Abstract
In this study, we report that the partitioning between mitochondria and cytoplasm of two variants, mCherry and DsRed Express (DRE), of the red fluorescent protein, DsRed, fused to one of the six matrix targeting sequences (MTSs) can be affected by both MTS and amino acid substitutions in DsRed. Of the six MTSs tested, MTSs from superoxide dismutase and DNA polymerase gamma failed to direct mCherry, but not DRE to mitochondria. By evaluating a series of chimeras between mCherry and DRE fused to the MTS of superoxide dismutase, we attribute the differences in the mitochondrial partitioning to differences in the primary amino acid sequence of the passenger polypeptide. The impairment of mitochondrial partitioning closely parallels the number of mCherry-specific mutations, and is not specific to mutations located in any particular region of the polypeptide. These observations suggest that both MTS and the passenger polypeptide affect the efficiency of mitochondrial import and provide a rationale for the observed diversity in the primary amino acid sequences of natural MTSs.
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Abbreviations
- aa:
-
Amino acid(s)
- COX:
-
Cytochrome c oxidase
- DRE:
-
DsRed Express
- FP:
-
Fluorescent protein
- GFP:
-
Green fluorescent protein
- hSOD:
-
Human mitochondrial superoxide dismutase
- LRS:
-
Leaky ribosomal scanning
- MDH2:
-
Mitochondrial isoform of malate dehydrogenase
- MPI:
-
Mitochondrial propensity index
- mRFP1:
-
Monomeric red fluorescent protein
- MTS:
-
Matrix targeting sequence
- OTC:
-
Ornithine transcarbamylase
- PolG:
-
DNA polymerase γ
- TFAM:
-
Mitochondrial transcription factor A
References
Taylor SW, Fahy E, Ghosh SS (2003) Global organellar proteomics. Trends Biotechnol 21(2):82–88
Hurt EC, Pesold-Hurt B, Schatz G (1984) The cleavable prepiece of an imported mitochondrial protein is sufficient to direct cytosolic dihydrofolate reductase into the mitochondrial matrix. FEBS Lett 178(2):306–310
Horwich AL et al. (1985) A leader peptide is sufficient to direct mitochondrial import of a chimeric protein. Embo J 4(5):1129–1135
Wilcox AJ et al. (2005) Effect of protein structure on mitochondrial import. Proc Natl Acad Sci USA 102(43):15435–15440
Sato T et al. (2005) Comparison of the protein-unfolding pathways between mitochondrial protein import and atomic-force microscopy measurements. Proc Natl Acad Sci USA 102(50):17999–18004
Matz MV et al. (1999) Fluorescent proteins from nonbioluminescent Anthozoa species. Nat Biotechnol 17(10):969–973
Jakobs S. et al. (2000) EFGP and DsRed expressing cultures of Escherichia coli imaged by confocal, two-photon and fluorescence lifetime microscopy. FEBS Lett 479(3):131–135
Gross LA et al. (2000) The structure of the chromophore within DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci USA 97(22):11990–11995
Baird GS, Zacharias DA, Tsien RY (2000) Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci USA 97(22):11984–11989
Shaner NC et al. (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22(12):1567–1572
Bevis BJ, Glick BS (2002) Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed). Nat Biotechnol 20(1):83–87
Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2(12):905–909
Alexeyev MF, Winkler HH (1999) Gene synthesis, bacterial expression and purification of the Rickettsia prowazekii ATP/ADP translocase. Biochim Biophys Acta 1419(2):299–306
Kozak M (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J Mol Biol 196(4):947–950
Sambrook J, Russel DW (2001) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, New York
Ho SN et al. (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77(1):51–59
Smith E et al. (2005) Leaky ribosomal scanning in mammalian genomes: significance of histone H4 alternative translation in vivo. Nucleic Acids Res 33(4):1298–1308
Eilers M, Schatz G (1986) Binding of a specific ligand inhibits import of a purified precursor protein into mitochondria. Nature 322(6076):228–232
Rassow J et al. (1989) Translocation arrest by reversible folding of a precursor protein imported into mitochondria. A means to quantitate translocation contact sites. J Cell Biol 109(4 Pt 1):1421–1428
Lu B et al. (2006) Tid1 isoforms are mitochondrial DnaJ-like chaperones with unique carboxyl termini that determine cytosolic fate. J Biol Chem 281(19):13150–13158
Karniely S, Regev-Rudzki N, Pines O (2006) The presequence of fumarase is exposed to the cytosol during import into mitochondria. J Mol Biol 358(2):396–405
Strobel G et al. (2002) Competition of spontaneous protein folding and mitochondrial import causes dual subcellular location of major adenylate kinase. Mol Biol Cell 13(5):1439–1448
Acknowledgments
The research in MA’s lab was supported in part by a grant from the United Mitochondrial Disease foundation. GLW is supported by the National Institutes of Health grants ES03456 and AG19602
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Pastukh, V., Shokolenko, I.N., Wilson, G.L. et al. Mutations in the passenger polypeptide can affect its partitioning between mitochondria and cytoplasm. Mol Biol Rep 35, 215–223 (2008). https://doi.org/10.1007/s11033-007-9073-7
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DOI: https://doi.org/10.1007/s11033-007-9073-7