Effect of mutations on the binding and translocation functions of a chloroplast transit peptide.

We studied transport and binding to intact chloroplasts of 10 mutants in three regions of the transit peptide of a precursor to the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxy-lyase (transphosphorylating), E.C.4.1.1.39]. Transport was assayed in a reconstituted system using isolated pea chloroplasts and radioactively labeled precursor. Binding to the chloroplast envelope was assayed in a similar manner using chloroplasts pretreated with nigericin. Most mutants showed a dramatically decreased capacity of binding, although some of them transported relatively well. The accumulation of the mutant proteins inside the chloroplast as a function of time was examined. Although the authentic small subunit precursor was imported rapidly, uptake of most mutant precursors was considerably slower and continued until the last time point examined. In terms of assigning functions to individual regions, we found that at least the middle region and parts of the amino and the carboxyl termini of the transit peptide are more important for receptor binding than for translocation. A two-step processing mechanism has been postulated for the maturation of the small subunit precursor. This model predicts the occurrence of processing intermediates. When precursors carrying carboxyl-terminal deletions were presented to the chloroplast, no defined intermediates could be detected. Instead, a number of proteins, probably resulting from aberrant processing, accumulated simultaneously inside the chloroplasts.     

The RNA-binding proteins HYL1 and SE promote accurate in vitro processing of pri-miRNA by DCL1

The results of genetic studies in Arabidopsis indicate that three proteins, the RNase III DICER-Like1 (DCL1), the dsRNA-binding protein HYPONASTIC LEAVES1 (HYL1), and the C2H2 Zn-finger protein SERRATE (SE), are required for the accurate processing of microRNA (miRNA) precursors in the plant cell nucleus. To elucidate the biochemical mechanism of miRNA processing, we developed an in vitro miRNA processing assay using purified recombinant proteins. We find that DCL1 alone releases 21-nt short RNAs from dsRNA as well as synthetic miR167b precursor RNAs. However, correctly processed miRNAs constitute a minority of the cleavage products. We show that recombinant HYL1 and SE proteins accelerate the rate of DCL1-mediated cleavage of pre- and pri-miR167b substrates and promote accurate processing.     

Structural diversity and differential light control of mRNAs coding for angiosperm glyceraldehyde-3-phosphate dehydrogenases

Subunits A and B of chloroplast glyceraldehyde-3-phosphate dehydrogenase are synthesized as higher molecular weight precursors when polyadenylylated mRNA from angiosperm seedlings is translated in vitro by wheat germ ribosomes. The in vivo levels of mRNA coding for these precursors are strongly light dependent, and the increase in translational activity stimulated by continuous white light, relative to dark-grown seedlings, is at least 5- to 10-fold for the seven plant species investigated. As opposed to this, light does not seem to change mRNA levels coding for cytosolic glyceraldehyde-3-phosphate dehydrogenase, and the polypeptides synthesized in vitro have the same size as the authentic subunits. In addition, precursors of the chloroplast enzyme were identified for 12 different angiosperm species and compared with their respective subunits synthesized in vivo. The patterns of the in vitro and in vivo products correlate in several major characteristics. They both display a remarkable interspecific heterogeneity with respect to size and number of polypeptides. The peptide extensions of the enzyme precursors calculated from these data vary between 4,000 and 12,000 daltons and seem to fall into three major size classes. The present data demonstrate that chloroplast glyceraldehyde-3-phosphate dehydrogenase, like its cytosolic counterpart, is encoded in the nucleus. Yet, the two dehydrogenases are controlled differently at both the ontogenetic and phylogenetic levels. They follow separate biosynthetic pathways with respect to light regulation, post-translational processing, and transport and also exhibit different evolutionary rates. The fast evolutionary change observed for the chloroplast enzyme contrasts sharply with the conservative structure and sequence of the cytosolic enzyme.     

Secretion and processing of insulin precursors in yeast.

