Crystal Structures of Mitochondrial Processing Peptidase Reveal the Mode for Specific Cleavage of Import Signal Sequences

The stromal processing peptidase (SPP) cleaves a large diversity of chloroplast precursor proteins, removing an N-terminal transit peptide. We predicted previously that this key step of the import pathway is mediated by features of the transit peptide that determine precursor binding and cleavage followed by transit peptide conversion to a degradable substrate. Here we performed competition experiments using synthesized oligopeptides of the transit peptide of ferredoxin precursor to investigate the mechanism of these processes. We found that binding and processing of ferredoxin precursor depend on specific interactions of SPP with the region consisting of the C-terminal 12 residues of the transit peptide. Analysis of four other precursors suggests that processing depends on the same region, although their transit peptides are highly divergent in primary sequence and length. Upon processing, SPP terminates its interaction with the transit peptide by a second cleavage, converting it to a subfragment form. From the competition experiments we deduce that SPP releases a subfragment consisting of the transit peptide without its original C terminus. Interestingly, examination of the ATP-dependent metallopeptidase activity responsible for degradation of transit peptide subfragments suggests that it may recognize other unrelated peptides and, hence, act separately from SPP as a novel stromal oligopeptidase.Chloroplasts are structurally and functionally complex organelles found in green plants and algae. They not only carry out photosynthesis but also perform other essential metabolic reactions required for plant growth and development. The vast majority of chloroplast proteins, however, is encoded by the nuclear genome. These proteins are synthesized in the cytoplasm as precursors containing an N-terminal chloroplast targeting signal, the transit peptide, that mediates post-translational import into the chloroplast. It is predicted from the genome sequence of Arabidopsis thaliana that up to 14% of nuclear genes encode precursor proteins with a transit peptide (1, 2). Thus, ϳ3500 different precursor proteins may be targeted to the chloroplast. The import pathway for these precursors depends on direct interactions between the transit peptide and import receptors of the outer envelope, translocation across the two envelope membranes, and removal of the transit peptide in the stromal compartment of the organelle (3-6).The cleavage of transit peptides during import is likely to be essential for chloroplast biogenesis and function. Given the central role of the chloroplast in plant development and the enormous amount of protein that must be imported, proteolytic processing of precursor proteins is one of the most important post-translational modifications that occur in a plant cell. Moreover, it was shown for the precursors of some highly abundant chloroplast proteins, e.g. ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit, ferredoxin, and plastocyanin, that the processing step is a prerequisite to adopt their acti...

Section: Spp Recognizes a Binding Site At The C Terminus Of A Transitmentioning

confidence: 99%

Determinants for Removal and Degradation of Transit Peptides of Chloroplast Precursor Proteins

Richter

Lamppa

2002

“…In addition, mutations that expedite trimer assembly interfered with Yfh1 precursor processing (supplemental Fig. S1), probably by inducing a protein fold incompatible with efficient cleavage by mitochondrial processing peptidase, whose activity depends on substrate conformation (64). Therefore, the rate-limiting step in Yfh1 assembly responds both to structural requirements for efficient maturation of the protein precursor as well as the need to prevent Yfh1 aggregation during mitochondrial iron overload.…”

Section: Discussionmentioning

confidence: 99%

Assembly of the Iron-binding Protein Frataxin in Saccharomyces cerevisiae Responds to Dynamic Changes in Mitochondrial Iron Influx and Stress Level

Gakh

Smith

Isaya

2008

Defects in frataxin result in Friedreich ataxia, a genetic disease characterized by early onset of neurodegeneration, cardiomyopathy, and diabetes. Frataxin is a conserved mitochondrial protein that controls iron needed for iron-sulfur cluster assembly and heme synthesis and also detoxifies excess iron. Studies in vitro have shown that either monomeric or oligomeric frataxin delivers iron to other proteins, whereas ferritin-like frataxin particles convert redox-active iron to an inert mineral. We have investigated how these different forms of frataxin are regulated in vivo. In Saccharomyces cerevisiae, only monomeric yeast frataxin (Yfh1) was detected in unstressed cells when mitochondrial iron uptake was maintained at a steady, low nanomolar level. Increments in mitochondrial iron uptake induced stepwise assembly of Yfh1 species ranging from trimer to >24-mer, independent of interactions between Yfh1 and its major ironbinding partners, Isu1/Nfs1 or aconitase. The rate-limiting step in Yfh1 assembly was a structural transition that preceded conversion of monomer to trimer. This step was induced, independently or synergistically, by mitochondrial iron increments, overexpression of wild type Yfh1 monomer, mutations that stabilize Yfh1 trimer, or heat stress. Faster assembly kinetics correlated with reduced oxidative damage and higher levels of aconitase activity, respiratory capacity, and cell survival. However, deregulation of Yfh1 assembly resulted in Yfh1 aggregation, aconitase sequestration, and mitochondrial DNA depletion. The data suggest that Yfh1 assembly responds to dynamic changes in mitochondrial iron uptake or stress exposure in a highly controlled fashion and that this may enable frataxin to simultaneously promote respiratory function and stress tolerance.

