A mutation in the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase that reduces the rate of its incorporation into holoenzyme

A mutant of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), in which Arg53 is replaced by Glu, was synthesized and imported into isolated chloroplasts. The mutant protein was efficiently imported into the chloroplast and correctly processed to the mature size. Like the wild type protein, it was stable over a period of at least 2 h. Unlike the wilk-type protein however, most of the mutant protein was not assembled with holo-Rubisco at the end of a 10-min import reaction. It migrated instead as a diffused band on a non-denaturing gel, slower than the precursor protein, but faster than the holoenzyme. The level of the unassembled mutant protein in the stroma decreased with time, while its level in the assembled fraction has increased, indicating that this protein is a slowly-assembled, rather than a non-assembled, mutant of the small suubunit of Rubisco. Accumulation of the mutant protein in the holoenzyme fraction was dependent on ATP and light. The transient species, migrating faster than the holoenzyme but slower than the precursor protein, may represent an intermediate in the assembly process of the small subunit of RubiscoAbbreviations LSU large subunit of Rubisco - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - SSU small subunit of Rubisco     

Structure of a slow processing precursor penicillin acylase from Escherichia coli reveals the linker peptide blocking the active-site cleft

Penicillin G acylase is a periplasmic protein, cytoplasmically expressed as a precursor polypeptide comprising a signal sequence, the A and B chains of the mature enzyme (209 and 557 residues respectively) joined by a spacer peptide of 54 amino acid residues. The wild-type AB heterodimer is produced by proteolytic removal of this spacer in the periplasm. The first step in processing is believed to be autocatalytic hydrolysis of the peptide bond between the C-terminal residue of the spacer and the active-site serine residue at the N terminus of the B chain. We have determined the crystal structure of a slowly processing precursor mutant (Thr263Gly) of penicillin G acylase from Escherichia coli, which reveals that the spacer peptide blocks the entrance to the active-site cleft consistent with an autocatalytic mechanism of maturation. In this mutant precursor there is, however, an unexpected cleavage at a site four residues from the active-site serine residue. Analyses of the stereochemistry of the 260-261 bond seen to be cleaved in this precursor structure and of the 263-264 peptide bond have suggested factors that may govern the autocatalytic mechanism.     

Deletion of the carboxyl-terminal portion of the transit peptide affects processing but not import or assembly of the small subunit of ribulose-1,5-bisphosphate carboxylase

Import of the small subunit of ribulose-1,5-biphosphate carboxylase/oxygenase into the chloroplast has been proposed to involve two proteolytic cleavages which convert the 20-kDa precursor (pSSU) into the mature 14-kDa subunit (SSU) via an 18-kDa intermediate. A deletion mutant (PSd48/57) of pSSU which lacks 10 amino acids in a conserved region in the carboxyl-terminal portion of the transit peptide is converted into a series of 16-18-kDa polypeptides in addition to the mature 14-kDa SSU when imported into isolated pea chloroplasts. We examined import and processing of this mutant pSSU to determine whether the 16-18-kDa SSUs undergo further maturation in the chloroplast stroma to yield 14-kDa SSU. The ratio of incorrectly processed to 14-kDa SSU is stable up to 60 min following import. This indicates that processing of PSd48/57 involves a single proteolytic cleavage which occurs during or immediately following transit across the chloroplast envelope. The carboxyl-terminal portion of the transit peptide confers either sequence specificity for the processing protease or provides a three-dimensional structure necessary for consistent cleavage at the mature amino terminus of SSU. Incorrectly processed SSUs were incorporated into the holoenzyme demonstrating that removal of the entire transit sequence is not necessary for assembly of the holoenzyme.     

