The Presequence of Fumarase is Exposed to the Cytosol during Import into Mitochondria

https://doi.org/10.1016/j.jmb.2006.02.023Get rights and content

The majority of mitochondrial proteins can be imported into mitochondria following termination of their translation in the cytosol. Import of fumarase and several other proteins into mitochondria does not appear to occur post-translationally according to standard in vivo and in vitro assays. However, the nature of interaction between the translation and translocation apparatuses during import of these proteins is unknown. Therefore, a major question is whether the nascent chains of these proteins are exposed to the cytosol during import into mitochondria. We asked directly if the presequence of fumarase can be cleaved by externally added mitochondrial processing peptidase (MPP) during import, using an in vitro translation–translocation coupled reaction. The presequence of fumarase was cleaved by externally added MPP during import, indicating a lack of, or a loose physical connection between, the translation and translocation of this protein. Exchanging the authentic presequence of fumarase for that of the more efficient Su9-ATPase presequence reduced the exposure of fumarase precursors to externally added MPP en route to mitochondria. Therefore, exposure to cytosolic MPP is dependent on the presequence and not on the mature part of fumarase. On the other hand, following translation in the absence of mitochondria, the authentic fumarase presequence and that of Su9-ATPase become inaccessible to added MPP when attached to mature fumarase. Thus, folding of the mature portion of fumarase, which conceals the presequence, is the reason for its inability to be imported in classical post-translational assays.

Another unique feature of fumarase is its distribution between the mitochondria and the cytosol. We show that in vivo the switch of the authentic presequence with that of Su9-ATPase caused more fumarase molecules to be localized to the mitochondria. A possible mechanism by which the cytosolic exposure, the targeting efficiency, and the subcellular distribution of fumarase are dictated by the presequence is discussed.

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;, trp163;, his3200;, leu21), 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).

References (29)

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