Homologues of yeast and bacterial rotenone‐insensitive NADH dehydrogenases in higher eukaryotes: two enzymes are present in potato mitochondria

Abstract

SummaryTwo different cDNAs, homologous to genes for rotenone-insensitive NADH dehydrogenases of bacteria and yeast, were isolated from potato. The encoded proteins, called NDA and NDB, have calculated molecular masses of 55 and 65 kDa, respectively. The N-terminal parts show similarity to mitochondrial targeting peptides and the polypeptides are in vitro imported into potato mitochondria. Import processing to a smaller polypeptide is seen for the NDA but not the NDB protein. After import, NDA is intramitochondrially sorted to the matrix side of the inner membrane, whereas NDB becomes exposed to the intermembrane space. Imported proteins are associated to membranes upon digitonin permeabilization. On expression in Escherichia coli, NDB is released from the bacterial membrane in the absence of divalent cations whereas detergents are necessary for solubilization of NDA. Both deduced amino-acid sequences contain the dual motifs for nucleotide binding with the characteristics of the core criteria, similar to the bacterial homologues. Unique among NADH dehydrogenases, the NDB amino-acid sequence contains a nonconserved insert, which is similar to EF-hand motifs for calcium binding. Phylogenetic analyses group the rotenone-insensitive NADH dehydrogenases largely by species, but suggest ancient gene duplications.

Identification of AtNDI1, an Internal Non-Phosphorylating NAD(P)H Dehydrogenase in Arabidopsis Mitochondria

“…However, the insertion sequence and EF hand motif was also absent from the AT4g21490 sequence and the NDA-like NAD(P)H DH sequences. It has been proposed that the EF hand motif could play a role in binding the enzymes to the inner membrane (Rasmusson et al, 1999). However, this hypothesis is not consistent with the non-phosphorylating NAD(P)H DHs found in other species (specifically yeast enzymes) as suggested by Kerscher (2000).…”

“…The highest similarity was seen between AtNDI1 and AT2g29990, with 82% amino acid residue identity. The plant protein sequences separate into two groups that either show a high similarity to the previously characterized potato StNDA or StNDB sequences (Rasmusson et al, 1999). The NDA-like sequences have an average of 74% identity (ranging from 70%-82%) compared with an average of 58% identity seen between the plant NDB-like enzymes (ranging from 47%-65%).…”

“…1). This "insert" region contains two motifs that have homology to Ca 2ϩ -binding EF hand motif (Marsden et al, 1990), of which only the first is well conserved in potato NDB (Rasmusson et al, 1999) and Arabidopsis, and only the second is well conserved in NcNDE1 (Melo et al, 1999). The presence of this motif in the plant NDB-like NAD(P)H DH sequences is not unexpected because the corresponding activities are sensitive to calcium levels (Møller, 2001).…”

Identification and Characterization of Respirasomes in Potato Mitochondria

“…Due to the presence of numerous alternative oxidoreductases, the respiratory chain of plant mitochondria is very much branched (Vanlerberghe and McIntosh, 1997;Rasmusson et al, 1999;Moller, 2001;Moore et al, 2003). Furthermore, the protein complexes of the respiratory chain include plantspecific protein subunits (Braun and Schmitz, 1995;Eubel et al, 2003;Heazlewood et al, 2003aHeazlewood et al, , 2003bMillar et al, 2003).…”

A simple method for the addition of rotenone in Arabidopsis thaliana leaves

“…9 This is because the existence of additional NADH dehydrogenases involved in NADH oxidation in the plant organelle. 10 To further analyze the usefulness of the proposed method, we also determined the oxygen consumption rate in Arabidopsis leaves. Oxygen uptake was compared in control and rotenone/ Triton X-100 treated leaves.…”