Determinants for cleavage of the chlorophyll a/b binding protein precursor: a requirement for a basic residue that is not universal for chloroplast imported proteins

We demonstrate that the precursor of the major light-harvesting chlorophyll a/b binding protein (LHCP of Photosystem II), encoded by a Type I gene, contains distinct determinants for processing at two sites during in vitro import into the chloroplast. Using precursors from both pea and wheat, it is shown that primary site processing, and release of a approximately 26-kD peptide, depends on an amino-proximal basic residue. Substitution of an arginine at position -4 resulted in an 80% reduction in processing, with the concomitant accumulation of a high molecular weight intermediate. Cleavage occurred normally when arginine was changed to lysine. The hypothesis that a basic residue is a general requirement for transit peptide removal was tested. We find that the precursors for the small subunit of Rubisco and Rubisco activase do not require a basic residue within seven amino acids of the cleavage site for maturation. In the wheat LHCP precursor, determinants for efficient cleavage at a secondary site were identified carboxy to the primary site, beyond what is traditionally called the transit peptide, within the sequence ala-lys-ala-lys (residues 38-41). Introduction of this sequence into the pea precursor, which has the residues thr-thr-lys-lys in the corresponding position, converted it to a substrate with an efficiently recognized secondary site. Our results indicate that two different forms of LHCP can be produced with distinct NH2-termini by selective cleavage of a single precursor polypeptide.     

Recognition of mitochondrial protein precursor lacking arginine at position -2 by mitochondrial processing peptidase: processing of bovine cytochrome P450(SCC) precursor

Mitochondrial processing peptidase (MPP) specifically cleaves off the N-terminal presequence of the mitochondrial protein precursor. Previous studies demonstrated that Arg at position -2 from the cleavage site, which is found among many precursors, plays a critical role in recognition by MPP. We analyzed the structural elements of bovine cytochrome P450 side-chain cleavage enzyme precursor [pre-P450(SCC)], which has Ala at position -2, for recognition by MPP. Replacement of Ala position -2 of pre-P450(SCC) with Arg resulted in an increase in the cleavage rate. Replacement with Gly caused a reduction in the cleavage rate and the appearance of an additional cleavage site downstream of the authentic site. A pre-P450(SCC) mutant with Met at position -2 retained cleavage efficiency equal to that of the wild type. These results indicate that -2 Ala of pre-P450(SCC) is recognized by MPP as a determinant for precise cleavage, and that the amino acid at -2 is required to have a straight methylene chain for interaction with the S(2) site. The preference for distal basic residues, a hydrophobic residue at +1, and hydroxyl residues at +2 and +3, was almost the same as those of the precursors with Arg at -2, indicating that the recognition mechanism of pre-P450(SCC) by MPP is essentially the same as that of the precursors with Arg at position -2.     

Mitochondrial import of Omi: the definitive role of the putative transmembrane region and multiple processing sites in the amino-terminal segment

The mitochondrial serine protease Omi/HtrA2 has a proapoptotic role in mammalian cells. However, neither the topology nor the processing of Omi in mitochondria is clearly understood. To determine the topology of Omi in the mitochondrial IMS, EGFP fusions were expressed with the entire N-terminal segment of full-length Omi (FL-Omi) (133-EGFP), and that without the transmembrane region (DeltaTM-EGFP) in the cells. Immunocytochemical staining and alkaline extraction experiments revealed that the TM determines the topology of Omi in the IMS and anchors the pro form into the inner membrane. As a result, the protease and the PDZ domains are exposed to the IMS. Mature Omi largely exists in the IMS as a soluble form. The processing sites of the precursor protein were examined by in vitro import experiments. The import of the processing mutants revealed importance of Arg80, Arg91, and Arg93 residues for the processing of the N-terminal segment of FL-Omi. These results suggest that the N-terminal segment of FL-Omi contains multiple processing sites processed by matrix processing proteases.     

Essential function in chloroplast recognition of the ferredoxin transit peptide processing region

Summary Plant ferredoxin is a nuclear-encoded chloroplast protein that is synthesized in the cytoplasm as a transit peptide-containing precursor molecule. To identify functional regions in the pre-ferredoxin transit peptide we constructed mutants with deletions of increasing length from the processing site toward the amino-terminus of the precursor. The mutant proteins were tested in an in vitro chloroplast binding and import assay. Deletion of the amino acids adjacent to the processing site completely abolishes binding and import. This region contains a sequence motif that is conserved among different precursor species. By constructing and testing mutants in the amino-terminal region of the mature part of the precursor protein, we found that this region of the molecule can greatly influence the import reaction.     

