Polyclonal and monoclonal antibodies that recognize the 35-, 36-, and 37-kDa alternative oxidase proteins of Sauromatum guttatum (Schott) were used to isolate a cDNA clone, pAOSG81, from an S. guttatum cDNA expression library. A fusion protein with an apparent molecular mass of 48 kDa was expressed from a pUell9 derivative of pAOSG81 (pAOSG81-119) in Escherichia coli cells and was recognized by the monoclonal antibodies. When the in vitro translated and immunoprecipitated products made from mRNA hybridselected by pAOSG81 were analyzed, a single band corresponding to a protein with an apparent molecular mass of42 kDa was observed. DNA sequence characterization showed that pAOSG81 contains the entire coding region of a protein with a calculated molecular mass of 38.9 kDa, a putative 63-amino acid transit peptide, and a 9-amino acid match to the authentic N-terminal sequence of the 36-kDa alternative oxidase protein.Analyses of the deduced amino acid sequence indicate: (i) that the transit peptide is predicted to form amphiphilic helices, and (ii) that three regions of the processed protein are likely to form transmembrane a-helices. We conclude from these data that pAOSG81 represents a nuclear gene, aoxl, encoding a precursor protein of one or more of the alternative oxidase proteins of S. guttatum.
Chemical inhibition of the mitochondrial electron transport chain (mtETC) by antimycin A (AA) or the TCA cycle by monofluoroacetate (MFA) causes increased expression of nucleus-encoded alternative oxidase (AOX) genes in plants. In order to better understand the mechanisms of this mitochondrial retrograde regulation (MRR) of gene expression, constructs containing deleted and mutated versions of a promoter region of the Arabidopsis thaliana AOX1a gene (AtAOX1a) controlling expression of the coding region of the enhanced firefly luciferase gene were employed to identify regions of the AtAOX1a promoter important for induction in response to mtETC or TCA cycle inhibition. Transient transformation coupled with in vitro and in vivo assays as well as plants containing transgenes with truncated promoter regions were used to identify a 93 base pair portion of the promoter, termed the MRR region, that was necessary for induction. Further mutational analyses showed that most of the 93 bp MRR region is important for both AA and MFA induction. Sub-regions within the MRR region that are especially important for strong induction by both AA or MFA were identified. Specific mutations in a W-box and Dof motifs in the MRR region indicate that these specific motifs are not important for induction. Recent evidence suggests that MRR of AOX genes following inhibition of the mtETC is via a separate signaling pathway from MRR resulting from metabolic shifts, such as those that result from MFA treatment. Our data suggest that these signaling pathways share regulatory regions in the AtAOX1a promoter. Arabidopsis proteins interacted specifically with a probe containing the MRR region, as shown by electrophoretic mobility shift assays and Southwestern blotting. These interactions were eliminated under reducing conditions.
Alternative respiratory pathway capacity increases during the development of the thermogenic appendix of a voodoo lily inflorescence. The levels of the alternative oxidase proteins increased dramatically between D-4 (4 days prior to the day of anthesis) and D-3 and continued to increase until the day of anthesis (D-day). The level of salicylic acid (SA) in the appendix is very low early on D-1, but increases to a high level in the evening of D-1. Thermogenesis occurs after a few hours of light on D-day. Therefore, the initial accumulation of the alternative oxidase proteins precedes the increase in SA by 3 days, indicating that other regulators may be involved. A 1.6-kb transcript encoding the alternative oxidase precursor protein accumulated to a high level in the appendix tissue by D-1. Application of SA to immature appendix tissue caused an increase in alternative pathway capacity and a dramatic accumulation of the alternative oxidase proteins and the 1.6-kb transcript. Time course experiments showed that the increase in capacity, protein levels, and transcript level corresponded precisely. The response to SA was blocked by cycloheximide or actinomycin D, indicating that de novo transcription and translation are required. However, nuclear, in vitro transcription assays indicated that the accumulation of the 1.6-kb transcript did not result from a simple increase in the rate of transcription of aox1.
Perturbation of mitochondrial function causes altered nuclear gene expression in plants. To study this response, called mitochondrial retrograde regulation, and developmental gene expression, a transgenic Arabidopsis thaliana (Col-0) line containing a firefly luciferase gene controlled by a promoter region of the Arabidopsis alternative oxidase 1a gene (AtAOX1a) was created. The transgene and the endogenous gene were developmentally induced in young cotyledons to a level higher than in older cotyledons and leaves. Analysis of transgene expression suggests that this is true for emerging leaves as well. Antimycin A (AA), a mitochondrial electron transport chain inhibitor, and monofluroacetate (MFA), a TCA cycle inhibitor, induced expression of the transgene and the endogenous gene in parallel. The following comparative responses of the transgene to inhibitors were observed: (a) the response in cotyledons to AA treatment differed greatly in magnitude from the response in leaves; (b) the induction kinetics in cotyledons following MFA treatment differed greatly from the kinetics in leaves; and (c) the induction kinetics following MFA treatment differed from the kinetics of AA in both leaves and cotyledons. The transgenic line was used in a genetic screen to isolate mutants with greatly decreased transgene and AtAOX1a induction in response to AA. Some of these mutant lines showed greatly decreased induction by MFA, but one did not. Taken altogether, the data provide genetic evidence that suggests that induction of the AtAOX1a gene by distinct mitochondrial perturbations are via distinct, but overlapping signaling pathways that are tissue specific.
