Functional analysis in Saccharomyces cerevisiae of naturally occurring amino acid substitutions in human dihydrolipoamide dehydrogenase
Functional analysis in Saccharomyces cerevisiae of naturally occurring amino acid substitutions in human dihydrolipoamide dehydrogenaseMargaret M. Lanterman, J. Richard Dickinson1 and Dean J. Danner*
Department of Genetics and Molecular Medicine, Emory University School of Medicine, 1462 Clifton Road, Atlanta, GA 30322, USA and 1School of Pure and Applied Biology, University of Wales, College of Cardiff, Cardiff CF1 3TL, UK
Received May 24, 1996;Revised and Accepted July 22, 1996
Dihydrolipoamide dehydrogenase is a common component of mammalian multienzyme complexes that decarboxylate [alpha]-ketoacids and catabolize glycine. The common function is to reoxidize a reduced lipoate component of each complex, thereby preparing that lipoate for another round of catalysis. Regions within dihydrolipoamide dehydrogenase involved in association with other proteins of the complexes are poorly defined, and despite high amino acid sequence conservation through evolution, it is unknown if dihydrolipoamide dehydrogenases are functionally equivalent across species. To address this issue, we asked whether the human enzyme could restore function to the [alpha]-ketoacid dehydrogenase complexes in a yeast strain deficient in endogenous dihydrolipoamide dehydrogenase. This dihydrolipoamide dehydrogenase null mutant will not grow on non-fermentable carbon sources. The human enzyme expressed from a CEN plasmid complemented the growth phenotype and restored full activity to the pyruvate and [alpha]-ketoglutarate dehydrogenase complexes. Human dihydrolipoamide dehydrogenases with selected amino acid substitutions were then tested in the null strain for their ability to restore function. Substitutions tested represented naturally occurring candidate mutations identified in an individual with inactive dihydrolipoamide dehydrogenase. A K37E change had full function while a P453L change resulted in reduced dihydrolipoamide dehydrogenase abundance in the mitochondria and no detectable catalytic activity.
Dihydrolipoamide dehydrogenase [E3] (EC 1.8.1.4) is a flavoprotein with a structure that is conserved in evolution from bacteria to plants and humans (1 ,2 ). In eukaryotic cells this protein functions as a homodimer within the mitochondria where it participates as a component of the [alpha]-ketoacid dehydrogenase complexes and the glycine cleavage system (1 ,3 ,4 ). Catalytically, E3 oxidizes the reduced dihydrolipoate covalently bound to the acyl transferase component of the ketoacid complexes and the hydrogen carrier protein of the glycine cleavage system. Lipoate reduction occurs along with the oxidative decarboxylation of the complex-specific substrate. For lipoate reoxidation, E3 activity utilizes non-covalently bound FAD as an intermediate leading to the ultimate reduction of NAD+ (4 ).
Current knowledge for a structure-function relationship in E3 derives mainly from X-ray crystallographic analysis of the yeast and bacterial proteins (5 ,6 ). Additional information comes from the comparison of this information with the crystal structure and amino acid sequence of glutathione reductase (4 ). Based on these analyses, FAD binding is thought primarily to involve residues within the first 150 amino acids of the protein (4 ). Two amino acids are implicated as having a critical role in catalytic activity, H452 and E457 (7 ). Naturally occurring mutations are reported for an individual expressing an E3 deficiency and thus suggest other functionally important residues. Two examples of patient identified amino acid substitutions are K37E and P453L, hypothesized as causal of the observed E3 inactivity (8 ). Based on earlier reports (4 ,7 ), Liu et al. suggested that the K37E substitution affected FAD-binding while the P453L change altered the catalytic site. However, confirmation of these two amino acid substitutions as causal of the enzyme dysfunction has yet to be determined.
