A matrix-located processing peptidase of plant mitochondria.

Nuclear-encoded mitochondrial precursor proteins are proteolytically processed inside the mitochondrion after import. The general mitochondrial processing activity in plant mitochondria has been shown to be integrated into the cytochrome bc1 complex of the respiratory chain. Here we investigate the occurrence of an additional, matrix-located processing activity by incubation of the precursors of the soybean mitochondrial proteins, alternative oxidase, the FAd subunit of the ATP synthetase and the tobacco F1 beta subunit of the ATP synthase, with the membrane and soluble components of mitochondria isolated from soybean cotyledons and spinach leaves. A matrix-located peptidase specifically processed the precursors to the predicted mature form in a reaction which was sensitive to orthophenanthroline, a characteristic inhibitor of mitochondrial processing peptidase (MPP). The specificity of the matrix peptidase was illustrated by the inhibition of processing of the alternative oxidase precursor in both soybean and spinach matrix extracts upon altering a single amino acid residue in the targeting presequence (-2 Arg to Gly). Additionally, there was no evidence for general proteolysis of precursor proteins incubated with the matrix. The purity of the matrix fractions was ascertained by spectrophotometric and immunological analyses. The results demonstrate that there is a specific processing activity in the matrix of soybean and spinach in addition to the previously well characterized membrane-bound MPP integrated into the cytochrome bcl complex of the respiratory chain.

The occurrence of hsp70 in the outer membrane of plant mitochondria.

Hsp70 was localized to the mitochondrial outer membranes of bean and cauliflower mitochondria. Western blotting showed that the outer membrane hsp70 was antigenically distinct from the mitochondrial-matrix hsp70, but was similar to the cytosolic form. The protein was resistant to solubilization with 200 mM sodium carbonate which showed the hsp70 was tightly bound to the outer membrane. Proteinase K studies suggested that the hsp70 was partially exposed to the cytosol with approximately 17% of the protein protease-accessible. It is suggested that the position of the outer membrane hsp70 could relate to a precursor unfolding function during protein import into mitochondria.

Identification of a 42-kDa plant mitochondrial outer membrane protein, MOM42, involved in the import of precursor proteins into plant mitochondria.

A 42-kDa plant outer mitochondrial membrane protein, MOM42, has been identified as an essential component of the plant mitochondrial precursor protein translocation apparatus. Immunological cross-reactivity has been detected between antibodies raised against both Neurospora and yeast mitochondrial outer membrane proteins and plant mitochondrial outer membrane proteins. Immunocompetition studies showed that import of precursors to Rieske FeS protein, ATPase su9-DHFR, and the adenine nucleotide transporter was inhibited in the presence of antibody to MOM42. The inhibition of Rieske Fes and su9-DHFR import was greater than that of the adenine nucleotide transporter. The competition studies suggest that the MOM42 is involved in the translocation of bound precursor proteins. The import data and the Western blots suggest that components of the mitochondrial import system are highly conserved.

DNA profiling of Bursaphelenchus species.

A 1190-bp DNA fragment, designated X14, was isolated from a Bursaphelenchus xylophilus (isolate J10) genomic library. Used as a probe for DNA profiling, this fragment identifies the pinewood nematode (Bursaphelenchus spp.) at both the species and isolate level. The DNA profiles of a number of different species and isolates hybridised with the X14 probe, are presented. The nucleotide sequence of the 1190-bp probe was determined. Secondary structure analysis identified a large imperfect inverted repeat (IR) element within the probe which could form a large stem-loop structure in the RNA.

Detection and identification of bursaphelenchus species with DNA fingerprinting and polymerase chain reaction.

We have evaluated the potential of DNA-based methods to identify and differentiate Bursaphelenchus spp. and isolates. The isolation of a DNA probe, designated X14, and development of a DNA fingerprinting method for the identification and differentiation of Bursaphelenchus species and strains is described. Polymerase chain reaction (PCR) amplification of DNA isolated from Bursaphelenchus species using two primers derived from the sequence of the cloned repetitive DNA fragment X14 resulted in multiple band profiles. A 4-kb fragment thus amplified from B. xylophilus DNA was not amplified from B. mucronatus or B. fraudulentus DNA. In addition to this fragment, several other fragments are amplified from the three species. The banding patterns obtained allowed species identification and may have value in determining taxonomic affinities.

Specificity of leaf mitochondrial and chloroplast processing systems for nuclear-encoded precursor proteins.

The specificity of the mitochondrial and chloroplast processing enzymes for the nuclear-encoded precursor proteins was investigated. Mitochondrial precursor proteins of the Nicotiana plumbaginifolia and the Neurospora crassa beta subunits of F1-ATPase and the Neurospora Rieske FeS precursor protein were processed to the correct mature size by matrix extracts isolated from spinach leaves, yeast, rat liver and beef heart. The mitochondrial extracts failed to process chloroplast precursor proteins of the stromal small subunit of ribulose 1,5-bisphosphate carboxylase and the thylakoid 33 kDa protein of the oxygen-evolving complex. Both mitochondrial F1 beta precursors were specifically processed by a soluble stromal extract from chloroplasts. However, no processing of the Rieske FeS precursor protein was observed under the same conditions with the chloroplast extract. The cleavage of the mitochondrial F1 beta precursors by the chloroplast extract was shown to be sensitive to the metal chelators EDTA and ortho-phenanthroline. The cleavage site of the mitochondrial F1 beta precursor by the chloroplast soluble extract appears to be located at the N-terminus.