In many organisms, an increasing number of proteins seem to play two or more unrelated roles. Here we report that maize sucrose synthase (SUS) is distributed in organelles not involved in sucrose metabolism and may have novel roles beyond sucrose degradation. Bioinformatics analysis predicts that among the three maize SUS isoforms, SH1 protein has a putative mitochondrial targeting peptide (mTP). We validated this prediction by the immunodetection of SUS in mitochondria. Analysis with isoform-specific antisera revealed that both SH1 and SUS1 are represented in mitochondria, although the latter lacks a canonical mTP. The SUS2 isoform is not detectable in mitochondria, despite its presence in the cytosol. In maize primary roots, the mitochondrion-associated SUS (mtSUS; which includes SH1 and SUS1) is present mostly in the root tip, indicating tissue-specific regulation of SUS compartmentation. Unlike the glycolytic enzymes that occur attached to the outside of mitochondria, SH1 and SUS1 are intramitochondrial. The low abundance of SUS in mitochondria, its high K m value for sucrose, and the lack of sucrose in mitochondria suggest that mtSUS plays a non-sucrolytic role. Co-immunoprecipitation studies indicate that SUS interacts with the voltage-dependent anion channel in an isoform-specific and anoxia-enhanced manner and may be involved in the regulation of solute fluxes into and out of mitochondria. In several plant species, at least one of the SUS proteins possesses a putative mTP, indicating the conservation of the noncatalytic function across plant species. Taken together, these observations suggest that SUS has a novel noncatalytic function in plant cells.In both plant and animal cells, a small number of catalytic and structural proteins play additional roles that in some cases are regulatory in nature (1-3). These "moonlighting" proteins possess the ability to assemble into multiprotein complexes and mediate sophisticated biological functions such as integrating signals. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 2 is the most studied among such versatile proteins (4). Besides its pivotal role in energy metabolism, GAPDH is shown to be involved in membrane fusion, microtubule assembly, RNA transport, DNA replication and repair, and cell death (5). The functional diversity of GAPDH is facilitated by its ability to interact with other proteins and translocate to multiple subcellular compartments. Sucrose synthase (SUS) catalyzes the reversible conversion of sucrose and UDP into fructose and UDP-glucose and is a key player in plant sucrose catabolism. In maize, the following three genes encoding sucrose synthase are known: sus1, sus2, and sh1. The isoforms encoded by these genes are symbolized SUS1, SUS2, and SUS-SH1, respectively (6). In this study, for the sake of simplicity, we call the SUS-SH1 isoform SH1. All the three isoforms are predominantly recovered as soluble (cytosolic) proteins from plant cells, in accordance with their predicted secondary structures. The well documented function of this en...
The cyanide-resistant alternative oxidase of plant mitochondria is a homodimeric protein whose activity can be regulated by a redox-sensitive intersubunit sulfhydryl/disulfide system and by ␣-keto acids. After determining that the Arabidopsis alternative oxidase possesses the redox-sensitive sulfhydryl/disulfide system, site-directed mutagenesis of an Arabidopsis cDNA clone was used to individually change the two conserved Cys residues, Cys-128 and Cys-78, to Ala. Using diamide oxidation and chemical cross-linking of the protein expressed in Escherichia coli, Cys-78 was shown to be: 1) the Cys residue involved in the sulfhydryl/disulfide system; and 2) not required for subunit dimerization. The C128A mutant was stimulated by pyruvate, while the C78A mutant protein had little activity and displayed no stimulation by pyruvate. Mutating Cys-78 to Glu produced an active enzyme which was insensitive to pyruvate, consistent with ␣-keto acid activation occurring through a thiohemiacetal. These results indicate that Cys-78 serves as both the regulatory sulfhydryl/disulfide and the site of activation by ␣-keto acids. In light of these results, the previously observed effects of sulfhydryl reagents on the alternative oxidase of isolated soybean mitochondria were re-examined and were found to be in agreement with a single sulfhydryl residue being the site both of ␣-keto acid activation and of the regulatory sulfhydryl/disulfide system.In all plants and many fungi, a cyanide-resistant, alternative respiratory pathway branches from the cytochrome pathway at the ubiquinone pool in mitochondria (1). Electron flow through this alternative pathway is not coupled to ATP synthesis at two of the three proton translocation sites. In specific thermogenic floral tissues of some plants, a high rate of electron flow through the alternative pathway generates heat and volatilizes compounds that attract insect pollinators (2). In non-thermogenic plant tissues, the alternative pathway may allow continued turnover of carbon skeletons through glycolysis and the tricarboxylic acid cycle when the ATP concentration is high and inhibits ATP synthase activity (3) so that the pathway can function as an energy overflow mechanism (4). However, because the alternative pathway can be active even when the cytochrome pathway is not saturated (5-7), it is likely that the two pathways are regulated in concert to balance ubiquinone pool oxidation/reduction and carbon skeleton turnover in response to cytosolic ATP levels (8, 9).In recent years, a great deal of progress has been made in understanding the post-translational regulation of the alternative oxidase, the terminal oxidase that comprises the alternative respiratory pathway. The alternative oxidase is a homodimeric protein in the inner mitochondrial membrane and the oxidation/reduction state of an intersubunit disulfide bond has been demonstrated to regulate its activity (10, 11). A second mechanism of activation of the alternative oxidase involves ␣-keto acids, notably pyruvate (12), which significan...
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