Studies on the import and processing of the alternative oxidase precursor by isolated soybean mitochondria

Abstract

Import of the synthetic precursor of the alternative oxidase from soybean was shown to be dependent on a membrane potential and ATP. The membrane potential in soybean mitochondria may be formed either by respiration through the cytochrome pathway, or through the alternative oxidase pathway with NAD(+)-linked substrates. Import of the alternative oxidase precursor in the presence of succinate as respiratory substrate was inhibited by KCN. Import in the presence of malate was insensitive to KCN and SHAM added separately, but was inhibited by KCN and SHAM added together (inhibitors of the cytochrome and alternative oxidases respectively). Import of the alternative oxidase was accompanied by processing of the precursor to a single 32 kDa product in both cotyledon and root mitochondria. This product had a different mobility than the two alternative oxidase bands detected by immunological means (34 and 36 kDa), suggesting that the enzyme had been modified in situ. When the cDNA clone of the alternative oxidase was modified by a single mutation (-2 Arg changed to -2 Gly), the processing of the precursor was inhibited.

Intracellular gene transfer: Reduced hydrophobicity facilitates gene transfer for subunit 2 of cytochromecoxidase

“…[ 35 S]-labeled precursor proteins were synthesized from cDNA clones, and in vitro import assays were carried out as previously described and modified by Daley at al. (16,23). Osmotic swelling to rupture the outer mitochondrial membrane of mitochondria was performed as described by Weinhues et al (24).…”

Plant mitochondria contain the protein translocase subunits TatB and TatC

“…The clones for AOX (X68702), Tim23 (At1g72750) and Rip (At5g13430) have been described previously (Murcha et al, 2003;Whelan et al, 1995;Carrie et al, 2015). [ 35 S]Met-labeled precursor proteins were synthesized using Flexi Rabbit Reticulocyte Lysate (Promega) as previously outlined (Chang et al, 2014).…”

“…[ 35 S]Met-labeled precursor proteins were synthesized using Flexi Rabbit Reticulocyte Lysate (Promega) as previously outlined (Chang et al, 2014). In vitro mitochondrial imports were then performed using isolated mitochondria as previously described (Whelan et al, 1995;Lister et al, 2007). All in vitro imports were obtained using radiography and images were scanned using a Typhoon scanner (GE Healthcare).…”

Dual Location of the Mitochondrial Preprotein Transporters B14.7 and Tim23-2 in Complex I and the TIM17:23 Complex in Arabidopsis Links Mitochondrial Activity and Biogenesis

“…protein import was affected in the tim23-2 KO and OE lines in Arabidopsis. Three proteins that contain N-terminal cleavable targeting signals and are imported via the general import pathway were used including the alternative oxidase (AOX) (Whelan et al, 1995), the F A d subunit of ATP synthase (F A d) (Smith et al, 1994), and the dual-targeted protein monodehydroascorbate reductase (MDHAR) (Chew et al, 2003). Protein import via the carrier import pathway was also tested using the adenine nucleotide carrier (ANT) and the phosphate carrier (Pic) (Murcha et al, 2005b).…”

“…[ 35 S]Met-labeled precursor proteins of AOX (X68702) (Whelan et al, 1995), F A d subunit of ATP synthase (X74296) (Smith et al, 1994), ANT (X57556), Pic (AB016063) (Murcha et al, 2005a), and MDHAR (At3g09940) (Chew et al, 2003) synthesized using the rabbit reticulocyte T N T in vitro transcription/translation lysate (Promega). Import of radiolabeled precursor protein into isolated mitochondria was performed as previously described at increasing time points.…”