyrinogen III oxidase linked to green fluorescent protein. However, the same construct is targeted exclusively into chloroplasts in vivo indicating that the in vitro mitochondrial import reactions are unphysiological, possibly because essential specificity factors are absent in these assays. Finally, we show that disruption of potential amphipathic helices in one presequence does not block import into pea mitochondria, indicating that other features are recognized.     

Varying competence for protein import into chloroplasts during the cell cycle in Chlamydomonas.

By studying the import of radioactively labelled small subunit of ribulose-1,5-bisphosphate carboxylase (pSS) into chloroplasts of the green alga C. reinhardtii cw-15 protein delivery to chloroplasts was found to vary during the cell cycle. Chloroplasts were isolated from highly synchronous cultures at different time points during the cell cycle. When pSS was imported into 'young' chloroplasts isolated early in the light period about three times less pSS was processed to small subunit SS than in 'mature' chloroplasts from the middle of the light period. In 'young' chloroplasts also, less pSS was bound to the envelope surface. During the second half of the light period the import competence of isolated chloroplasts decreased again when based on chlorophyll content or cell volume, but did not change significantly when related to chloroplast number. Measurements of pSS binding to the surface of chloroplasts of different age indicated that the adaptation of protein import competence during the cell cycle is due to a variation of the number of binding sites per chloroplast surface area, rather than to modulation of the binding constant.     

O-phospho-L-serine and the thiocarboxylated sulfur carrier protein CysO-COSH are substrates for CysM, a cysteine synthase from Mycobacterium tuberculosis

The kinetic pathway of CysM, a cysteine synthase from Mycobacterium tuberculosis, was studied by transient-state kinetic techniques. The expression of which is upregulated under conditions of oxidative stress. This enzyme exhibits extensive homology with the B-isozymes of the well-studied O-acetylserine sulfhydrylase family and employs a similar chemical mechanism involving a stable alpha-aminoacrylate intermediate. However, we show that specificity of CysM for its amino acid substrate is more than 500-fold greater for O-phospho-L-serine than for O-acetyl-L-serine, suggesting that O-phospho-L-serine is the likely substrate in vivo. We also investigated the kinetics of the carbon-sulfur bond-forming reaction between the CysM-bound alpha-aminoacrylate intermediate and the thiocarboxylated sulfur carrier protein, CysO-COSH. The specificity of CysM for this physiological sulfide equivalent is more than 3 orders of magnitude greater than that for bisulfide. Moreover, the kinetics of this latter reaction are limited by association of the proteins, while the reaction with bisulfide is consistent with a rapid equilibrium binding model. We interpret this finding to suggest that the CysM active site with the bound aminoacrylate intermediate is protected from solvent and that binding of CysO-COSH produces a conformational change allowing rapid sulfur transfer. This study represents the first detailed kinetic characterization of sulfide transfer from a sulfide carrier protein.     

Import of a precursor protein into chloroplasts is inhibited by the herbicide glyphosate

Import of the precursor to 5-enolpyruvylshikimate-3-phosphate synthase (pEPSPS) into chloroplasts is inhibited by the herbicide glyphosate. Inhibition of import is maximal at glyphosate concentrations of ≥10 μm and occurs only when pEPSPS is present as a ternary complex of enzyme–shikimate-3-phosphate–glyphosate. Glyphosate alone had no effect on the import of pEPSPS since it is not known to interact with the enzyme in the absence of shikimate-3-phosphate. Experiments with wild-type and glyphosate-resistant mutant forms of pEPSPS show that inhibition of import is directly proportional to the binding constants for glyphosate. Inhibition of import is thus a direct consequence of glyphosate binding to the enzyme–shikimate-3-phosphate complex. The potential for non-specific effects of glyphosate on the chloroplast transport mechanism has been discounted by showing that import of another chloroplast-designated protein was unaffected by high concentrations of glyphosate and shikimate-3-phosphate. The mechanism of import inhibition by glyphosate is consistent with a precursor unfolding/refolding model.     

The importance of the transit peptide and the transported protein for protein import into chloroplasts

Summary We compared the transport in vitro of fusion proteins of neomycin phosphotransferase II (NPTII) with either the transit peptide of the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase or the transit peptide and the 23 aminoterminal amino acids of the mature small subunit. The results showed that the transit peptide is sufficient for import of NPTII. However, transport of the fusion protein consisting of the transit peptide linked directly to NPTII was very inefficient. In contrast, the fusion protein containing a part of the mature SSU was imported with an efficiency comparable to that of the authentic SSU precursor. We conclude from these results that other features of the precursor protein in addition to the transit peptide are important for transport into chloroplasts. In order to identify functional regions in the transit peptide, we analyzed the transport of mutant fusion proteins. We found that the transport of fusion proteins with large deletions in the aminoterminal, or central part was drastically reduced. In contrast, duplication of a part of the transit peptide led to a marked increase in transport.     

A purified precursor polypeptide requires a cytosolic protein fraction for import into mitochondria.

The beta-subunit of mitochondrial ATPase is coded by a nuclear gene, synthesized outside the mitochondria as a larger precursor and imported into mitochondria. The beta-subunit precursor was purified from yeast, both as a homogeneous, unlabeled polypeptide and in radiochemically pure form. Both precursor preparations were cleaved to the mature beta-subunit by partially purified processing protease from the mitochondrial matrix. However, import of the radiochemically pure precursor into isolated yeast mitochondria required a cytosolic fraction from yeast or reticulocytes. The cytosolic factor was non-dialyzable and trypsin-sensitive; its apparent mol. wt. was approximately 40 000 as judged by gel filtration. Import of some proteins into mitochondria thus requires proteins of the 'soluble' cytoplasm.     

