Molecular features, processing and import of the Rieske iron-sulfur protein from potato mitochondria

Abstract

The mitochondrial iron-sulfur protein (also termed Rieske iron-sulfur protein) of cytochrome c reductase was purified from potato tubers and identified with heterologous antibodies. The sequences of the N-terminus of this 25 kDa protein and of an internal peptide were determined to design oligonucleotide mixtures for screening a cDNA library. One class of cDNA clones containing an open reading frame of 265 amino acids was isolated. The encoded protein contains the peptide sequences of the 25 kDa protein and shares about 50% sequence identity with the Rieske iron-sulfur proteins from fungi and around 43% with those from mammals. In vitro transcription and translation of the cDNA reveals that the iron-sulfur protein is made as a larger precursor of 30 kDa which is processed by the cytochrome c reductase/processing peptidase complex from potato. The processing product obtained after in vitro processing has the same size as the mature protein imported into isolated mitochondria. The presequence, which targets the protein to the organelle, is 53 amino acids long and has molecular features different from those found in presequences of fungal iron-sulfur proteins, which are processed in two steps. Our results indicate that, unlike in yeast and Neurospora, the presequence of the iron-sulfur protein from potato is removed by a single processing enzyme in one step.

Modifications to the Arabidopsis Defense Proteome Occur Prior to Significant Transcriptional Change in Response to Inoculation withPseudomonas syringae

“…A spot containing the mature form of the mitochondrial Rieske Fe-S protein, a component of the mitochondrial cytochrome bc 1 complex (Emmermann et al, 1994), increased after all bacterial challenges. Remarkably, this spot was not present in the mock MgCl 2 treatment, suggesting that this represents a rapid and highly specific mitochondrial PAMP response.…”

Molecular Features and Mitochondrial Import Pathway of the 14-Kilodalton Subunit of Cytochrome c Reductase from Potato

“…107, 1995 In plants little is known about the small subunits of the bcj complex. Cyt c reductase from potato (Solanum tuberosum) comprises 10 subunits (Berry et al, 1991;Braun and Schmitz, 1992;Braun et al, 1994) and was shown to include the activity of the general mitochondrial processing peptidase, which cleaves off the presequences of nuclear-encoded mitochondrial precursors upon their import into the organelle (Braun et al, 1992a(Braun et al, , 1994. Here we report the identification, topography, sequence, and import pathway of the 14-kD protein from higher plants.…”

“…Antibodies were raised against the 14-kD protein from potato and used for immunological identification of the corresponding subunits of Cyt c reductase from wheat. The proteolytic fragmentation of the 14-kD proteins from potato and wheat, the separation of the generated peptides by reverse-phase HPLC, and the N-terminal sequencing of peptides by cyclic Edman degradation are described elsewhere (Braun et al, 1994).…”

“…Two oligonucleotide mixtures with the lowest degeneracy were derived from internal sequences of the 14-kD protein of Cyt c reductase from potato (Braun et al, 1994). The mixtures contained the full complement of sequences that could potentially encode the two octapeptides GluAsp-Leu-Gln-Ala-Met-Gln-Thr (384 combinations) and Glu-Ile-Val-Asp-Ala-Arg-Asn-Gln (2304 combinations).…”

“…Both systems allowed translation of the 14-kD protein but the reticulocyte lysate turned out to be more efficient. The isolation of mitochondria from potato for experiments on in vitro import of the 14-kD protein was described by Emmermann et al (1994). The conditions for the import experiments are detailed elsewhere (Braun and Schmitz, 1995).…”