Crystal structure of the gamma-glutamyltranspeptidase precursor protein from Escherichia coli. Structural changes upon autocatalytic processing and implications for the maturation mechanism

Gamma-glutamyltranspeptidase (GGT) is an extracellular enzyme that plays a key role in glutathione metabolism. The mature GGT is a heterodimer consisting of L- and S-subunits that is generated by posttranslational cleavage of the peptide bond between Gln-390 and Thr-391 in the precursor protein. Thr-391, which becomes the N-terminal residue of the S-subunit, acts as the active residue in the catalytic reaction. The crystal structure of a mutant GGT, T391A, that is unable to undergo autocatalytic processing, has been determined at 2.55-A resolution. Structural comparison of the precursor protein and mature GGT demonstrates that the structures of the core regions in the two proteins are unchanged, but marked differences are found near the active site. In particular, in the precursor, the segment corresponding to the C-terminal region of the L-subunit occupies the site where the loop (residues 438-449) forms the lid of the gamma-glutamyl group-binding pocket in the mature GGT. This result demonstrates that, upon cleavage of the N-terminal peptide bond of Thr-391, the newly produced C terminus (residues 375-390) flips out, allowing the 438-449 segment to form the gamma-glutamyl group-binding pocket. The electron density map for the T391A protein also identified a water molecule near the carbonyl carbon atom of Gln-390. The spatial arrangement around the water and Thr-391 relative to the scissile peptide bond appears suitable for the initiation of autocatalytic processing, as in other members of the N-terminal nucleophile hydrolase superfamily.     

Crystal structures of mitochondrial processing peptidase reveal the mode for specific cleavage of import signal sequences

BACKGROUND: Mitochondrial processing peptidase (MPP) is a metalloendopeptidase that cleaves the N-terminal signal sequences of nuclear-encoded proteins targeted for transport from the cytosol to the mitochondria. Mitochondrial signal sequences vary in length and sequence, but each is cleaved at a single specific site by MPP. The cleavage sites typically contain an arginine at position -2 (in the N-terminal portion) from the scissile peptide bond in addition to other distal basic residues, and an aromatic residue at position +1. Mitochondrial import machinery recognizes amphiphilic helical conformations in signal sequences. However, it is unclear how MPP specifically recognizes diverse presequence substrates. RESULTS: The crystal structures of recombinant yeast MPP and a cleavage-deficient mutant of MPP complexed with synthetic signal peptides have been determined. MPP is a heterodimer; its alpha and beta subunits are homologous to the core II and core I proteins, respectively, of the ubiquinol-cytochrome c oxidoreductase complex. Crystal structures of two different synthetic substrate peptides cocrystallized with the mutant MPP each show the peptide bound in an extended conformation at the active site. Recognition sites for the arginine at position -2 and the +1 aromatic residue are observed. CONCLUSIONS: MPP bound two mitochondrial import presequence peptides in extended conformations in a large polar cavity. The presequence conformations differ from the amphiphilic helical conformation recognized by mitochondrial import components. Our findings suggest that the presequences adopt context-dependent conformations through mitochondrial import and processing, helical for recognition by mitochondrial import machinery and extended for cleavage by the main processing component.     

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

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

Complete processing of a small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from pea requires the amino acid sequence Ile-Thr-Ser

Chloroplast import and processing of two precursor proteins with mutations in the carboxyl-terminal region of the transit peptide were examined in vitro. Deletion mutations were introduced into the 57-amino acid transit peptide of a chloroplast protein, the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, from pea. A mutant, PSd48/57, in which nine carboxyl-terminal amino acids of the transit peptide had been deleted, was imported and processed to a series of 13- to 18-kDa polypeptides including the 14-kDa mature small subunit. In contrast, processing of a mutant, PSd45/57, in which an additional three amino acids had been removed, resulted in a series of polypeptides which did not include the mature small subunit. Whereas PSd48/57 was imported as efficiently as the wild-type precursor, import of PSd45/57 was only 25% as efficient as that of the authentic precursor. The mutant precursor proteins PSd48/57 and PSd45/57 are distinguished by a three-amino acid sequence, Ile-Thr-Ser, located in the carboxyl-terminal region of the transit peptide. We show that all or part of this sequence is required for correct processing.     

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.     

In vitro processing of the proproteins GrdE of protein B of glycine reductase and PrdA of D-proline reductase from Clostridium sticklandii: formation of a pyruvoyl group from a cysteine residue.

