Membrane Potential-Driven Protein Import into Mitochondria: The Sorting Sequence of Cytochrome b2
 Modulates  the Δψ-Dependence of Translocation of the 
 Matrix-targeting Sequence

Mol Biol Cell. 2000 November; 11(11): 3977–3991.

PMCID: PMC15051

Membrane Potential-Driven Protein Import into Mitochondria

The Sorting Sequence of Cytochrome b₂ Modulates the Δψ-Dependence of Translocation of the Matrix-targeting Sequence

Andreas Geissler,^*^† Thomas Krimmer,^*^† Ulf Bömer,^* Bernard Guiard,^‡ Joachim Rassow,^*^§ and Nikolaus Pfanner^*^‖

^*Institut für Biochemie und Molekularbiologie and ^†Fakultät für Biologie, Universität Freiburg, D-79104 Freiburg, Germany; ^‡Centre de Génétique Moleculaire CNRS, Université Pierre et Marie Curie, 91190 Gif-sur-Yvette, France; and ^§Institut für Mikrobiologie, Universität Hohenheim, D-70593 Stuttgart, Germany

Thomas D. Fox, Monitoring Editor

^‖Corresponding author. E-mail address: pfanner/at/uni-freiburg.de

Received March 20, 2000; Revised July 20, 2000; Accepted September 13, 2000.

See the article"The basal flux of Akt in the mitochondria is mediated by heat shock protein-90" in J Neurochem, volume 5 on page 1289.

This article has been cited by other articles in PMC.

Abstract

The transport of preproteins into or across the mitochondrial inner membrane requires the membrane potential Δψ across this membrane. Two roles of Δψ in the import of cleavable preproteins have been described: an electrophoretic effect on the positively charged matrix-targeting sequences and the activation of the translocase subunit Tim23. We report the unexpected finding that deletion of a segment within the sorting sequence of cytochrome b₂, which is located behind the matrix-targeting sequence, strongly influenced the Δψ-dependence of import. The differential Δψ-dependence was independent of the submitochondrial destination of the preprotein and was not attributable to the requirement for mitochondrial Hsp70 or Tim23. With a series of preprotein constructs, the net charge of the sorting sequence was altered, but the Δψ-dependence of import was not affected. These results suggested that the sorting sequence contributed to the import driving mechanism in a manner distinct from the two known roles of Δψ. Indeed, a charge-neutral amino acid exchange in the hydrophobic segment of the sorting sequence generated a preprotein with an even better import, i.e. one with lower Δψ-dependence than the wild-type preprotein. The sorting sequence functioned early in the import pathway since it strongly influenced the efficiency of translocation of the matrix-targeting sequence across the inner membrane. These results suggest a model whereby an electrophoretic effect of Δψ on the matrix-targeting sequence is complemented by an import-stimulating activity of the sorting sequence.

INTRODUCTION

The mitochondrial outer and inner membranes contain protein complexes that are responsible for the import of nuclear-encoded preproteins (Ryan and Jensen, 1995 blue right-pointing triangle

; Schatz and Dobberstein, 1996 blue right-pointing triangle

; Neupert, 1997 blue right-pointing triangle

; Pfanner et al., 1997 blue right-pointing triangle

). Three preprotein translocases have been identified. The transport of preproteins across the outer membrane is mediated by the translocase of the outer membrane (TOM) that contains receptors for preproteins and a general import pore. Two translocases of the inner membrane (TIM) exist. The TIM23 complex is responsible for import of the major class of cleavable mitochondrial preproteins. Each cleavable preprotein carries an amino-terminal extension (presequence) that directs the protein across the outer and inner membranes into the matrix and is termed the matrix-targeting sequence. The TIM23 complex consists of two integral membrane proteins, Tim23 and Tim17, that constitute the import channel and a peripherally attached import motor, which are formed by a dynamic complex between the matrix heat shock protein Hsp70 and Tim44 (Schatz, 1996 blue right-pointing triangle

; Jensen and Johnson, 1999 blue right-pointing triangle

; Voos et al., 1999 blue right-pointing triangle

; Bauer et al., 2000 blue right-pointing triangle

). The second TIM is the TIM22 complex that mediates the insertion of a class of hydrophobic preproteins without presequence into the inner membrane. The metabolite carriers of the inner membrane are typical representatives of these preproteins that contain internal targeting information in the mature protein part (Davis et al., 1998 blue right-pointing triangle