A series of dibasic insulin precursors including proinsulin was expressed and secreted from Saccharomyces cerevisiae. Recombinant plasmids were constructed to encode fusion proteins consisting of a modified mating factor alpha 1 leader sequence and an insulin precursor. The leader sequence serves to direct the fusion protein into the secretory pathway of the cell and to expose it to the Lys-Arg processing enzyme system. The secreted peptides were purified from the fermentation broth and characterized by sequencing and amino acid analysis. Processing at one or both dibasic sequences was shown in proinsulin and in other insulin precursors containing a short spacer peptide in place of the C peptide. In contrast, no processing was observed in the absence of a spacer peptide in the insulin precursor molecule, e.g., B-Lys-Arg-A (where A and B are the A and B chain of human proinsulin, respectively). This type of single-chain insulin precursors isolated from such constructions could be enzymatically converted into insulin by treatment with trypsin and carboxypeptidase B. The above results suggest that the C-peptide region of proinsulin serves to direct the trypsin-like converting enzyme to process at the two dibasic sequences. We propose that in hormone precursors in general the spacer peptides serve to expose dibasic sequences for processing.     

Efficient in vitro import of a cytosolic heat shock protein into pea chloroplasts

In order to further our understanding of the targeting of nuclear-encoded proteins into intracellular organelles, we have investigated the import of chimeric precursor proteins into pea chloroplasts. Two different chimeric precursor proteins were produced by in vitro expression of chimeric genes. One chimeric precursor contained the transit peptide of the small subunit of soybean ribulose 1,5-bisphosphate carboxylase and the mature peptide of the same protein from pea. The second contained the same transit peptide plus 13 amino acids of the pea mature peptide fused to a cytosolic heat shock protein. The extent of import and binding of the two chimeric proteins was examined by using quantitative assays and was compared to the import of pea small subunit precursor. Both precursor proteins imported well into pea chloroplasts, although the extent of import observed with the chimeric small-subunit-heat shock precursor was less than that observed with the soybean-pea small subunit precursor. The heat shock protein alone did not import into nor bind to chloroplasts. The binding of both the chimeric small-subunit-heat shock protein and the soybean-pea small subunit precursor to chloroplasts was physiologically significant, as shown by the fact that when chloroplasts with bound precursors were isolated, these bound precursors could subsequently be imported.     

Two mitochondrial matrix proteases act sequentially in the processing of mammalian matrix enzymes.

The imported precursors of the mammalian matrix enzymes malate dehydrogenase [(S)-malate:NAD+ oxidoreductase, EC 1.1.1.37] and ornithine transcarbamylase (carbamoyl-phosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) are cleaved to their mature subunits in two steps, each catalyzed by matrix-localized processing proteases. The number and properties of these proteases are the subjects of this report. We have identified and characterized two distinct protease activities in a crude matrix fraction from rat liver: processing protease I, which cleaves these precursors to the corresponding intermediate form; and processing protease II, which cleaves the intermediate forms to mature subunits. Protease I is insensitive to chelation by EDTA and to inactivation with N-ethylmaleimide; protease II is inhibited by 5 mM EDTA and is inactivated by treatment with N-ethylmaleimide. We have prepared from mitochondrial matrix an 800-fold-enriched protease I fraction free of protease II activity by using the following steps: ion exchange, hydroxyapatite, molecular sieving, and hydrophobic chromatography. Using similar procedures, we also have prepared an approximately 2000-fold-enriched protease II fraction, which has a trace amount of contaminating protease I. This enriched protease II fraction has little or no cleavage activity toward mitochondrial precursors but rapidly and efficiently converts intermediate forms to mature size. Finally, we show that protease I alone is sufficient to cleave the precursor of a third nuclear-encoded mitochondrial protein subunit--the beta subunit of propionyl-CoA carboxylase [propanoyl-CoA:carbon dioxide ligase (ADP-forming), EC 6.4.1.3]--to its mature size.     