GrdE and PrdA of Clostridium sticklandii are subunits of glycine reductase and D-proline reductase, respectively, that are processed post-translationally to form a catalytic active pyruvoyl group. The cleavage occurred on the N-terminal side of a cysteine residue, which is thus the precursor of a pyruvoyl moiety. Both proproteins could be over-expressed in Escherichia coli and conditions were developed for in vitro processing. GrdE could be expressed as full-size protein, whereas PrdA had to be truncated N-terminally to achieve successful over-expression. Both proproteins were cleaved at the in vivo observed cleavage site after addition of 200 mM NaBH4 in Tris buffer (pH 7.6) at room temperature as analysed by SDS/PAGE and MS. Cleavage of GrdE was observed with a half-time of approximately 30 min. Cys242, as the precursor of the pyruvoyl group in GrdE, was changed to alanine, serine, or threonine by site-directed mutagenesis. The Cys242-->Ser and Cys242-->Thr mutant proteins were also cleaved under similar conditions with extended half-times. However, the Cys242-->Ala mutant protein was not cleaved indicating a pivotal role of the thiol group of cysteine or hydroxyl group of serine and threonine during the processing of pyruvoyl group-dependent reductases.     

Identification of a new active site for autocatalytic processing of penicillin acylase precursor in Escherichia coli ATCC11105

Penicillin acylase (PA) from Escherichia coli ATCC11105 is a periplasmic heterodimer consisting of a 24 kDa small subunit and a 65 kDa large subunit. It is synthesized as a single 96 kDa precursor and then matures to functional PA via a posttranslational processing pathway. The GST-PA fusion protein expression system was established for monitoring the precursor PA processing in vitro. The purified PA precursor was processed into mature PA the same way as in vivo, but pH dependently. From the primary sequence analysis, we identified a putative conserved lysine residue (K299) responsible for the pH dependent processing. The substitution of K299 residue by site-directed mutagenesis affected both the enzyme activity and the precursor PA processing in vivo. Furthermore, it was shown that the processing rates of wild-type and mutant precursor PAs depended on the pKa values of their side chain R group. These results demonstrated that the lysine residue (K299) was involved in the precursor processing of PA together with N-terminal serine residue (S290) of the large subunit.     

Mutation of the Oct-1 POU-specific recognition helix leads to altered DNA binding and influences enhancement of adenovirus DNA replication.

To assess which residues of Oct-1 POU-specific (POUs) are important for DNA recognition and stimulation of adenovirus DNA replication we have mutated 10 residues of the POUs helix-turn-helix motif implicated in DNA contact. Seven of these turned out to have reduced DNA binding affinity. Of these, three alanine substituted proteins were found to have a changed specificity using a binding site selection procedure. Mutation of the first residue in the recognition helix, Gln44, to alanine led to a loss of specificity for the first two bases, TA, of the wild-type recognition site TATGC(A/T)AAT. Instead of the A, a T was selected, suggesting a new contact and a novel specificity. A change in specificity was also observed for the T45A mutant, which could bind to TATAC(A/T)AAT, a site hardly recognized by the wild-type protein. Mutation of residue Arg49 led to a relaxed specificity for three consecutive bases, TGC. This residue, which is critical for high affinity binding, is absent from the structurally homologous lambdoid helix-turn-helix motifs. Employing a reconstituted system all but two mutants could stimulate adenovirus DNA replication upon saturation. Mutation of residues Gln27 and Arg49 impairs the ability of the Oct-1 POU domain protein to enhance replication, with a concomitant loss of DNA contacts. Since the POU domain binds the precursor terminal protein-DNA polymerase complex and guides it to the origin, lack of stimulation may be caused by incorrect targetting of the DNA polymerase due to loss of specificity.     