; Koehler et al., 1999 blue right-pointing triangle

; Truscott and Pfanner, 1999 blue right-pointing triangle

; Bauer et al., 2000 blue right-pointing triangle

; Kerscher et al., 2000 blue right-pointing triangle

Two import driving forces have been found for the translocation of preproteins into mitochondria (Schleyer et al., 1982 blue right-pointing triangle

; Pfanner and Neupert, 1986 blue right-pointing triangle

; Eilers et al., 1987 blue right-pointing triangle

; Kang et al., 1990 blue right-pointing triangle

; Scherer et al., 1990 blue right-pointing triangle

; Martin et al., 1991 blue right-pointing triangle

; Gambill et al., 1993 blue right-pointing triangle

). The membrane potential Δψ across the inner membrane is required for transport of preproteins via both the TIM23 complex and the TIM22 complex. The ATP-dependent import motor consisting of matrix Hsp70 and Tim44 is needed for preproteins imported by the TIM23 complex. In the case of cleavable preproteins, Δψ promotes the transport of the amino-terminal matrix-targeting sequence (Schleyer and Neupert, 1985 blue right-pointing triangle

; Martin et al., 1991 blue right-pointing triangle

), while Hsp70 is crucial for import of the mature portion of a preprotein by direct binding to the unfolded polypeptide chain (Kang et al., 1990 blue right-pointing triangle

; Ostermann et al., 1990 blue right-pointing triangle

; Scherer et al., 1990 blue right-pointing triangle

; Gambill et al., 1993 blue right-pointing triangle

). Two roles for Δψ in the import of cleavable preproteins have been assigned. 1) An electrophoretic effect of Δψ on the positively charged matrix-targeting sequence has been concluded from several observations: studies with synthetic presequence peptides indicated that the positively charged residues are driven in by the electrical gradient (Roise and Schatz, 1988 blue right-pointing triangle

; Roise, 1992 blue right-pointing triangle

; de Kruijff, 1994 blue right-pointing triangle

); the electrical component of the proton–motive force across the inner membrane is essential for protein import, while the [open triangle]

pH is dispensable (Martin et al., 1991 blue right-pointing triangle

); a matrix-targeting sequence with a low positive net charge required a high Δψ for import, while a matrix-targeting sequence with a high positive net charge could be imported at a lower Δψ (Martin et al., 1991 blue right-pointing triangle

). 2) Δψ supports the dimerization of Tim23, a likely prerequisite for the interaction of a matrix-targeting sequence with the TIM23 complex (Bauer et al., 1996 blue right-pointing triangle

The TIM23 complex does not only transport preproteins into the matrix. A number of preproteins destined for the intermembrane space or inner membrane are imported via this translocase (Bömer et al., 1997 blue right-pointing triangle

; Kurz et al., 1999 blue right-pointing triangle

). These preproteins contain an additional sorting sequence besides the matrix-targeting sequence. The intermembrane space protein cytochrome b₂ represents a typical example. Its preprotein carries a second cleavable segment, the sorting sequence, which is located between the matrix-targeting signal and the mature protein (Hurt and van Loon, 1986 blue right-pointing triangle

; Hartl et al., 1987 blue right-pointing triangle

; Glick et al., 1992 blue right-pointing triangle

; Koll et al., 1992 blue right-pointing triangle

; Gärtner et al., 1995a blue right-pointing triangle

; Gruhler et al., 1995 blue right-pointing triangle

). While the matrix-targeting signal is directed into the matrix space and cleaved off, the adjacent sorting sequence is arrested in the inner membrane and prevents a complete translocation of the preprotein across the inner membrane. Subsequently, the inner membrane peptidase I (Pratje and Guiard, 1986 blue right-pointing triangle

; Schneider et al., 1991 blue right-pointing triangle

; Kalousek et al., 1993 blue right-pointing triangle

) cleaves off the sorting sequence and releases the mature protein to the intermembrane space. A number of alterations of the sorting sequence, such as partial deletions and amino acid substitutions, have been described that inactivate its sorting function and cause a complete translocation of the mutant cytochrome b₂ into the matrix (Koll et al., 1992 blue right-pointing triangle

; Beasley et al., 1993 blue right-pointing triangle

; Schwarz et al., 1993 blue right-pointing triangle

; Voos et al., 1993 blue right-pointing triangle

; Gärtner et al., 1995a blue right-pointing triangle

; Merlin et al., 1997 blue right-pointing triangle

; Bömer et al., 1997 blue right-pointing triangle

, 1998

In this report, we studied the import of mutant forms of the precursor of cytochrome b₂ and made the surprising finding that a sequence beyond the matrix-targeting signal, i.e. the sorting sequence, strongly influenced the Δψ-dependence of protein import. We analyzed the properties of this Δψ-dependence and found that it cannot be attributed to the two roles of Δψ known so far. The sorting sequence contributes to the import driving mechanism in a novel manner and thus modulates the effectiveness of Δψ action.