Plasmodium falciparum cysteine protease falcipain-2 cleaves erythrocyte membrane skeletal proteins at late stages of parasite development

Plasmodium falciparum-derived cysteine protease falcipain-2 cleaves host erythrocyte hemoglobin at acidic pH and specific components of the membrane skeleton at neutral pH. Analysis of stage-specific expression of these 2 proteolytic activities of falcipain-2 shows that hemoglobin-hydrolyzing activity is maximum in early trophozoites and declines rapidly at late stages, whereas the membrane skeletal protein hydrolyzing activity is markedly increased at the late trophozoite and schizont stages. Among the erythrocyte membrane skeletal proteins, ankyrin and protein 4.1 are cleaved by native and recombinant falcipain-2 near their C-termini. To identify the precise peptide sequence at the hydrolysis site of protein 4.1, we used a recombinant construct of protein 4.1 as substrate followed by MALDI-MS analysis of the cleaved product. We show that falcipain-2-mediated cleavage of protein 4.1 occurs immediately after lysine 437, which lies within a region of the spectrin-actin-binding domain critical for erythrocyte membrane stability. A 16-mer peptide containing the cleavage site completely inhibited the enzyme activity and blocked falcipain-2-induced fragmentation of erythrocyte ghosts. Based on these results, we propose that falcipain-2 cleaves hemoglobin in the acidic food vacuole at the early trophozoite stage, whereas it cleaves specific components of the red cell skeleton at the late trophozoite and schizont stages. It is the proteolysis of skeletal proteins that causes membrane instability, which, in turn, facilitates parasite release in vivo.     

Brief Report: Processing of a plant peptide hormone precursor facilitated by posttranslational tyrosine sulfation

Most peptide hormones and growth factors are matured from larger inactive precursor proteins by proteolytic processing and further posttranslational modification. Whether or how posttranslational modifications contribute to peptide bioactivity is still largely unknown. We address this question here for TWS1 (Twisted Seed 1), a peptide regulator of embryonic cuticle formation in Arabidopsis thaliana. Using synthetic peptides encompassing the N- and C-terminal processing sites and the recombinant TWS1 precursor as substrates, we show that the precursor is cleaved by the subtilase SBT1.8 at both the N and the C termini of TWS1. Recognition and correct processing at the N-terminal site depended on sulfation of an adjacent tyrosine residue. Arginine 302 of SBT1.8 was found to be required for sulfotyrosine binding and for accurate processing of the TWS1 precursor. The data reveal a critical role for posttranslational modification, here tyrosine sulfation of a plant peptide hormone precursor, in mediating processing specificity and peptide maturation.     

Protein cleavage during virus assembly: a novel specificity of assembly dependent cleavage in bacteriophage T4.

Cleavage of precursor proteins occurs during assembly of numerous viruses. Seven bacteriophage T4 head-related proteins areknown to be cleaved during morphogenesis. Sequences surrounding the cleavage sites in T4 head precursors P23 and IPIII are reported here. We previously determined the sequences of precursor and processed forms of IPII and IPI. Cleavage occurs at a glutamyl-alanyl bond in each protein. By comparison of sequences around five cleaved and four uncleaved Glu-Ala bonds in head precursors, it appears that cleavage is limited to the Thr or Ala, and X2 to hydrophilic residues. The results suggest the viral-induced assembly protease recognizes and cleaves an extended primary structure in the structurally dissimilar precursors.     

The Drosophila DPP signal is produced by cleavage of its proprotein at evolutionary diversified furin-recognition sites

