Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Nov;6(11):160246.
doi: 10.1098/rsob.160246.

The functional readthrough extension of malate dehydrogenase reveals a modification of the genetic code

Julia Hofhuis  1 , Fabian Schueren  1 , Christopher Nötzel  1 , Thomas Lingner  2 , Jutta Gärtner  1 , Olaf Jahn  3 , Sven Thoms  4
Affiliations

The functional readthrough extension of malate dehydrogenase reveals a modification of the genetic code

Julia Hofhuis et al. Open Biol. 2016 Nov.

Abstract

Translational readthrough gives rise to C-terminally extended proteins, thereby providing the cell with new protein isoforms. These may have different properties from the parental proteins if the extensions contain functional domains. While for most genes amino acid incorporation at the stop codon is far lower than 0.1%, about 4% of malate dehydrogenase (MDH1) is physiologically extended by translational readthrough and the actual ratio of MDH1x (extended protein) to 'normal' MDH1 is dependent on the cell type. In human cells, arginine and tryptophan are co-encoded by the MDH1x UGA stop codon. Readthrough is controlled by the 7-nucleotide high-readthrough stop codon context without contribution of the subsequent 50 nucleotides encoding the extension. All vertebrate MDH1x is directed to peroxisomes via a hidden peroxisomal targeting signal (PTS) in the readthrough extension, which is more highly conserved than the extension of lactate dehydrogenase B. The hidden PTS of non-mammalian MDH1x evolved to be more efficient than the PTS of mammalian MDH1x. These results provide insight into the genetic and functional co-evolution of these dually localized dehydrogenases.