MATERIALS AND METHODS

Yeast Strains

Unless stated otherwise, the experiments in this study were performed using mitochondria isolated from the Saccharomyces cerevisiae wild-type strain PK82 (MATα his4–713 lys2 ura3–52 [open triangle]

trp1 leu2–3112) (Gambill et al., 1993 blue right-pointing triangle

). For the experiments shown in Figure Figure6,6, the strains PK82 and PK83 (MATα ade2–101 lys2 ura3–52 leu2–3112 [open triangle]

trp1 ssc1–3(LEU2)) (Gambill et al., 1993 blue right-pointing triangle

), MB3–46 (MATα ade2–101 his3- [open triangle]

200 leu2- [open triangle]

1 lys2–801 ura3::LYS2 tim23–2) (Dekker et al., 1993 blue right-pointing triangle

), and the corresponding wild-type strain MB3 (MATα ade2–101 his3- [open triangle]

200 leu2- [open triangle]

1 lys2–801 ura3::LYS2) were used.

Figure 6

The requirement of mtHsp70 and Tim23 for the import of b₂-DHFR preproteins. (A) Requirement for mtHsp70. pb₂-DHFR, pb₂( [open triangle]

47–65)-DHFR, and pb₂(QIC)-DHFR were imported into mitochondria isolated from the scc1–3 mutant strain and (more ...)

Construction of b₂-Dihydrofolate Reductase Fusion Proteins

For in vitro transcription of pb₂(167)-dihydrofolate reductase (DHFR), pb₂( [open triangle]

47–65)-DHFR, and pb₂(K48I,R49C)-DHFR, pGEM4Z plasmids containing the respective open reading frames were used (Rassow et al., 1989 blue right-pointing triangle

; Koll et al., 1992 blue right-pointing triangle

; Bömer et al., 1997 blue right-pointing triangle

). To construct the other fusion proteins, oligonucleotide-directed missense mutagenesis polymerase chain reaction (PCR) was used with the following primers and templates: forward primer 5′-GTCGTTCGAACAAGACT CGCAAAT-ATGCACACAGTCATG-3′ and reverse primer 5′-CATGACTGTG TGCATATTTGCGAGTCTTGTTCGAACGAC-3′ on pb₂(K48I,R49C)-DHFR (Bömer et al., 1997 blue right-pointing triangle

) as a template to generate pb₂(QIC)-DHFR; forward primer 5′- GTCATGGACTGCCTTGCAGGTCGGTGCAA-TTCTAG-3′ and reverse primer 5′- CTAGAATTGCACCGACCTGCAAGGCAGTCCATGAC-3′ on pb₂(QIC)-DHFR as a template to generate pb₂(QIC-Q)-DHFR; forward primer 5′-CAAAAT CCAAGTCG-TTCCAACAAAACTCAAGAAAACGCAC-3′ and reverse primer 5′-GTGCGTTTTCTTGAGTTTTGTTGGAACGACTTGGATTTTG-3′ on pb₂(167)-DHFR (Rassow et al., 1989 blue right-pointing triangle

; Voos et al., 1993 blue right-pointing triangle

) as a template to generate pb₂(E43Q,D45N)-DHFR; forward primer 5′-GTTCGAACAAGACTCAGTCGGTGCAATTCTAG-3‘ and reverse primer 5‘-CTAGAATTGCACCGACTGAGTCTTGTTCGAAC-3‘ on pb₂(167)-DHFR to generate pb₂( [open triangle]

47–57)-DHFR. After the PCR reaction, the template DNA was digested with DpnI. To construct pb₂(A63P)-DHFR, a PCR was performed on pb₂(167)-DHFR using 5′-GAATTGGATTTAGGTGACACTATA-3′ as a forward primer and 5′-GAACTAGRAGCGGGTAGAATTGCACCG-3′ as a reverse primer. The resulting product was used as a forward primer in a subsequent PCR with 5′-CAAGCTCTAATACGACTCACTATA-3′ as a reverse primer, using pb₂(167)-DHFR as a template. After digestion with DpnI, the product was cut with EcoRI and MscI and was ligated into EcoRI- and MscI-digested pb₂(167)-DHFR. All constructs were transformed into the Escherichia coli strain XL-1 blue (Stratagene, La Jolla, CA). Formation of the correct products was confirmed by DNA sequencing.