Processing of precursor proteins by plant mitochondria.

Precursor proteins from Neurospora crassa were correctly processed by a matrix extract from Vicia faba and cauliflower mitochondria. Processing yielded mature protein of the same molecular mass as mature Neurospora protein. The processing activity has two components. One is antigenically related to and of the same molecular mass as the processing enhancing protein of Neurospora. The second component was not recognized by antibody to the matrix processing protease from Neurospora mitochondria. The second component also houses the protease activity. Similar results were obtained using precursors to both the F1 beta subunit of the mitochondrial F0F1 ATPase and subunit V of the Rieske FeS complex from Neurospora. The beta subunit of the F0F1 ATPASE was processed to the mature form. Subunit V of the Rieske FeS complex was processed to the intermediate form only. Additional processing seen during import into plant mitochondria is not catalyzed by these proteins.

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

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

A water-soluble form of porin from the mitochondrial outer membrane of Neurospora crassa. Properties and relationship to the biosynthetic precursor form.

Mitochondrial porin, the outer membrane pore-forming protein, was isolated in the presence of detergents and converted into a water-soluble form. This water-soluble porin existed under nondenaturing conditions as a mixture of dimers and oligomers. The proportion of dimers increased with decreasing porin concentration during conversion. Water-soluble porin inserted spontaneously into artificial bilayers as did detergent-solubilized porin. Whereas the latter form had no specific requirements for the lipid composition of the bilayer, water-soluble porin inserted only into membranes containing a sterol, and only in the presence of very low concentrations of Triton X-100 (0.001% w/v) in the solution bathing the bilayer. The channels formed by water-soluble porin were indistinguishable from those formed by detergent-purified porin with respect to specific conductance and voltage dependence of conductance. Water-soluble porin bound tightly in a saturable fashion to isolated mitochondria. The bound form was readily accessible to added protease, indicating its presence on the mitochondrial surface. The number of binding sites was in the range of 5-10 pmol/mg of mitochondrial protein. Water-soluble porin apparently binds to a site on the assembly pathway of the porin precursor, since mitochondria whose binding sites were saturated with the water-soluble form did not import porin precursor synthesized in a cell-free system.

Transport of cytoplasmically synthesized proteins into the mitochondria in a cell free system from Neurospora crassa.

Synthesis and transport of mitochondrial proteins were followed in a cell-free homogenate of Neurospora crassa in which mitochondrial translation was inhibited. Proteins synthesized on cytoplasmic ribosomes are transferred into the mitochondrial fraction. The relative amounts of proteins which are transferred in vitro are comparable to those transferred in whole cells. Cycloheximide and puromycin inhibit the synthesis of mitochondrial proteins but not their transfer into mitochondria. The transfer of immunoprecipitable mitochondrial proteins was demonstrated for matrix proteins, carboxyatractyloside-binding protein and cytochrome c. Import of proteins into mitochondria exhibits a degree of specificity. The transport mechanism differentiates between newly synthesized proteins and preexistent mitochondrial proteins, at least in the case of matrix proteins. In the cell-free homogenate membrane-bound ribosomes are more active in the synthesis of mitochondrial proteins than are free ribosomes. The finished translation products appear to be released from the membrane-bound ribosomes into the cytosol rather than into the membrane vesicles. The results suggest that the transport of cytoplasmically synthesized mitochondrial proteins is essentially independent of cytoplasmic translation; that cytoplasmically synthesized mitochondrial proteins exist in an extramitochondrial pool prior to import; that the site of this pool is the cytosol for at least some of the mitochondrial proteins; and that the precursors in the extramitochondrial pool differ in structure or conformation from the functional proteins in the mitochondria.

Higher-plant mitochondrial ribosomes contain a 5S ribosomal ribonucleic acid component.

Ribosomes from higher-plant mitochondria contain 5S rRNA, in contrast with the mitochondrial ribosomes of animals and fungi, in which such a component has not been detected. In common with the ribosomes of prokaryotes and chloroplasts, higher-plant mitochondrial ribosomes do not appear to contain an RNA equivalent to the 5.8 S rRNA that is found in eukaryoytes hydrogen-bonded to the largest of the cytoplasmic rRNA species.

The metabolism of indoleacetic acid by barley grains.

It has been shown that indoleacetic acid (IAA) does not occur in developing grains of Hordeum vulgare L. (barley), but that an unidentified indolic compound does. This compound, designated 'A', was also found to be a product of the metabolism of exogenous IAA by barley. The expression of the gibberellic acid effect was delayed for at least 8 h if grains were imbibed in a solution of IAA, and during this time, the IAA was metabolised. The enzyme system involved could be peroxidase, which was active in the grains at all stages of their development and at maturity, and partially purified extracts of peroxidase were found to have considerable IAA oxidase activity.