An internal targeting signal directing proteins into the mitochondrial intermembrane space - PubMed (original) (raw)

An internal targeting signal directing proteins into the mitochondrial intermembrane space

K Diekert et al. Proc Natl Acad Sci U S A. 1999.

Abstract

Import of most nucleus-encoded preproteins into mitochondria is mediated by N-terminal presequences and requires a membrane potential and ATP hydrolysis. Little is known about the chemical nature and localization of other mitochondrial targeting signals or of the mechanisms by which they facilitate membrane passage. Mitochondrial heme lyases lack N-terminal targeting information. These proteins are localized in the intermembrane space and are essential for the covalent attachment of heme to c type cytochromes. For import of heme lyases, the translocase of the mitochondrial outer membrane complex is both necessary and sufficient. Here, we report the identification of the targeting signal of mitochondrial heme lyases in the third quarter of these proteins. The targeting sequence is highly conserved among all known heme lyases. Its chemical character is hydrophilic because of a large fraction of both positively and negatively charged amino acid residues. These features clearly distinguish this signal from classical presequences. When inserted into a cytosolic protein, the targeting sequence directs the fusion protein into the intermembrane space, even in the absence of a membrane potential or ATP hydrolysis. The heme lyase targeting sequence represents the first topogenic signal for energy-independent transport into the intermembrane space and harbors two types of information. It assures accurate recognition and translocation by the translocase of the mitochondrial outer membrane complex, and it is responsible for driving the import reaction by undergoing high-affinity interactions with components of the intermembrane space.

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Figures

Figure 1

Figure 1

CCHL does not contain essential import information in its N-terminal half. The precursor proteins CCHL (A), CCHL Δ2–170 (B), and CCHL Δ177–346 (C; see Materials and Methods) were synthesized by in vitro transcription and translation in reticulocyte lysate by using [35S]methionine as a label. The black bar on top of each panel represents the region of CCHL present in these proteins; the thin line corresponds to deleted segments. The radiolabeled proteins were added to isolated mitochondria in import buffer (36). After incubation for 10 min at 25°C, mitochondria were reisolated by centrifugation (10 min at 9,000 × g). Mitochondria were resuspended in a small volume of SoH buffer (0.6 M sorbitol_/20 mM Hepes-KOH, pH 7.2). Samples were diluted 10-fold into SoH buffer or water in the presence or absence of proteinase K as indicated. The hypotonic condition results in swelling of the organelles and leads to selective rupture of the outer membrane allowing added proteinase K to degrade proteins exposed in the intermembrane space (D, CC1HL) but not of proteins of the matrix (Tim44p and Mge1p). After incubation for 30 min at 0°C, protease digestion was halted by the addition of PMSF, and proteins were precipitated with trichloroacetic acid. Proteins were separated by SDS/_PAGE, blotted on nitrocellulose, and quantified by PhosphorImager analysis. In addition, an autoradiograph is shown. The material that is not digested after swelling represents aggregated preprotein. The rightmost lanes contain 50% of input preprotein as a standard (St.). Import (i.e., protease-resistant protein relative to bound material) varied by not more than 15% in various experiments.

Figure 2

Figure 2

The third quarter of CCHL contains both necessary and sufficient targeting information. The import of the indicated CCHL mutant proteins was estimated as described in the legend of Fig. 1. The amount of radiolabeled protein was quantitated by PhosphorImager analysis and is given above the autoradiographs relative to the amount of preprotein bound to mitochondria (set to 100%). The rightmost lanes contain 50% of input preprotein (St., standard).

Figure 3

Figure 3

The import information of N. crassa CCHL is contained within two conserved small segments. Import of the indicated CCHL mutant proteins was estimated and analyzed as described in the legend of Fig. 2. St., standard representing 50% of input preprotein.

Figure 4

Figure 4

The targeting sequence of heme lyases can direct a cytosolic protein into the intermembrane space. The indicated regions of N. crassa CCHL (A and B) or of S. cerevisiae CC1HL (C) were inserted into the cytosolic protein DHFR. Import of these fusion proteins or of CC1HL (D) and further analysis of import were performed as described in the legend of Fig. 2. Other parts of the heme lyases did not support the import of the corresponding fusion proteins (not shown). The DHFR sequence is indicated by the hatched boxes; the N. crassa CCHL and S. cerevisiae CC1HL sequences are given as black and grey bars, respectively. St., standard representing 50% of input preprotein.

Figure 5

Figure 5

Import mediated by the heme lyase targeting sequence occurs along the authentic heme lyase import pathway. The fusion proteins DCD 171–279 and DC1D 101–169 were imported into isolated mitochondria. (A) The organelles were pretreated with 50 μg_/ml trypsin (27). (B) ATP was hydrolyzed by treatment with 20 units/ml apyrase (ref. ; import reactions contained 20 μM oligomycin and 5 μM carboxy-atractyloside). (C) The membrane potential was depleted by the addition of the uncoupling reagent carbonyl cyanide m-chlorophenylhydrazone (50 μM; CCCP; ref. 26). Under any of these conditions, import of presequence-containing preproteins into the matrix was strongly impaired (not shown). Further treatment of the samples and analysis of import were performed as described in Fig. 2. The result for only one of the heme lyase/_DHFR fusion proteins is shown.

Figure 6

Figure 6

The heme lyase targeting signal encompasses two signature motifs that are highly conserved in these proteins. The sequence alignment of known heme lyases was prepared by using the

multalin

program (54). No bacterial homologues have been identified. The heme lyase targeting signal determined in this study is underlined. The secondary structure of these sequences was predicted by using the

phdsec

program (55) revealing similar results for CCHL and CC1HL proteins. H, α-helical; E, extended β-stranded. Nc, N. crassa; Sp, Schizosaccharomyces pombe; Sc, S. cerevisiae; Ca, Candida albicans; Hs, Homo sapiens; Mm, Mus musculus; Ce, Caenorhabditis elegans.

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References

    1. Neupert W. Annu Rev Biochem. 1997;66:861–915. - PubMed
    1. Schatz G, Dobberstein B. Science. 1996;271:1519–1526. - PubMed
    1. Pfanner N, Craig E A, Honlinger A. Annu Rev Cell Dev Biol. 1997;13:25–51. - PubMed
    1. von Heijne G. EMBO J. 1986;5:1335–1342. - PMC - PubMed
    1. Roise D, Schatz G. J Biol Chem. 1988;263:4509–4511. - PubMed

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