Human proteins can function in yeast (9 ,10 ) including proteins that function in mitochondria (11 ). Interaction of a human protein with a mitochondrial multi-protein complex in yeast also has been shown (12 ,13 ); however, a human protein interacting with several mitochondrial multienzyme complexes has yet to be demonstrated. The ability of human E3 [hE3] to function in yeast requires correct mitochondrial targeting, protein import and processing, and finally association with three different [alpha]-ketoacid dehydrogenase multienzyme complexes. This association is not identical for all of the complexes. E3 binds to the branched chain acyltransferase component of the branched chain [alpha]-ketoacid dehydrogenase [BCKD] complex (14 ), and to the decarboxylase component of the [alpha]-ketoglutarate [KGD] complex (15 ). For E3 to associate with the pyruvate dehydrogenase [PD] complex requires a separate E3-binding protein (16 ,17 ). Based on these various protein-protein interactions required by the different complexes, full complementation of the LPD null yeast strain by hE3 was not a trivial assumption.
Here we show that hE3 is able to complement a yeast strain deficient in endogenous E3 by interacting with the various yeast [alpha]-ketoacid dehydrogenase complexes. Further, the E3-null model was then used directly to test the effect of the two reported amino acid substitutions on E3 activity.
To develop a yeast cell in which human E3 mutations could be tested for function, the strain MML22 (lpd1::URA3) was constructed. Genotypes of all yeast strains used in these studies are shown in Table 1 . The LPD1 gene of S. cerevisiae strain DMA2 was disrupted by homologous recombination with a targeting vector that substituted the URA3 gene for the middle third of the LPD1 gene. Structure of the mutant allele was confirmed by Southern blot and PCR analysis (data not shown). As seen in Figure 1 , immunoblots demonstrated the wild-type parental strain (DMA2) expresses E3 while MML22 expresses no detectable endogenous yeast E3. In the transformed cells overexpression of either human or yeast E3 resulted in high levels of these proteins (note exposure time in Fig. 1 ). Since antihuman E3 antibody was used, cross reactivity with the yeast protein might account for some of the intensity difference observed.
Restriction endonucleases and DNA-modifying enzymes were purchased from Promega, Boehringer-Mannheim or US Biochemical. [1-14C] Pyruvic acid and sodium salt (10-30 mCi mmol) was purchased from Amersham, and [1-14C] [alpha]-ketoglutaric acid (52 mCi/mmol) from DuPont. Taq DNA polymerase was purchased from Boehringer-Mannheim.
S.cerevisiae strains used here were derived from SJR336 (gift from Dr Sue Jinks-Robertson, Emory University). Genotypes for constructed strains are described in Table 1 .
pBluescript-E3 was a gift from Dr Mulchand S. Patel (SUNY Buffalo; 21 ), pRS315-GAL1/10 a gift from Dr C. Glover (University of Georgia), pYME3 a gift from Dr Lester Reed (University of Texas at Austin; 22 ), YEplac195 a gift from Dr R. Daniel Gietz (University of Manitoba, Canada) and pSR244 a gift from Dr Sue Jinks-Robertson (Emory University).
Bacterial cultures were grown in LB broth or on plates (1.5% agar) with 100 [mu]g ampicillin/ml. Yeast were grown in SD-leu (2% glucose), SGly-leu (3% glycerol) selective media (23 ), YPGlyGlu (1% yeast extract, 2% peptone, with varying amounts of glycerol and glucose as noted in the figure legends) or YPD (1% yeast extract, 2% peptone, 2% glucose). Plates for growth of yeast strains were made with 2% agar. Large volume yeast cultures (200 ml) were grown in baffled 1 l flasks.
