Targeting and processing of a chimeric protein with the N-terminal presequence of the precursor to glyoxysomal citrate synthase.

Glyoxysomal citrate synthase in pumpkin is synthesized as a precursor that has a cleavable presequence at its N-terminal end. To investigate the role of the presequence in the transport of the protein to the microbodies, we generated transgenic Arabidopsis plants that expressed beta-glucuronidase with the N-terminal presequence of the precursor to the glyoxysomal citrate synthase of pumpkin. Immunogold labeling and cell fractionation studies showed that the chimeric protein was transported into microbodies and subsequently was processed. The chimeric protein was transported to functionally different microbodies, such as glyoxysomes, leaf peroxisomes, and unspecialized microbodies. These observations indicated that the transport of glyoxysomal citrate synthase is mediated by its N-terminal presequence and that the transport system is functional in all plant microbodies. Site-directed mutagenesis of the conserved amino acids in the presequence caused abnormal targeting and inhibition of processing of the chimeric protein, suggesting that the conserved amino acids in the presequence are required for recognition of the target or processing.     

The N-terminal portion of mature aldehyde dehydrogenase affects protein folding and assembly

Human liver cytosolic (ALDH1) and mitochondrial (ALDH2) aldehyde dehydrogenases are both encoded in the nucleus and synthesized in the cytosol. ALDH1 must fold in the cytosol, but ALDH2 is first synthesized as a precursor and must remain unfolded during import into mitochondria. The two mature forms share high identity (68%) at the protein sequence level except for the first 21 residues (14%); their tertiary structures were found to be essentially identical. ALDH1 folded faster in vitro than ALDH2 and could assemble to tetramers while ALDH2 remained as monomers. Import assay was used as a tool to study the folding status of ALDH1 and ALDH2. pALDH1 was made by fusing the presequence of precursor ALDH2 to the N-terminal end of ALDH1. Its import was reduced about 10-fold compared to the precursor ALDH2. The exchange of the N-terminal 21 residues from the mature portion altered import, folding, and assembly of precursor ALDH1 and precursor ALDH2. More of chimeric ALDH1 precursor was imported into mitochondria compared to its parent precursor ALDH1. The import of chimeric ALDH2 precursor, the counterpart of chimeric ALDH1 precursor, was reduced compared to its parent precursor ALDH2. Mature ALDH1 proved to be more stable against urea denaturation than ALDH2. Urea unfolding improved the import of precursor ALDH1 and the chimeric precursors but not precursor ALDH2, consistent with ALDH1 and the chimeric ALDHs being more stable than ALDH2. The N-terminal segment of the mature protein, and not the presequence, makes a major contribution to the folding, assembly, and stability of the precursor and may play a role in folding and hence the translocation of the precursor into mitochondria.     

cDNA cloning of rat liver 2,4-dienoyl-CoA reductase

cDNA clones of 2,4-dienoyl-CoA reductase were isolated from rat liver cDNA libraries constructed in phages lambda gt11 and lambda gt10. Hybrid selected translation analysis revealed that 2,4-dienoyl-CoA reductase was translated as a polypeptide with a molecular weight of about 36,000, which was about 3,000 molecular weight units larger than mature reductase. Sequencing analysis revealed that the open reading frame encoded a polypeptide consisting of 335 amino acid residues (predicted molecular weight = 36,132), which contained an N-terminal extension peptide of 34 amino acid residues (presequence) in addition to the mature enzyme. Thus, 2,4-dienoyl-CoA reductase is synthesized as a larger precursor polypeptide, and the N-terminal extension peptide may be acting as the mitochondrial import signal.     