Stimulation of transit-peptide release and ATP hydrolysis by a cochaperone during protein import into chloroplasts

Three components of the chloroplast protein translocon, Tic110, Hsp93 (ClpC), and Tic40, have been shown to be important for protein translocation across the inner envelope membrane into the stroma. We show the molecular interactions among these three components that facilitate processing and translocation of precursor proteins. Transit-peptide binding by Tic110 recruits Tic40 binding to Tic110, which in turn causes the release of transit peptides from Tic110, freeing the transit peptides for processing. The Tic40 C-terminal domain, which is homologous to the C terminus of cochaperones Sti1p/Hop and Hip but with no known function, stimulates adenosine triphosphate hydrolysis by Hsp93. Hsp93 dissociates from Tic40 in the presence of adenosine diphosphate, suggesting that Tic40 functions as an adenosine triphosphatase activation protein for Hsp93. Our data suggest that chloroplasts have evolved the Tic40 cochaperone to increase the efficiency of precursor processing and translocation.     

Three sets of translocation intermediates are formed during the early stage of protein import into chloroplasts

During the early stage of protein import into chloroplasts, precursor proteins synthesized in the cytosol irreversibly bind to chloroplasts to form the early translocation intermediate under stringent energy conditions. Many efforts have been made to identify the components involved in protein import by analyzing the early intermediate. However, the state of the precursor within the intermediate has not been well investigated so far. In this study, an attempt was made to evaluate the extent of translocation of the precursor by determining the state of the precursor in the early intermediate under various conditions and analyzing the fragments generated by limited proteolysis of the precursors docked to chloroplasts. Our results indicate that three different sets of early intermediate are formed based on temperature and the hydrolysis of GTP/ATP. These have been identified based on the size of proteolytic fragments of the precursor as "energy-dependent association," "insertion," and "penetration" states. These findings suggest two individual ATP-hydrolyzing steps during the early stage of protein import, one of which is temperature-sensitive. Our results also demonstrate that translocation through the outer envelope membrane is mainly dependent on internal ATP.     

The triose phosphate-3-phosphoglycerate-phosphate translocator from spinach chloroplasts: nucleotide sequence of a full-length cDNA clone and import of the in vitro synthesized precursor protein into chloroplasts.

The nucleotide sequence of several cDNA clones coding for the phosphate translocator from spinach chloroplasts has been determined. The cDNA clones were selected from a lambda gt10 library prepared from poly(A)+ mRNA of spinach leaves using oligonucleotide probes modeled from amino acid sequences of tryptic peptides prepared from the isolated translocator protein. A 1439 bp insert of one of the clones codes for the entire 404 amino acid residues of the precursor protein corresponding to a mol. wt of 44,234. The full-length clone includes 21 bp at the transcribed non-coding 5' region with the ribosome initiation sequence ACAATGG, a 1212 bp coding region and 199 bp at the non-coding 3' region excluding the poly(A) tail which starts 17 bp downstream from a putative polyadenylation signal, AATAAT. According to secondary structure predictions the mature part of the chloroplast phosphate translocator exhibits high hydrophobicity and consists of at least seven membrane-spanning segments. Using plasmid-programmed wheat germ lysate the precursor protein was synthesized in vitro and could be imported into spinach chloroplasts where it is inserted into the inner envelope membrane.     

Point mutations destabilizing a precursor protein enhance its post-translational import into mitochondria.

In order to study the role of protein unfolding during post-translational protein import into mitochondria, we destabilized the structure of a mitochondrial precursor protein by site-directed mutagenesis. The precursor consisted of the first 16 residues of the yeast cytochrome oxidase subunit IV precursor fused to mouse dihydrofolate reductase. Labilization of the folded precursor structure was monitored by increased susceptibility to protease and diminished ability of methotrexate to block import of the precursor into isolated yeast mitochondria. On comparing the original precursor with two mutant forms that were destabilized to different degrees, increased labilization correlated with an increased rate and efficiency of import into mitochondria. This supports the view that the precursor must unfold in order to enter the mitochondria.     

Unfolding and refolding of a purified precursor protein during import into isolated mitochondria.

A purified mitochondrial precursor protein unfolds to a protease-sensitive conformation at the surface of isolated mitochondria before being imported into the organelles. This unfolding is stimulated by a potential across the mitochondrial inner membrane, but does not require ATP. In contrast, import of the surface-bound unfolded precursor requires ATP, but no potential; it is accompanied by a refolding inside the mitochondria.     

Mitochondrial protein import: involvement of the mature part of a cleavable precursor protein in the binding to receptor sites.

The precursor of F0-ATPase subunit 9 was bound to mitochondria in the absence of a mitochondrial membrane potential (delta psi). Binding was mediated by a protease-sensitive component on the mitochondrial surface. When delta psi was reestablished, bound precursor was directly imported without prior release from the mitochondrial membranes. A chimaeric protein consisting of the complete subunit 9 precursor fused to cytosolic dihydrofolate reductase (DHFR) was also specifically bound to mitochondria in the absence of delta psi. Two other fusion proteins, consisting either of the entire presequence of subunit 9 and DHFR or of part of the presequence and DHFR, were imported in the presence of delta psi. In the absence of delta psi, however, specific binding to mitochondria did not take place. We suggest that the hydrophobic mature part of subunit 9 is involved in the delta psi-independent binding of the subunit 9 precursor to receptor sites on the mitochondrial surface.