Strain DMA2 was constructed by making gal80::HIS3 disruptions in strain SJR336. The NcoI-SmaI gal80::HIS3 fragment of pSR244 was used for transformation. pSR244 is a pGEM3Zf(+) backbone with the HindIII GAL80 gene fragment disrupted by replacing the internal 600 bp BglII GAL80 fragment with the 1.7 kb BamHI fragment of the HIS3 gene. Strain MML22 was constructed by making an lpd1::URA3 disruption in DMA2. The KpnI lpd1::URA3 fragment of pMLe3::URA3 was used for transformation. pYME3 was used to construct pMLe3::URA3 by replacing the 840 bp HincII fragment of LPD1 with the 1.2 kb URA3SmaI fragment from YEplac195 (24 ). pYME3 is pGEM7Zf(+) with cDNA corresponding to nucleotides 65-1503 of the LPD1 gene as insert.
For cloning of the full-length coding region of LPD1, primers sense (5'-GGAATTCACAATGTTAAGAATC-3') and antisense (5'-GACTAGTTTTCAACAATGAATAGC-3') were used to amplify the yeast LPD1 gene and the product was cut with EcoRI and SpeI to ligate into EcoRI and XbaI cut pGEM3 vector, creating pML3E3. The LPD1 gene disruption with URA3 was confirmed by PCR amplification using an LPD1 specific primer with a URA3 specific primer [i.e. LPD1 sense with antisense URA3 (5'-GATTTTTCCATGGAGGGCAC-3') and E3 antisense with URA3 sense (5'-TGTCAGATCCTGTAGAGACC-3')].
pRS315-GAL1/10 has a GAL1/10 expression cassette inserted into the EcoRI and BamHI sites in the multicloning region of the CEN plasmid pRS315 (25 ). Constructed plasmids for the human and yeast E3 contain the entire open reading frames of the previously reported cDNA clones (21 ,22 ). YpML7 was constructed by inserting an EcoRI (partial digest) and XhoI human cDNA fragment for E3 (from pBluescript-E3) into the SalI and EcoRI sites of the multicloning region in pRS315-GAL1/10. YpML8 was constructed by inserting the EcoRI-SalI yeast LPD1 insert from pML3E3 into the EcoRI and SalI sites of the multicloning region in pRS315-GAL1/10. YpML10 was constructed as YpML7, using pMLK37E as the source of EcoRI/XhoI insert. YpML11 was constructed by cutting pMLP453Lmat with HindIII/ApaI and ligating into HindIII/ApaI cut YpML7. All transformations were done by the lithium acetate method (26 ). Because pRS315-GAL1/10 vector bears the LEU2 gene, selection for transformants was done by plating on SD-leu.
Mitochondria were isolated from a cell extract produced by glass bead disruption of cells grown to stationary phase in 150-200 ml YPGlyGlu (27 ). The buffer was yeast busting buffer [YBB] (0.25 M mannitol, 1 mM EDTA and 50 mM Tris-HCl pH 7.4). Mitochondrial pellets were resuspended in 30 mM KxPO4, pH 7.5 and samples for mitochondrial protein determination were incubated at 95oC in 1% SDS for 5 min prior to the addition of BCA protein assay reagent (Pierce). Based on this protein analysis, remaining mitochondria were diluted to a final concentration of 1.5-2.0 mg protein/ml for enzyme assays. The final concentration of cofactors were 0.1 mM thiamin pyrophosphate (TPP), 1.0 mM MgCl2, 2.5 mM NAD+, 0.13 mM CoA, 0.32 mM dithiothreitol (DTT) for PD complex activity; and 0.2 mM TPP, 0.2 mM MgCl2, 0.5 mM NAD+, 1.3 mM CoA, 1.65 mM DTT for KGD complex activity. All reactions were incubated in 30 mM KxPO4 pH 7.5. Mitochondria (100 [mu]l) and cofactors (50 [mu]l) were added to a 1.5 ml tube with the lid cut off. This reaction tube was placed in a 1.5 * 7 cm scintillation vial containing 750 [mu]l [beta]-phenylethylamine as the CO2 trap, and the vial sealed with a serum cap. After a 10 min preincubation at 37oC, the reaction was started with injection of 100 [mu]l substrate, containing 0.1 [mu]Ci [1-14C]-substrate. The final concentration of substrate for all assays was 0.1 mM. Reactions were incubated at 37oC for 20-30 min, and terminated with the addition of 100 [mu]l 15% TCA to release CO2. This CO2 was collected for 1 h at 37oC. The reaction tube was discarded, EcoLume (ICN) was added to the scintillation vial, and 14C counts representing released CO2 were assessed by liquid scintillation counting. Since yeast PD is not regulated by phosphorylation as in mammalian tissue, conditions to fully activate the complex are not necessary during enzyme assays (28 ).