Maturation of bone morphogenetic proteins (BMPs) requires cleavage of their precursor proteins by furin-type proprotein convertases. Here, we find that cleavage sites of the BMP2/4/decapentaplegic (DPP) subfamily have been evolutionary diversified and can be categorized into 4 different types. Cnidaria BMP2/4/DPP is considered to be a prototype containing only 1 furin site. Bilateria BMP2/4/DPP acquired an additional cleavage site with either the combination of minimal–optimal or optimal–optimal furin sites. DPPs belonging to Diptera, such as Drosophila and mosquito, and Lepidoptera of silkworm contain a third cleavage site between the 2 optimal furin sites. We studied how the 3 furin sites (FSI–III) of Drosophila DPP coordinate maturation of ligands and contribute to signals in vivo. Combining mutational analysis of furin-recognition sites and RNAi experiments, we found that the Drosophila DPP precursor is initially cleaved at an upstream furin-recognition site (FSII), with consequent cleavages at 2 furin sites (FSI and FSIII). Both Dfurin1 and Dfurin2 are involved in the processing of DPP proproteins. Biochemical and genetic analyses using cleavage mutants of DPP suggest the first cleavage at FSII to be critical and sufficient for long-range DPP signaling. Our data suggest that the Drosophila DPP precursor is cleaved in a different manner from vertebrate BMP4 even though they are functional orthologs. This indicates that the furin-cleavage sites in BMP2/4/DPP precursors are tolerant to mutations acquired through evolution and have adapted to different systems in diversified species.     

A protease responsible for post-translational cleavage of a conserved Asn-Gly linkage in glycinin, the major seed storage protein of soybean

The assembly of 11S globulin seed storage proteins in plants is regulated in part by the activity of a protease that cleaves between asparagine and glycine residues. Post-translational cleavage of subunit precursors into acidic and basic polypeptides is associated with the ability of subunits in trimers to aggregate into hexamers in vitro. An activity is present in extracts from immature soybean seeds that specifically cleaves immature 11S seed storage proteins of soybean and Vicia faba into the polypeptides of the mature proteins. Sequence microanalysis has been used to demonstrate that proglycinin and prolegumin are cut at the legitimate site when proteins synthesized in vitro are used as substrates. A single amino acid change in the cleavage site renders the substrate uncleavable. The protease responsible for this activity also hydrolyzes a synthetic octapeptide whose sequence reproduces four amino acids on either side of the glycinin subunit G4 cleavage site. This assay permitted the purification and characterization of the protease. It is a glycosylated enzyme with an acidic pH optimum and a molecular mass of about 45 kDa in solution.     

Posttranslational processing of progrowth hormone-releasing hormone

The prepro-GH-releasing hormone (prepro-GHRH; 12.3 kDa) precursor, like other neuropeptide precursors, undergoes proteolytic cleavage to give rise to mature GHRH, which is the primary stimulatory regulator of pituitary GH secretion. In this study we present the first model of in vitro pro-GHRH processing. Using pulse-chase analysis, we demonstrate that at least five peptide forms in addition to GHRH are produced. The pro-GHRH (after removal of its signal peptide, 10.5 kDa) is first processed to an 8.8-kDa intermediate form that is cleaved to yield two products: the 5.2-kDa GHRH and GHRH-related peptide (GHRH-RP; 3.6 kDa). GHRH-RP is a recently described peptide derived from proteolytic processing of pro-GHRH that activates stem cell factor, a factor known to be essential for hemopoiesis, spermatogenesis, and melanocyte function. Further cleavage results in a 3.5-kDa GHRH and a 2.2-kDa product of GHRH-RP. Like GHRH, there is GHRH-RP immunostaining in hypothalamic neurons in the median eminence as detected by immunohistochemistry and immunoelectron microscopy. Based on deduced amino acid sequences of the pro-GHRH processing products, several peptides were synthesized and tested for their ability to stimulate the cAMP second messenger system. GHRH, GHRH-RP, and one of these peptides [prepro-GHRH-(75-92)-NH2] all significantly stimulated the PKA pathway. This work delineates a new model of pro-GHRH processing and demonstrates that novel peptides derived from this processing may have biological action.     

Implications of the picornavirus capsid structure for polyprotein processing.

Mature picornaviral proteins are derived by progressive, posttranslational cleavage of a precursor polyprotein. These cleavages play a role in the control of virus functions. Although the processed termini are separated by as much as 75 A in the native virus capsid, the fold and arrangement of polypeptide chains in a protomer before proteolysis are likely to be similar to that found in the mature virus. The three-dimensional structures of rhinovirus and Mengo virus suggest that the cleavage sites within the protomeric precursor are in structurally flexible regions. The final proteolytic processing event, maturation of the virion peptide VP0 (also called peptide 1AB) appears to occur by an unusual autocatalytic serine protease-type mechanism possibly involving viral RNA basic groups that would serve as proton-abstractors during the cleavage reaction.     