Keywords: LDHBx; MDH1x; genetic code; peroxisome; redox shuttle; translational readthrough.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Translational readthrough of human malate dehydrogenase. (a) The SCC of MDH1 promotes a high level of translational readthrough. Venus/hRluc dual reporter assay with MDH1 wild-type and mutant SCCs in HeLa cells. Red line indicates background level of readthrough as observed with a construct containing two consecutive UAA stop codons separating Venus and luciferase tags. Mutations are indicated in red. Alterations of the SCC dramatically reduce readthrough efficiency. N = 3; *p < 0.01, #p < 0.05 versus WT (UGA CUA) (Student's t-test). (b) Full-length MDH1 is extended by readthrough. Geneticin (100 µg ml−1) induces MDH1 readthrough. Western blot of MDH1x (UGA) or MDH1x-UGG (stop codon replaced by Trp codon UGG) containing an N-terminal HA- and a C-terminal myc-tag. Molecular mass marker in kilodaltons; n.t., not transfected. (c) Quantification of (b). MDH1x readthrough is 4.3 ± 0.82%, treatment with geneticin induced readthrough to 13.1 ± 1.17% (ImageJ, N = 3, *p = 0.002). (d) Dual reporter assay with MDH1x wild-type SCC (SCC0) and MDH1x SCC containing the complete (SCCx) or 31/57 nucleotides (SCCxΔ26, deletion of the last 26 nucleotides) of the extension. Readthrough does not differ significantly between the constructs, suggesting that the SCC is the main contributor to MDH1x readthrough. SCCxScr, MDH1 SCC with a scrambled sequence of the 50 nucleotides following the SCC. N = 4. Error bars, s.e.m.
Figure 2.
Figure 2.
MDH1 readthrough in several cell types and in comparison to LDHB. (a) MDH1 stop codon readthrough in various mammalian cell lines. HeLa, U373 and U118 cells were transfected with MDH1 and LDHB SCC reporter constructs and analysed by dual reporter assays. Readthrough is expressed as hRLuc/Venus signal. MDH1 readthrough is significantly higher compared with LDHB readthrough in all cell lines (*p = 0.001 (U373), p = 0.002 (HeLa), p = 0.001 (U118); Student's t-test). MDH1 and LDHB readthrough are highest in U118 cells. MDH1: #p = 0.01 (U118 versus HeLa), p = 0.01 (U118 versus U373). LDHB: p = 3 × 10−7 (U118 versus HeLa), p = 8 × 10−5 (U118 versus U373); Student's t-test; N = 5. (b) Geneticin (100 µg ml−1) induces MDH1 readthrough in U373, HeLa and U118 cells. N = 3. MDH1 versus LDHB: p = 0.002 (U373), 4 × 10−6 (HeLa) and 0.01 (U118); Student's t-test. Error bars, s.e.m.
Figure 3.
Figure 3.
Tryptophan and arginine are incorporated during readthrough at the MDH1 stop codon. (a,b) LC-MS analysis (base peak intensity chromatogram, BPI) of the tryptic peptides derived from the gel regions that are marked by rectangles in the colloidal Coomassie-stained gel lane shown in the inset. WB: specific detection of myc-tagged proteins by western blotting used to identify gel regions of interest. Intense bands at an apparent molecular weight of approximately 50 kDa and approximately 20 kDa correspond to antibody heavy and light chains, respectively. (c,d) Mass-selective chromatographic display of the readthrough-related tryptic peptides. Extracted ion chromatograms (XIC) show the peptides resulting from incorporation of Trp (triply charged molecular ion, green trace) or of Arg (doubly charged molecular ion, red trace). Analysis of the MDH1x-derived proteins (d) not only confirmed the presence of Trp as seen in the control scenario with Trp-coding (c), but additionally revealed the incorporation of Arg that is undetectable in the control. Signal intensities of the two peptides do not reflect the ratio of incorporation of Trp and Arg, respectively. The generated peptides considerably differ in their ionization behaviour, which is particularly due to the emergence of an additional tryptic cleavage site upon incorporation of Arg. For generation of XIC, a mass tolerance window of 20 ppm was applied to continuum data without lock mass correction. Major signals are labelled with the corresponding amino acid sequence together with the fragment ions detected by mass spectrometric sequencing. For the sake of clarity, only C-terminal y-ions and N-terminal b-ions are depicted and neutral loss of ammonia or water is not considered.
Figure 4.
Figure 4.
Functional translational readthrough of MDH1. Immunofluorescence with anti-MDH1 and anti-Pex14 in untransfected HeLa (a) and HEK (b) cells. Endogenous MDH1 shows mainly cytosolic localization. Removal of cytosol (-CYT) after digitonin treatment reveals colocalization of MDH1 with the peroxisomal markers Pex14 and PMP70. (c) Peroxisomal targeting of MDH1x depends on the stop codon. Direct immunofluorescence microscopy of transfected HeLa cells: MDH1 localizes mainly to the cytosol. Removal of cytosol (-CYT) after digitonin permeabilization reveals peroxisomal localization of MDH1. (d) Exchange of UGA with the tighter stop codon UAA strongly reduces the amount of MDH1x in the peroxisome pre and post removal of cytosol (-CYT). Scale bars, 10 µm.
Figure 5.
Figure 5.
Assessing the ‘bird gap’: comparative analysis of the zebra finch and human hidden PTS1. (a) Alignment of MDH1x termini in mammals and non-mammalian vertebrates. MDH1x is conserved throughout vertebrates. Red box marks readthrough extension. Bracket on the right-hand side: Mammals. PTS1 score calculation suggests that mammalian MDH1x PTS1 is weaker than the PTS1 of non-mammalian vertebrate MDH1x. Non-mammalian species, however, do not have LDHBx (bird gap). (b) PTS1 of both human and zebra finch (Taeniopygia guttata) MDH1x localize to peroxisomes. PTS1 from peroxisomal membrane protein ACOX3 was used as a positive control. Scale bar, 10 µm. (c) Quantitative live analysis of peroxisomal protein import efficiency. Venus-tagged PTS1 was expressed in HeLa cells. After digitonin permeabilization of the plasma membrane, the cytoplasm was washed out by PBS. Hundred cells from images pre and post removal of cytoplasm were used to calculate import efficiency. (d) Analysis of Venus fluorescence before and after removal of cytoplasm revealed that targeting efficiency of zebra finch Venus-tagged MDH1x PTS1 is more efficient compared with human MDH1x PTS1. **p = 0.005 versus human PTS1 (Student's t-test), N = 5. Error bars, s.e.m.
See this image and copyright information in PMC

References

    1. Hofstetter H, Monstein HJ, Weissmann C. 1974. The readthrough protein A1 is essential for the formation of viable Q beta particles. Biochim. Biophys. Acta 374, 238–251. (doi:10.1016/0005-2787(74)90366-9) - DOI - PubMed
    1. Pelham HR. 1978. Leaky UAG termination codon in tobacco mosaic virus RNA. Nature 272, 469–471. (doi:10.1038/272469a0) - DOI - PubMed
    1. Weiner AM, Weber K. 1971. Natural readthrough at the UGA termination signal of Q-beta coat protein cistron. Nat. New Biol. 234, 206–209. (doi:10.1038/newbio234206a0) - DOI - PubMed
    1. Engelberg-Kulka H, Dekel L, Israeli-Reches M, Belfort M. 1979. The requirement of nonsense suppression for the development of several phages. Mol. Gen. Genet. 170, 155–159. (doi:10.1007/BF00337791) - DOI - PubMed
    1. Geller AI, Rich A. 1980. A UGA termination suppression tRNATrp active in rabbit reticulocytes. Nature 283, 41–46. (doi:10.1038/283041a0) - DOI - PubMed

Publication types