“…In fact, a crystal structure analysis of MPP localized the residues of the motif as part of an ␣-helix at the catalytic center, with the two histidines separated by one helical turn and complexed to a metal ion (presumably Zn 2ϩ ). The internal glutamic acid was near a water molecule that was also bound to the metal ion (35). A model for catalysis based on the metallopeptidase thermolysin proposes that the internal lanes 1 and 2).…”

Section: Discussionmentioning

confidence: 99%

“…SPP-(1-874) was sufficient to carry out precursor processing. The functional counterpart to SPP in mitochondria, MPP, is a heterodimeric complex of ϳ100 kDa cooperatively formed by ␣-and ␤-subunits of similar size (35). Sequence similarity to SPP, however, is restricted to the M16 peptidase region present in ␤-subunit (see above).…”

Section: Discussionmentioning

confidence: 99%

Structural Properties of the Chloroplast Stromal Processing Peptidase Required for Its Function in Transit Peptide Removal

Richter

Lamppa

2003

The stromal processing peptidase (SPP) catalyzes removal of transit peptides from a diversity of precursor proteins imported into chloroplasts. SPP contains an HXXEH zinc-binding motif characteristic of members of the metallopeptidase family M16. We previously found that the three steps of precursor processing by SPP (i.e. transit peptide binding, removal, and conversion to a degradable subfragment) are mediated by features that reside in the C-terminal 10 -15 residues of the transit peptide. In this study, we performed a mutational analysis of SPP to identify structural elements that determine its function. SPP loses the ability to proteolytically remove the transit peptide when residues of the HXXEH motif, found in an N-terminal region, are mutated. Deletion of 240 amino acids from its C terminus also abolishes activity. Interestingly, however, SPP can still carry out the initial binding step, recognizing the Cterminal residues of the transit peptide. Hence, transit peptide binding and removal are two separable steps of the overall processing reaction. Transit peptide conversion to a subfragment also depends on the HXXEH motif. The precursor of SPP, containing an unusually long transit peptide itself, is not proteolytically active. Thus, the SPP precursor is synthesized as a latent form of the metallopeptidase.Chloroplast biogenesis and function depends on the import of a large diversity of proteins from the cytoplasm. These proteins are synthesized as precursors containing an N-terminal targeting signal, called the transit peptide, that mediates multiple steps in the import pathway (for reviews, see Refs. 1-4). Whereas the chloroplast is unique to the plant cell, the mitochondrion and the endoplasmic reticulum (ER) 1 are two other major eukaryotic organelles that depend on massive protein translocation mediated by N-terminal targeting signals. It has been predicted for Arabidopsis thaliana that up to 11,000 different precursor proteins may be targeted to these three organelles, ϳ3,500 to the chloroplast alone (5). Distinct properties of the targeting signals ensure organelle-specific sorting and translocation of the precursor proteins. Ultimately, highly specialized proteolytic enzymes that reside in each organelle remove the targeting signals (for a review, see Ref. 6).We have identified a general stromal processing peptidase (SPP) that removes transit peptides from an array of precursors involved in different biosynthetic pathways and destined for different chloroplast compartments (7). The activity of SPP, which is a soluble, monomeric enzyme, depends on metal ions (8). Analysis of a full-length SPP cDNA from pea revealed that the enzyme contains an HXXEH zinc-binding motif characteristic of members of the metallopeptidase family M16, such as pitrilysin, insulin-degrading enzyme, and the ␤-subunit of the mitochondrial processing peptidase (MPP) (9, 10). Conservation extends beyond the zinc-binding motif, where 25-30% identity is found in an N-terminal region of ϳ200 residues of SPP. The residues of the HXX...