Primary structure of a key enzyme in plant tetrapyrrole synthesis: glutamate 1-semialdehyde aminotransferase.

The formation of delta-aminolevulinate from glutamate 1-semialdehyde (GSA) is catalyzed by glutamate 1-semialdehyde aminotransferase (EC 5.4.3.8). The active form of the barley enzyme appears to be a dimer of identical subunits with a molecular mass of 46 kDa. From the purified enzyme, amino acid sequences of the N-terminal ends of the mature protein as well as an internal peptide were determined. DNA primers deduced from these peptide sequences were used to amplify with the polymerase chain reaction a cDNA sequence encoding part of the enzyme. Screening a cDNA library with this DNA fragment identified a full-length clone encoding the 49,540-Da precursor of the GSA aminotransferase. The transit peptide for chloroplast import consists of 34 amino acids. GSA aminotransferase and a precursor form were expressed on a multicopy plasmid in Escherichia coli. Both recombinant gene products reacted with an antibody against the barley GSA aminotransferase. Active barley GSA aminotransferase expressed in E. coli was shown to be active in assays of bacterial cell extracts. As a gene symbol for barley GSA aminotransferase, Gsa is proposed.     

Organellar oligopeptidase (OOP) provides a complementary pathway for targeting peptide degradation in mitochondria and chloroplasts