NMR solution structure of the mitochondrial F1beta presequence from Nicotiana plumbaginifolia

We have isolated, characterized and determined the three-dimensional NMR solution structure of the presequence of ATPsynthase F1beta subunit from Nicotiana plumbaginifolia. A general method for purification of presequences is presented. The method is based on overexpression of a mutant precursor containing a methionine residue introduced at the processing site, followed by CNBr-cleavage and purification of the presequence on a cation-exchange column. The F1beta presequence, 53 amino acid residues long, retained its native properties as evidenced by inhibition of in vitro mitochondrial import and processing at micromolar concentrations. CD spectroscopy revealed that the F1beta presequence formed an alpha-helical structure in membrane mimetic environments such as SDS and DPC micelles (approximately 50% alpha-helix), and in acidic phospholipid bicelles (approximately 60% alpha-helix). The NMR solution structure of the F1beta presequence in SDS micelles was determined on the basis of 518 distance and 21 torsion angle constraints. The structure was found to contain two helices, an N-terminal amphipathic alpha-helix (residues 4-15) and a C-terminal alpha-helix (residues 43-53), separated by a largely unstructured 27 residue long internal domain. The N-terminal amphipathic alpha-helix forms the putative Tom20 receptor binding site, whereas the C-terminal alpha-helix is located upstream of the mitochondrial processing peptidase cleavage site.     

cDNA cloning and expression of a gene for 3-ketoacyl-CoA thiolase in pumpkin cotyledons

A cDNA clone for 3-ketoacyl-CoA thiolase (EC 2.3.1.16) was isolated from a gt11 cDNA library constructed from the poly(A)⁺ RNA of etiolated pumpkin cotyledons. The cDNA insert contained 1682 nucleotides and encoded 461 amino acid residues. A study of the expression in vitro of the cDNA and analysis of the amino-terminal sequence of the protein indicated that pumpkin thiolase is synthesized as a precursor which has a cleavable amino-terminal presequence of 33 amino acids. The amino-terminal presequence was highly homologous to typical amino-terminal signals that target proteins to microbodies. Immunoblot analysis showed that the amount of thiolase increased markedly during germination but decreased dramatically during the light-inducible transition of microbodies from glyoxysomes to leaf peroxisomes. By contrast, the amount of mRNA increased temporarily during the early stage of germination. In senescing cotyledons, the levels of the thiolase mRNA and protein increased again with the reverse transition of microbodies from leaf peroxisomes to glyoxysomes, but the pattern of accumulation of the protein was slightly different from that of malate synthase. These results indicate that expression of the thiolase is regulated in a similar manner to that of other glyoxysomal enzymes, such as malate synthase and citrate synthase, during seed germination and post-germination growth. By contrast, during senescence, expression of the thiolase is regulated in a different manner from that of other glyoxysomal enzymes.     

Functional Analysis of the N-Terminal Prepeptides of Watermelon Mitochondrial and Glyoxysomal Malate Dehydrogenases*

Mitochondrial and glyoxysomal malate dehydrogenase (mMDH; gMDH; L-malate: NAD⁺ oxidoreductase; EC 1.1.1.37) of watermelon (Citrullus vulgaris) cotyledons are synthesized with N-terminal cleavable presequences which are shown to specify sorting of the two proteins. The two presequences differ in length (27 or 37 amino acids) and primary structure. Precursor proteins of the two isoenzymes with site-directed mutations in their presequences and hybrid precursor proteins with reciprocally exchanged presequences were analyzed for proper import using two approaches, namely in vitro using isolated watermelon organelles or in vivo after synthesis in the heterologous host, Hansenula polymorpha. The mitochondrial presequence is essential and sufficient to target the mature glyoxysomal isoenzyme into mitochondria (Gietl et al., 1994). As to the function of the mitochondrial presequence a substitution of ^?3R (considered important for one step precursor cleavage in yeast and mammals) with ^?3L permitted import into mitochondria but cleavage of the transit peptide and conversion into active mature enzyme was impeded. Substitution of ^?13R^?12S (in a sequence reminiscent of the octapeptide motif serving as a substrate for the mammalian and yeast intermediate peptidase) into ^?13L¹²F permitted mitochondrial import and processing like the wild type transit peptide. Purified rat mitochondrial processing protease, which can effect single step cleavage of mitochondrial protein precursors, cleaves in vitro translated watermelon mMDH precursor into its mature form. The glyoxysomal presequence is essential and sufficient to target the mature mitochondrial isoenzyme into peroxisomes of Hansenula polymorpha, but these peroxisomes lack a processing enzyme to cleave the presequence (Gietl et al., 1994). We here show that isolated watermelon organelles also import the hybrid proteins in vitro and process the glyoxysomal presequence. Site directed mutations within the conserved RI-X₅-HL-motif impede efficiency of import and cleavage by watermelon organelles.     