Crystal structures of penicillin acylase enzyme-substrate complexes: structural insights into the catalytic mechanism

The crystal structure of penicillin G acylase from Escherichia coli has been determined to a resolution of 1.3 A from a crystal form grown in the presence of ethylene glycol. To study aspects of the substrate specificity and catalytic mechanism of this key biotechnological enzyme, mutants were made to generate inactive protein useful for producing enzyme-substrate complexes. Owing to the intimate association of enzyme activity and precursor processing in this protein family (the Ntn hydrolases), most attempts to alter active-site residues lead to processing defects. Mutation of the invariant residue Arg B263 results in the accumulation of a protein precursor form. However, the mutation of Asn B241, a residue implicated in stabilisation of the tetrahedral intermediate during catalysis, inactivates the enzyme but does not prevent autocatalytic processing or the ability to bind substrates. The crystal structure of the Asn B241 Ala oxyanion hole mutant enzyme has been determined in its native form and in complex with penicillin G and penicillin G sulphoxide. We show that Asn B241 has an important role in maintaining the active site geometry and in productive substrate binding, hence the structure of the mutant protein is a poor model for the Michaelis complex. For this reason, we subsequently solved the structure of the wild-type protein in complex with the slowly processed substrate penicillin G sulphoxide. Analysis of this structure suggests that the reaction mechanism proceeds via direct nucleophilic attack of Ser B1 on the scissile amide and not as previously proposed via a tightly H-bonded water molecule acting as a "virtual" base.     

Mutations at the transit peptide-mature protein junction separate two cleavage events during chloroplast import of the chlorophyll a/b-binding protein

We have shown previously that during in vitro import into chloroplasts, the precursor of the major light-harvesting chlorphyll a/b-binding protein (LHCP) generated from a wheat gene gives rise to two mature forms (25 and approximately 26 kDa) which are inserted into the thylakoids. However, during incubation of the LHCP precursor with a chloroplast-soluble extract in an organelle-free processing reaction, the NH2 terminus is cleaved, yielding only a 25-kDa peptide. In the present study, mutations at the transit peptide-mature protein junction were introduced in the LHCP precursor to investigate the relationship between the two peptides and the determinants of proteolytic processing. Mutant p delta 3 lacks 3 amino acids including Met34 at the primary cleavage site thought to give rise to the 26-kDa peptide. It is still processed during import and in the organelle-free reaction yielding in both assays only a 25-kDa peptide. Mutant p + 4 has 4 amino acids inserted immediately after Met34 and a proline that disrupts the alpha-helix predicted by the Garnier-Osguthorpe-Robson method (Garnier, J., Osguthorpe, D. J., and Robson, B. (1978) J. Mol. Biol. 120, 97-120) to extend through this region. Although p + 4 is imported, it is inefficiently processed; both a 25- and 26-kDa peptide are found, but at least 60% of the imported precursor remains uncleaved. Less than 5% is processed in the organelle-free assay. Replacement of the predicted alpha-helix in the mutant p + 4 alpha restores processing upon import into the chloroplast, but this mutant, which also has a 4-amino acid insert, yields only a 26-kDa peptide. p + 4 alpha is not processed in the organelle-free reaction. These results provide evidence that the two forms of LHCP obtained during import are the result of independent processing at two cleavage sites: the first site at Met34, and a second approximately 10 amino acids downstream within what has been designated the NH2 terminus of the mature protein. Whereas p delta 3 has the first site removed but retains a functional second site, in p + 4 alpha only the first site, or one very near it, is accessible to the processing enzyme during import. The conditions of the organelle-free reaction are specific for processing at only the secondary site. We discuss the implications of these findings in terms of the heterogeneity of LHCP in vivo.     

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.     

A signal sequence domain essential for processing, but not import, of mitochondrial pre-ornithine carbamyl transferase

Studies using deletion mutagenesis indicate that a processing recognition site lies proximal to the normal cleavage position between gln32 and ser33 of pre-ornithine carbamyl transferase (pOCT). pOCT cDNA was manipulated to delete codons specifying amino acids 22-30 of the signal sequence. The mutant precursor, designated pOCT delta 22-30, was imported to the matrix compartment by purified mitochondria, but remained largely unprocessed; the low level of processing that was observed did not involve the normal cleavage site. Several manipulations performed downstream of the normal pOCT processing site (deletion, substitution, and hybrid protein constructions) affected neither import nor correct processing. Our data suggest that domains specifying import and processing site recognition may be functionally segregated within the signal peptide; that processing is not requisite for import of pOCT; and that a proximal region, not involving the normal signal peptide cleavage site, is required for processing site recognition.