Both mitochondria and chloroplasts contain distinct proteolytic systems for precursor protein processing catalyzed by the mitochondrial and stromal processing peptidases and for the degradation of targeting peptides catalyzed by presequence protease. Here, we have identified and characterized a component of the organellar proteolytic systems in Arabidopsis thaliana, the organellar oligopeptidase, OOP (At5g65620). OOP belongs to the M3A family of peptide-degrading metalloproteases. Using two independent in vivo methods, we show that the protease is dually localized to mitochondria and chloroplasts. Furthermore, we localized the OPP homolog At5g10540 to the cytosol. Analysis of peptide degradation by OOP revealed substrate size restriction from 8 to 23 aa residues. Short mitochondrial targeting peptides (presequence of the ribosomal protein L29 and presequence of 1-aminocyclopropane-1-carboxylic acid deaminase 1) and N- and C-terminal fragments derived from the presequence of the ATPase beta subunit ranging in size from 11 to 20 aa could be degraded. MS analysis showed that OOP does not exhibit a strict cleavage pattern but shows a weak preference for hydrophobic residues (F/L) at the P1 position. The crystal structures of OOP, at 1.8–1.9 Å, exhibit an ellipsoidal shape consisting of two major domains enclosing the catalytic cavity of 3,000 ų. The structural and biochemical data suggest that the protein undergoes conformational changes to allow peptide binding and proteolysis. Our results demonstrate the complementary role of OOP in targeting-peptide degradation in mitochondria and chloroplasts.Protein turnover (degradation/biogenesis) in plant cells is a highly dynamic process, with as much as 50% of the total protein being replaced every 4–7 d (reviewed in ref. 1). In endosymbiotic organelles, mitochondria, and chloroplasts, the rate of protein degradation increases further during senescence and under conditions of environmental or developmental stress or insufficient carbon supply (reviewed in ref. 2). Highlighting the need for protein degradation is the fact that the genome of Arabidopsis thaliana encodes 723 putative proteases (3), the majority of which presently are uncharacterized.The machineries involved in protein degradation in plant cytoplasm and endosymbiotic organelles are highly conserved and reflect their evolutionary origin. Proteins in the cytoplasm and the nucleus are cleaved mostly by the 20S/26S proteasome. In turn, chloroplastic and mitochondrial protein degradation is carried out by proteases of bacterial origin belonging to the serine (Lon, ClpP, Deg) or metalloprotease (FtsH) type families (reviewed in refs. 4–6). To date ∼35 different proteases in the chloroplasts and mitochondria of A. thaliana have been identified as being involved in the removal of misfolded or oxidatively damaged proteins and in the degradation of unassembled subunits of protein complexes.Apart from the general degradation processes, chloroplasts and mitochondria also are sites of targeted proteolysis responsible for the maturation of precursor proteins. Approximately 3,000 chloroplastic and 1,000 mitochondrial proteins are synthesized in the cytosol as precursors carrying a targeting signal, allowing them to be imported posttranslationally to these organelles (7–9). For the proteins localized in the chloroplastic stroma or in the mitochondrial matrix, the targeting signal is an N-terminal extension termed “transit peptide” or “presequence,” respectively, which is cleaved off after import in a reaction termed “processing” (10). Most of the mitochondrial preproteins undergo processing by the mitochondrial processing peptidase (MPP), which in plants is integrated into the cytochrome bc1 complex of the respiratory chain (11). MPP is the only processing peptidase presently characterized in plant mitochondria. In some cases MPP cleavage is followed by additional trimming by octapeptidyl aminopeptidase 1 (Oct1), which cleaves off an N-terminal octapeptide (12, 13). Oct1 activity has been identified thus far only in yeast and mammals, but the gene coding for a putative ortholog also is present in the A. thaliana genome (5). Processing in the chloroplast stroma is carried out by the stromal processing peptidase (SPP), which cleaves off transit peptide and performs additional cleavage(s) within the transit peptide to generate shorter fragments (14, 15).In A. thaliana, mitochondrial presequences range from 19 to 109 aa (16); chloroplastic transit peptides usually are longer, ranging from 26 to 146 aa (9).Peptides generated as a result of preprotein processing can be degraded by the presequence protease (PreP), a metallopeptidase of the M16C family (reviewed in ref. 17). PreP initially was identified in plant mitochondria as the protease responsible for the degradation of the presequence of the ATPase beta subunit (pF₁β) and the chloroplastic transit peptide of the small subunit of ribulose-1,5-bisphosphate carboxylase oxygenase (18, 19). Consistent with this result, A. thaliana PreP (AtPreP) was shown to have a dual localization in mitochondrial matrix and chloroplastic stroma (18, 20). In addition to targeting peptides generated by processing, AtPreP1 degrades a wide range of unstructured peptides ranging from 10 from 65 aa (18). The crystal structure of AtPreP1 revealed that the enzyme consists of two bowl-shaped halves connected by a hinge region, forming a catalytic chamber of 10,000 ų, which is big enough to accommodate unstructured peptides up to 65 aa long; however, small folded proteins cannot be degraded. Residues localized in both halves of the enzyme contribute to proteolysis, suggesting that the structure must close for the cleavage to occur (21). Later studies led to the identification of AtPreP orthologs in yeast and mammals, both localized in mitochondrial matrix (22, 23). The human PreP also was shown to degrade amyloid-β peptides and has been associated with Alzheimer’s disease (23).In addition to AtPreP and its orthologs, it has been suggested that other proteases of the M3A family take part in the degradation of signal sequences. For instance, in Escherichia coli, oligopeptidase A (OpdA) degrades the 20-aa signal peptide of prolipoprotein (24). Mitochondria-localized peptidases of M3A family were found in yeast (PRD1) and mammals (neurolysin) (25, 26). Their function in mitochondria remains unknown, although it has been suggested that PRD1 cooperates with Cym1 (the PreP ortholog in yeast) in the degradation of mitochondrial presequences (27).The increase in abundance of peptides generated by either unspecific degradation or preprotein processing might influence organellar function and lead to various physiological consequences. For instance, in Caenorhabditis elegans peptides exported from mitochondria are involved in retrograde signaling as a part of the mitochondrial unfolded protein response (reviewed in ref. 28). Additionally, it has long been known that presequence peptides destabilize mitochondrial membranes, potentially leading to uncoupling of respiration or to dissipation of the membrane potential (29–32). Accumulation of presequences also was shown to impair mitochondrial precursor processing by direct inhibition of MPP activity (33). For these reasons, the turnover of peptides generated in mitochondria and chloroplasts must be regulated, either by active export or local degradation. In yeast, peptides up to 20 aa long can be exported out of mitochondria by an ABC-type transporter termed “Mdl1” (27, 34), but a similar system has not been identified in plants.Here we describe the identification and characterization of organellar oligopeptidase (OOP), an M3 plant peptidase localized within the mitochondria and chloroplasts. We have characterized the evolutionary origin, the intracellular localization, and the structural and catalytic properties of OOP. Our results provide biochemical evidence of mitochondrial presequence degradation by an M3 peptidase and also suggest that this protein has a broader role in general peptide degradation in these endosymbiotic organelles.     

