Reprogrammed expression of subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae

Abstract

A synthetic gene has been designed and constructed by total chemical synthesis as a first step in the functional relocation from the mitochondrion to the nucleus of a gene encoding subunit 9 of the yeast mitochondrial ATPase complex. This gene (NAP9) incorporates codons frequently used in nuclear genes of Saccharomyces cerevisiae and additionally includes a series of unique restriction enzyme cleavage sites to facilitate future systematic manipulations of the gene and its protein product. Following the expression of the NAP9 gene by transcription and translation in vitro, a radiolabelled protein was produced which displayed a gel electrophoretic mobility and solubility in chloroform/methanol characteristic of the authentic subunit 9 proteolipid encoded in vivo by the mitochondrial olil gene. In order to achieve import into mitochondria of yeast subunit 9, a fusion was made between the NAP9 gene and DNA encoding the cleavable presequence of the nuclearly encoded precursor to subunit 9 from Neurospora crassa. Following expression in vitro, the resultant fusion protein was imported and appropriately processed by isolated yeast mitochondria. The import of yeast subunit 9 was less efficient than that observed in parallel import experiments with yeast subunit 8 attached to the same presequence or with the naturally occurring intact N . crassa subunit 9 precursor. Yeast subunit 9 lacking a leader sequence is not imported into mitochondria but, unlike subunit 8, it does not embed itself into the outer membrane, in spite of its highly hydrophobic character.FoF1-ATPase complexes in energy-transducing membranes of mitochondria, chloroplasts and bacteria are comprised of two functionally distinct sectors: Fo, a membraneembedded sector containing hydrophobic proteins that constitute a proton channel, and F1, a soluble aggregate on which adenine nucleotide interconversions are catalyzed [l]. In the fully assembled FoF I-ATPase complex ATP synthesis is coupled to proton translocation. A key component of the proton channel is a dicyclohexylcarbodiimide-binding proteolipid [2], denoted subunit 9 in the mitochondrial ATPase complex (mtATPase) of the yeast Saccharomyces cerevisiae. As with the analogous proteolipids from other FoF,-ATPase complexes [2], the yeast subunit 9 (76 amino acids) [3] has two hydrophobic transmembrane stems separated by a charged hydrophilic loop that is thought to make functional contact with the F1 sector [4]. Aggregates of subunit 9, together with subunit 6 (259 amino acids), are considered to comprise the proton channel in yeast mitochondria [5]. One further Fo component in yeast is subunit 8 (48 amino acids), a proteolipid known to be responsible for the assembly of subunit 6 [5]. In S. cerevisiae, subunits 9, 6 and 8 are specified by the mitochondrial genes olil, oli2 and aapl, respectively [5].

Mitochondrial DNA mutations in human cancer

“…However, a promising therapeutic approach to patients with mtDNA mutation is based on allotopic gene expression (allotopic gene expression is expressing a mitochondrially encoded gene from nucleus transfected constructs as fusion with an Nterminus mitochondrial target sequence). This concept was first developed by Phillip Nagley in Australia in the late 1980s, in Saccharomyces cerevisiae genes (Gearing and Nagley, 1986;Farrell et al, 1988;Law et al, 1988Law et al, , 1990. The first successful mammalian allotopic expression of a mtDNA-encoded polypeptide was shown in 2002 by Manfredi et al (2002).…”

The Mitochondrial Protein Import Apparatus

“…In a series of experiments, it was demonstrated that the (normally mitoehondrially encoded) subunits 8 and 9 of yeast FoF~-ATPase could be imported and correctly processed by mitochondria (148)(149)(150). For this purpose, synthetic "genes" for these subunits were fused to DNA segments coding for a mitochondrial matrix-targeting signal (presequence) and the constructs were expressed in yeast.…”

Assembly of imported subunit 8 into the ATP synthase complex of isolated yeast mitochondria

“…For import experiments, mitochondria (200 pg protein) were incubated with lo6 dpm 35S-labelled precursor [19]; the incubation buffer (final volume 100 pl) contained 0.6 M sorbitol, 20 mM Hepes/KOH, 40 mM KC1, 8 mM methionine, 1 mM dithiothreitol, 10 mM MgC12, 10 mM succinate, 10 mM malate, 5 mM ATP, pH 7.4 and 40% (by vol.) postribosomal supernatant of rabbit reticulocyte lysate (Promega) prepared by spinning the lysate at 150000 x g for 30 min.…”

“…Following import incubations, mitochondrial proteins were analyzed by SDS/ PAGE either from whole mitochondria, or after lysis and immunoadsorption to collect mtATPase complexes. For the former purpose, the mitochondria in 20 pl of a single-import incubation mixture were simply re-isolated by centrifugation in an Eppendorf microfuge [19]. For the latter purpose, the residual 80-pl portion of the single-import incubation mixture was centrifuged to recover mitochondria, which were then solubilized in 40 p1 cholate extraction buffer (0.5% sodium cholate, 1 % octyl P-D-glucopyranoside, 10 mM p-aminobenzamidine/HCl, 10 mM c-amino-N-hexanoic acid, 2 mM phenylmethylsulphonyl fluoride, 1 mg/ml ATP, 278 mM mannitol, 222 mM sorbitol, 0.56 mM EDTA and 11 mM Tris/ HC1, pH 7.4).…”