Yeast were grown in 50 ml SD-leu for 18 h and collected by centrifugation. Cell lysates were made as described (18 ), using a vortex to disrupt the cells. Dihydrolipoamide dehydrogenase activity was assayed spectrophotometrically measuring the forward reaction with dihydrolipoamide and acetyl-NAD+ as substrate, as described(29 ), using 20-100 [mu]g cell lysate protein per assay.
Yeast mitochondrial proteins were resolved on 12.5% SDS-PAGE and transferred to nitrocellulose. Western blot analysis was done with a 1 h incubation in a 1:5000 dilution of antihuman E3 as primary antibody (gift from Dr B. Robinson, Hospital for Sick Children, Toronto, Canada). Secondary antibody incubation was for 30 min in a 1:50 000 dilution of goat anti-rabbit IgG(H+L) horseradish peroxidase conjugate (BioRad). Amersham ECL reagents and HyperFilm were used for detection.
Yeast genomic DNA was prepared (30 ) and digested overnight with NspI. After resolution of DNA fragments by electrophoresis in 0.8% agarose, the fragments were transferred to nylon membranes using a Turboblotter (Schleicher & Schuell). The NspI-EcoRI 1410 bp LPD1 fragment was labeled with 32P using Megaprime (Amersham). This probe was allowed to hybridize overnight at 68oC after which the membrane was washed twice in 7* SSPE, 0.25% SDS for 15 min at 25oC followed by two washes in 1* SSPE, 0.75% SDS for 15 min at 37oC. Hybridizing fragments were detected with a Molecular Dynamics Phosphorimager.
Mutation K37E (AAA to GAA) was made by two step PCR, starting with pBluescript-E3 as template. First, a 5' product using a T7 sense primer with an E3 antisense primer (5'-TGTTTCATTTTCCTCAATGC-3') and a 3' product using an E3 sense primer (5'-GCATTGAGGAAAATGAAACA-3') with a T3 antisense primer were amplified by PCR. These products were isolated in low melting point agarose, combined and used as template for a second PCR amplification with the E3 sense primer (5'-CATATGGATCCGGAAAAATGCAGAGC-3') and T3 antisense primer. The product was a full length cDNA encoding the K37E mutation. The 313 bp BamHI-NcoI fragment of this product was cloned into BamHI/NcoI cut pBluescript-E3 to create pMLK37E. Nucleotide sequence was confirmed by the use of Sequenase 2.0 (Amersham).
Similarly, the P453L (CCG to CTG) mutation was made by two step PCR using the T7 sense primer with the E3 antisense primer (5'-GCCTCTGATAAGGTCAGATGTGCA-3'), and the E3 sense primer (5'-ATCTGACCTTATCAGAGGCTTTTA-3') with a T3 antisense primer. As above the products were combined and used as template for amplification with T7 sense and T3 antisense primers. The 3' EcoRI-XhoI 777 bp fragment encoding E3-P453L was cloned into EcoRI/XhoI cut pMLE3mat (pBluescript with cDNA for mature human E3) to create pMLP453L. In making this construct, a silent A1526G substitution occurred that eliminated the 3' HindIII site. Nucleotide sequence was confirmed using Sequenase 2.0.
Special thanks to Sue Jinks-Robertson, Judy Fridovich-Keil and Grant MacGregor for their technical assistance and critical reading of this manuscript. MML was supported in part by a National Institutes of Health Predoctoral Training Grant GM08367 and the research was supported by an NIH grant DK38320.
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