The delta'-subunit of higher plant six-subunit mitochondrial F1-ATPase is homologous to the delta-subunit of animal mitochondrial F1-ATPase.

Mitochondrial F1-ATPases purified from several dicotyledonous plants contain six different subunits of alpha, beta, gamma, delta, delta' and epsilon. Previous N-terminal amino acid sequence analyses indicated that the gamma-, delta-, and epsilon-subunits of the sweet potato mitochondrial F1 correspond to the gamma-subunit, the oligomycin sensitivity-conferring protein and the epsilon-subunit of animal mitochondrial F1F0 complex (Kimura, T., Nakamura, K., Kajiura, H., Hattori, H., Nelson, N., and Asahi, T. (1989) J. Biol. Chem. 264, 3183-3186). However, the N-terminal amino acid sequence of the delta'-subunit did not show any obvious homologies with known protein sequences. A cDNA clone for the delta'-subunit of the sweet potato mitochondrial F1 was identified by oligonucleotide-hybridization selection of a cDNA library. The 1.0-kilobase-long cDNA contained a 600-base pair open reading frame coding for a precursor for the delta'-subunit. The precursor for the delta'-subunit contained N-terminal presequence of 21-amino acid residues. The mature delta'-subunit is composed of 179 amino acids and its sequence showed similarities of about 31-36% amino acid positional identity with the delta-subunit of animal and fungal mitochondrial F1 and about 18-25% with the epsilon-subunit of bacterial F1 and chloroplast CF1. The sweet potato delta'-subunit contains N-terminal sequence of about 45-amino acid residues that is absent in other related subunits. It is concluded that the six-subunit plant mitochondrial F1 contains the subunit that is homologous to the oligomycin sensitivity-conferring protein as one of the component in addition to five subunits that are homologous to subunits of animal mitochondrial F1.     

Putative ammo-terminal presequence for β-subunit of plant mitochondrial F1ATPase deduced from the amino-terminal sequence of the mature subunit

The α- and β-subunits of sweet potato mitochondrial F₁ATPase were purified from the F₁ complex by gel filtration and ion-exchange high-performance liquid chromatography. Isoelectric focussing and N-terminal amino acid sequencing indicated that the purified β-subunit contains at least two polypeptides similar to each other. The N-terminal 18 amino acid sequence of the β-subunit showed homology to the amino acid sequence of the tobacco mitochondrial F₁ATPase β-subunit precursor deduced from the nucleotide sequence [(1985) EMBO J. 4, 2159-2165] between residues 56 and 73, suggesting that the N-terminal 55 amino acids of the tobacco precursor constitute the presequence required for mitochondrial targetting.     

Changes in Targeting Efficiencies of Proteins to Plant Microbodies Caused by Amino Acid Substitutions in the Carboxy-terminal Tripeptide

It has been demonstrated that the carboxyl terminus of microbodyenzymes functions as a targeting signal to microbodies in higherplants. We have examined an ability of 24 carboxy-terminal aminoacid sequences to facilitate the transport of a cytosolic passengerprotein, ß-glucuroni-dase, into microbodies in greencotyledonary cells of trans-genic Arabidopsis. Immunoelectronmicroscopic analysis revealed that carboxy-terminal tripeptidesequences of the form [C/A/S/P]-[K/R]-[I/L/M] function as amicrobody-targeting signal, although tripeptides with prolineat the first amino acid position and isoleucine at the carboxylterminus show weak targeting efficiencies. All known micro-bodyenzymes that are synthesized in a form similar in size to themature molecule, except catalase, contain one of these tripeptidesequences at their carboxyl terminus. (Received April 14, 1997; Accepted April 8, 1997)     

