Journal of Molecular Biology
The Presequence of Fumarase is Exposed to the Cytosol during Import into Mitochondria
Introduction
Early studies have demonstrated that cycloheximide-treated yeast accumulate a large number of cytosolic polysomes on the surface of mitochondria and these polysomes are enriched in mRNA encoding mitochondrial proteins.1, 2, 3, 4 Moreover, mitochondrial proteins precursors are essentially undetectable in vivo.5 These findings indicate either highly efficient post-translational targeting and translocation of proteins into mitochondria, or a co-translational mode of import. The earlier possibility is supported by the finding that, if forced, the majority of mitochondrial proteins may be imported into mitochondria following the termination of their translation in the cytosol.6 Two models that are consistent with all the observations above and can explain the apparent coupling between translation and import have been presented; the first suggests that a polysome translating a nascent mitochondrial protein can randomly arrive at close proximity of the mitochondria and engage the outer membrane translocons, thereby allowing subsequent precursors to be imported during or immediately following translation termination.7, 8 Alternatively, mRNA encoding mitochondrial proteins have been suggested to be targeted to the mitochondrial surface even in the absence of translation.9, 10
There are a limited number of mitochondrial proteins that apparently cannot be imported post-translationally. These include the yeast proteins fumarase,11, 12 manganese-dependent superoxide dismutase 2 (Sod2p),13 and the major adenylate kinase (Aky2).14 Two major lines of evidence indicate that these proteins cannot be imported post-translationally: (i) in vitro, precursors fully synthesized in reticulocyte lysates could not be imported into isolated mitochondria;11, 15 (ii) in vivo, precursors accumulated, when import into mitochondria was blocked by addition of the ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), could not be chased into mitochondria upon restoration of the membrane potential.12, 13 Even though the requirement of the above proteins for “translation-coupled import” has been clearly established, what kind of interaction ensues between the translation apparatus and the translocation apparatus, and what makes these specific proteins behave differently from other mitochondrial proteins, is essentially unknown.
Here, we have asked whether the nascent chain of fumarase is exposed to the cytosol during import into the mitochondria and compared its behavior to that of Su9-DHFR, which, like most mitochondrial proteins, is known for its ability to be imported post-translationally. We show that the presequence of fumarase is exposed to the cytosol before translocation into mitochondria. In addition, we suggest that when translated to completion, folding of the mature portion of fumarase hinders its ability to be imported in classical post-translational assays. We have examined the role of the presequence versus the mature portion of the protein in the unique distribution of fumarase between the mitochondria and the cytosol.12, 16
Section snippets
The fumarase presequence is accessible to cleavage prior to import
Exposure of presequences during import was monitored by their sensitivity to externally added MPP in an in vitro translation–translocation-coupled reaction. The accessibility of the presequence of fumarase to cleavage was compared to that of the hybrid protein Su9(1-79)-dihydrofolate reductase (Su9DHFR), which, in contrast to fumarase, is a model protein for post-translational import into mitochondria. The translation reaction was initiated by adding fumarase and Su9DHFR mRNAs to reticulocyte
Discussion
Here, we studied the accessibility of the presequence of fumarase to the cytosolic environment during import into the mitochondria. We conclude that under conditions of translation in the presence of mitochondria, the fumarase nascent chain is exposed to the cytosol. (i) Previous data support the notion that the folding of fumarase in the cytosol is the driving force for its subcellular distribution, which is consistent with accessibility of its polypeptide chain to the cytosol during import.20
Strains and plasmids
The Saccharomyces cerevisiae strains used were YPH499 (MATa ade2-101; lys2-801;, ura3-52;, trp1-Δ63;, his3-Δ200;, leu2-Δ1), JN516 Δssa2-4 SSA1 (MATa leu2-3,112; his3-11; ura3-52; trp1Δ1 lys2; SSA1; ssa2∷LEU2 ssa3∷TRP1 ssa4∷LYS2)21 and a1-45ΔU (JB67 selected for loss of URA3) Δssa2-4 ssa1-t.s. (MATa leu2-3,112; his3-11; ura3-52; trp1Δ1; lys2; ssa1-45 ssa2∷LEU2 ssa3∷TRP1ssa4∷LYS2).21 BY4741 (Mat a his3Δ; leu2Δ0; met15Δ0; ura3Δ0) served as the wild-type ACO1 strain. Generation of an aconitase
Acknowledgements
We thank Yudit Karp for her dedicated assistance, Doron Rapaport for kindly providing purified recombinant MPP and for fruitful discussions over this study. We thank Eitan Bibi for critical reading of the manuscript. This research was supported by the Israel Science Foundation (ISF), German Israeli Foundation (GIF), and German Israeli Project Cooperation (DIP).
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2009, Journal of Biological ChemistryCitation Excerpt :There is no evidence for an export mechanism of matrix-located proteins out of the organelle. One possible mechanism, that could explain the data, involves reverse translocation and was first described for the tricarboxylic acid cycle enzyme, fumarase (18, 25, 27, 28, 33). According to this mechanism the extramitochondrial localization is achieved by retrograde movement of processed protein (MTS removed by MPP) through the TOM and TIM23 translocation channels back to the cytosol during translocation.
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2007, Journal of Biological ChemistryCitation Excerpt :Taken together, these results indicate that targeting to the mitochondria prevents the fumarase-TVS derivatives from being cleaved by the cytosolic TEV and rule out the possibility that the TEV protease is inactive in the cytosol or may not cleave these substrates in this compartment. Slowing Down Translocation Allows Cleavage of Fumarase by Cytosolic TEV—The observation that the fumarase nascent chain is not exposed to the cytosol during import (as measured by cytosolic TEV cleavage) appears to contradict previous in vitro results, which show that at least the presequence of fumarase is exposed to the cytosol before import (14). One possible explanation for this could be that in vitro, import into mitochondria is not as efficient and rapid as in vivo.