Internal cleavage of the inhibitory 7B2 carboxyl-terminal peptide by PC2: a potential mechanism for its inactivation.

The neuroendocrine protein 7B2 contains two domains, a 21-kDa protein required for prohormone convertase 2 (PC2) maturation and a carboxyl-terminal (CT) peptide that inhibits PC2 at nanomolar concentrations. To determine how the inhibition of PC2 is terminated, we studied the metabolic fate of the 7B2 CT peptide in RinPE-7B2, AtT-20/PC2-7B2, and alphaTC1-6 cells. Extracts obtained from cells labeled for 6 h with [3H]valine were subjected to immunoprecipitation using an antibody raised against the extreme carboxyl terminus of r7B2, and immunoprecipitated peptides were separated by gel filtration. All three cell lines yielded two distinct peaks at about 3.5 kDa and 1.5 kDa, corresponding to the CT peptide and a smaller fragment consistent with cleavage at an interior Lys-Lys site. These results were corroborated using a newly developed RIA against the carboxyl terminus of the CT peptide which showed that the intact CT peptide represented only about half of the stored CT peptide immunoreactivity, with the remainder present as the 1.5-kDa peptide. Both peptides could be released upon phorbol 12-myristate 13-acetate stimulation. We investigated the possibility that PC2 itself could be responsible for this cleavage by performing in vitro experiments. When 125I-labeled CT peptide was incubated with purified recombinant PC2, a smaller peptide was generated. Analysis of CT peptide derivatives for their inhibitory potency revealed that CT peptide 1-18 (containing Lys-Lys at the carboxyl terminus) represented a potent inhibitor, but that peptide 1-16 was inactive. Inclusion of carboxypeptidase E (CPE) in the reaction greatly diminished the inhibitory potency of the CT peptide against PC2, in line with the notion that the CT peptide cleavage product is not inhibitory after the removal of terminal lysines by CPE. In summary, our data support the idea that PC2 cleaves the 7B2 CT peptide at its internal Lys-Lys site within secretory granules; deactivation of the cleavage product is then accomplished by CPE, thus providing an efficient mechanism for intracellular inactivation of the CT peptide.     

Arginine in the leader peptide is required for both import and proteolytic cleavage of a mitochondrial precursor.

Most mitochondrial proteins are encoded in the nucleus and translated in the cytoplasm as larger precursors containing NH2-terminal "leader" peptides, which are strikingly basic in overall amino acid composition. Recent experiments indicate that these leader peptides are both necessary and sufficient to direct post-translational recognition and import of precursors by mitochondria. In this report, we demonstrate a critical role for one or more of the basic arginine residues in the leader peptide of the subunit precursor for the human mitochondrial matrix enzyme, ornithine transcarbamoylase (ornithine carbamoyltransferase, carbamoylphosphate: L-ornithine carbamoyltransferase, EC 2.1.3.3). The distal three of four basic residues, all arginines, in the leader peptide of ornithine transcarbamoylase were replaced at once with charge-neutral glycine residues. The altered ornithine transcarbamoylase precursor failed to be taken up by intact mitochondria in vitro. Moreover, it also failed to be proteolytically cleaved upon incubation with a mitochondrial matrix fraction containing the Zn2+-dependent protease, which normally cleaves the leader peptide.