Signals Required for the Import and Processing of the Alternative Oxidase into Mitochondria

Abstract

The critical residues involved in targeting and processing of the soybean alternative oxidase to plant and animal mitochondria was investigated. Import of various site-directed mutants into soybean mitochondria indicated that positive residues throughout the length of the presequence were important for import, not just those in the predicted region of amphiphilicity. The position of the positive residues in the C-terminal end of the presequence was also important for import. Processing assays of the various constructs with purified spinach mitochondrial processing peptidase showed that all the ؊2-position mutants had a drastic effect on processing. In contrast to the import assay, the position of the positive residue could be changed for processing. Deletion mutants confirmed the site-directed mutagenesis data in that an amphiphilic ␣-helix was not the only determinant of mitochondrial import in this homologous plant system. Import of these constructs into rat liver mitochondria indicated that the degree of inhibition differed and that the predicted region of amphiphilic ␣-helix was more important with rat liver mitochondria. Processing with a rat liver matrix fraction showed little inhibition. These results are discussed with respect to targeting specificity in plant cells and highlight the need to carry out homologous studies and define the targeting requirements to plant mitochondria.

Alternative oxidase: an inter-kingdom perspective on the function and regulation of this broadly distributed 'cyanide-resistant' terminal oxidase

“…1). This subcellular localisation in soybean (and likely other eukaryotes) requires the presence of a presequence for mitochondrial targeting (Tanudji et al 1999;Fig. 3), and provides an explanation as to why the N-terminal regions of eukaryotic AOXs are longer than those of prokaryotes (Fig.…”

Refining the Definition of Plant Mitochondrial Presequences through Analysis of Sorting Signals, N-Terminal Modifications, and Cleavage Motifs

“…Many mitochondrial presequences have a loosely conserved motif near the cleavage site comprising an Arg residue at the 22 and/or 23 position (von Heijne et al, 1989;Schneider et al, 1998). This Arg has been experimentally shown to be an important recognition site for the mitochondrial processing peptidase (MPP; Arretz et al, 1994;Ogishima et al, 1995;Tanudji et al, 1999). MPP is a heterodimeric enzyme that contains two similar subunits: a-MPP is involved in binding precursor proteins and b-MPP catalyzes the cleavage of the presequence Luciano et al, 1997).…”

New functions of the chloroplast Preprotein and Amino acid Transporter (PRAT) family members in protein import

“…16 Moreover, 2 unique Gly residues were identified at positions 4 and 6 in HP30, but no Gly residues were found in this region for HP30-2. 16 Because insertion of Gly residues into a mitochondrial targeting signal has been reported to abolish mitochondrial targeting ability, 37 these differences could regulate the faithful targeting of HP30 to chloroplasts. How HP20 is imported into chloroplasts has not been analyzed.…”