Processing of mitochondrial presequences

Mitochondrial proteins are synthesized as precursor proteins on either cytosolic or mitochondrial ribosomes. The synthesized precursors from both translation origins possess targeting signals that guide the protein to its final destination in one of the four subcompartments of the organelle. The majority of nuclear-encoded mitochondrial precursors and also mitochondrial-encoded preproteins have an N-terminal presequence that serves as a targeting sequence. Specific presequence peptidases that are found in the matrix, inner membrane and intermembrane space of mitochondria proteolytically remove the signal sequence upon import or sorting. Besides the classical presequence peptidases MPP, IMP and Oct1, several novel proteases have recently been described to possess precursor processing activity, and analysis of their functional relevance revealed a tight connection between precursor processing, mitochondrial dynamics and protein quality control. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.     

Discrete mutations in the presequence of potato formate dehydrogenase inhibit the in vivo targeting of GFP fusions into mitochondria

Most mitochondrial proteins are encoded by the nucleus, translated in the cytosol, and imported. Mitochondrial precursors generally contain their targeting information in a cleavable N-terminal presequence, which is rich in hydroxylated and positively charged residues and can form amphiphilic alpha-helices. We report the in vivo targeting of green fluorescent protein (GFP) by the FDH presequence, as well as several truncated or mutated variants. Some of these mutations modify the amphiphilicity of the predicted alpha-helix. The removal of the first two residues abolishes import and some single amino acid mutations strongly inhibit import. Such strong effects on import had not been observed in similar studies on other plant mitochondrial presequences, suggesting that the FDH presequence is a particularly good model for functional studies.     

Primary structure requirements for correct sorting of the yeast mitochondrial protein ADH III to the yeast mitochondrial matrix space.

Alcohol dehydrogenase isoenzyme III (ADH III) in Saccharomyces cerevisiae, the product of the ADH3 gene, is located in the mitochondrial matrix. The ADH III protein was synthesized as a larger precursor in vitro when the gene was transcribed with the SP6 promoter and translated with a reticulocyte lysate. A precursor of the same size was detected when radioactively pulse-labeled proteins were immunoprecipitated with anti-ADH antibody. This precursor was rapidly processed to the mature form in vivo with a half-time of less than 3 min. The processing was blocked if the mitochondria were uncoupled with carbonyl cyanide m-chlorophenylhydrazone. Mutant enzymes in which only the amino-terminal 14 or 16 amino acids of the presequence were retained were correctly targeted and imported into the matrix. A mutant enzyme that was missing the amino-terminal 17 amino acids of the presequence produced an active enzyme, but the majority of the enzyme activity remained in the cytoplasmic compartment on cellular fractionation. Random amino acid changes were produced in the wild-type presequence by bisulfite mutagenesis of the ADH3 gene. The resulting ADH III protein was targeted to the mitochondria and imported into the matrix in all of the mutants tested, as judged by enzyme activity. Mutants containing amino acid changes in the carboxyl-proximal half of the ADH3 presequence were imported and processed to the mature form at a slower rate than the wild type, as judged by pulse-chase studies in vivo. The unprocessed precursor appeared to be unstable in vivo. It was concluded that only a small portion of the presequence contains the necessary information for correct targeting and import. Furthermore, the information for correct proteolytic processing of the presequence appears to be distinct from the targeting information and may involve secondary structure information in the presequence.     

Mutagenesis and computer modelling approach to study determinants for recognition of signal peptides by the mitochondrial processing peptidase

Determinants for the recognition of a mitochondrial presequence by the mitochondrial processing peptidase (MPP) have been investigated using mutagenesis and bioinformatics approaches. All plant mitochondrial presequences with a cleavage site that was confirmed by experimental studies can be grouped into three classes. Two major classes contain an arginine residue at position -2 or -3, and the third class does not have any conserved arginines. Sequence logos revealed loosely conserved cleavage motifs for the first two classes but no significant amino acid conservation for the third class. Investigation of processing determinants for a class III precursor, Nicotiana plumbaginifolia F1beta precursor of ATP synthase (pF1beta), was performed using a series of pF1beta presequence mutants and mutant presequence peptides derived from the C-terminal portion of the presequence. Replacement of -2 Gln by Arg inhibited processing, whereas replacement of either the most proximally located -5 Arg or -15 Arg by Leu had only a low inhibitory effect. The C-terminal portion of the pF1beta presequence forms a helix-turn-helix structure. Mutations disturbing or prolonging the helical element upstream of the cleavage site inhibited processing significantly. Structural models of potato MPP and the C-terminal pF1beta presequence peptide were built by homology modelling and empirical conformational energy search methods, respectively. Molecular docking of the pF1beta presequence peptide to the MPP model suggested binding of the peptide to the negatively charged binding cleft formed by the alpha-MPP and beta-MPP subunits in close proximity to the H111XXE114H115X(116-190)E191 proteolytic active site on beta-MPP. Our results show for the first time that the amino acid at the -2 position, even if not an arginine, as well as structural properties of the C-terminal portion of the presequence are important determinants for the processing of a class III precursor by MPP.     

Cloning and sequence analysis of cDNA encoding aspartate aminotransferase isozymes from Panicum miliaceum L., a C4 plant.

The cytosolic and mitochondrial isozymes of aspartate aminotransferase (AspAT) function in the C4 dicarboxylate cycle of photosynthesis. We constructed a cDNA library from leaf tissues of Panicum miliaceum, an NAD-malic-enzyme-type C4 plant and screened the library for AspAT isozymes. A full-length cDNA clone for cytosolic AspAT was isolated. This clone contains an open reading frame that encodes 409 amino acids. We also isolated two cDNA clones for different precursors of mitochondrial AspAT. Comparing these two sequences in the coding regions, we found 12 amino acid substitutions out of 28 base substitutions. The encoded amino acid sequences predict that mitochondrial AspAT are synthesized as precursor proteins of 428 amino acid residues, which each consist of a mature enzyme of 400 amino acid residues and a 28-amino-acid presequence. This prediction coincides with the observation that the in vitro translation product of the mRNA for mitochondrial AspAT was substantially larger than the mature form. A comparison of the amino acid sequences of the AspAT isozymes from P. miliaceum with the published sequences for the enzymes from various animals and microorganisms reveals that functionally and/or structurally important residues are almost entirely conserved in all AspAT species.     

Purification and characterization of precursors of mouse protamine mP2

Mouse protamine 2 (62 residues) is synthesized as a precursor pmP2 (106 residues) which is subsequently processed during the end of spermiogenesis. Three proteins with molecular and electrophoretic properties similar to those of pmP2 were isolated from testis purified nuclei. The comparison of their amino acid composition and of their N-terminal sequence with those of pmP2 shows that the three isolated proteins are products of pmP2 precursor processing. The three intermediate proteins pmP2/5, pmP2/11 and pmP2/20 which contain 102, 96 and 87 residues respectively, are generated from pmP2 precursors after N-terminal excision of 4, 10 and 19 residues, respectively.     

Complete nucleotide sequence of hepatic 5-aminolaevulinate synthase precursor

Chick embryo liver mitochondrial matrix protein, 5-aminolaevulinate synthase, is synthesised initially as a larger cytosolic precursor. In this report we present the complete nucleotide sequence of a cDNA clone coding for the precursor together with corresponding confirmatory amino acid sequence of peptides derived from purified mature mitochondrial enzyme. The deduced amino acid sequence shows that the precursor consists of mature enzyme of 579 amino acids and an N-terminal extension of 56 amino acids. The latter presequence is highly basic in character as found with other mitochondrial preproteins.