Import of Preproteins into Isolated Mitochondria

Mitochondria were isolated from yeast cells grown on YPG (1% yeast extract, 2% bactopeptone, and 3% glycerol) according to published procedures (Daum et al., 1982 blue right-pointing triangle

; Hartl et al., 1987 blue right-pointing triangle

; Kang et al., 1990 blue right-pointing triangle

; Gambill et al., 1993 blue right-pointing triangle

), were resuspended in SEM buffer (250 mM sucrose, 1 mM EDTA, and 10 mM Mops-KOH, pH 7.2) to a concentration of 5 mg/ml, and were stored at −80°C. Radiolabeled mitochondrial preproteins were synthesized by in vitro translation in rabbit reticulocyte lysate (Amersham Pharmacia Biotech, Uppsala, Sweden) in the presence of [³⁵S]methionine/cysteine after in vitro transcription using SP6 polymerase (Stratagene) (Söllner et al., 1991 blue right-pointing triangle

For the in vitro import assay, mitochondria (25–50 μg of protein) were diluted with import buffer (1% [wt/vol] fatty acid-free bovine serum albumin [BSA], 250 mM sucrose, 80 mM KCl, 5 mM MgCl₂, 2 mM ATP, 2 mM NADH, and 10 mM Mops-KOH, pH 7.2) to a final volume of 100 μl. The samples were preincubated for 5 min at 25°C before the import reaction was started by adding 2–4 μl of reticulocyte lysate containing ³⁵S-labeled preproteins. For the accumulation of import intermediates, the import was performed in the presence of 5 μM of methotrexate (Sigma Chemical, St. Louis, MO) when indicated. After incubation for 2.5–6 min at 25°C, the import reaction was stopped by the addition of 1 μM valinomycin to dissipate the membrane potential [open triangle]

ψ. The samples were treated with proteinase K (40 μg/ml) for 15 min, followed by the addition of 1 mM phenylmethylsulfonyl fluoride and incubation for 10 min on ice. The mitochondria were subsequently reisolated, washed with SEM buffer, and subjected to SDS-PAGE.

Carbonyl Cyanide m-Chlorophenylhydrzone Titration

The mitochondria were partially uncoupled by the addition of the protonophore carbonyl cyanide m-chlorophenylhydrzone (CCCP) (Martin et al., 1991 blue right-pointing triangle

; Gärtner et al., 1995b blue right-pointing triangle

). CCCP was added (from a stock solution concentrated 100-fold in ethanol) before the preincubation at 25°C and before import. All samples were made chemically identical by adding the corresponding amount of solvent to the control samples. To prevent the generation of an electrochemical potential by a reversed action of the F_oF₁-ATPase, 20 μM oligomycin was added to the import buffer.

Intramitochondrial Localization of Imported Proteins

To determine the localization of the imported proteins, import was performed as described above. After import, the samples were diluted with five volumes of EM buffer (1 mM EDTA, and 10 mM Mops-KOH, pH 7.2) to rupture the outer membrane by swelling. After 15 min of incubation on ice, five volumes of S₅₀₀EM buffer (500 mM sucrose, 1 mM EDTA, and 10 mM Mops-KOH, pH 7.2), were added to reestablish the original osmotic conditions. For nonswelling conditions, the import mix was diluted twice with SEM buffer. Mitochondria of all samples were reisolated by centrifugation resuspended in 100 μl of SEM buffer, and proteinase K treatment was carried out as described above. After SDS-PAGE, blotting of the proteins to nitrocellulose membrane and subsequent immunodecoration with control antibodies were performed.

Coimmunoprecipitation

The interaction of imported protein with mtHsp70 was analyzed by coimmunoprecipitation (Voisine et al., 1999 blue right-pointing triangle

). pb₂(

47–65)-DHFR was imported into mitchondria in the presence of different concentrations of CCCP for 5 min at 25°C. The mitochondria were reisolated, washed with SEM, and lysed in buffer A (0.1% [vol/vol] Triton X-100, 100 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM PMSF, and 5 mM EDTA). After a clarifying spin (16,000 × g for 5 min), the supernatants were transferred to antibodies directed against mtHsp70 that were bound to protein-A sepharose. The samples were incubated for 1 h at 4°C rotating end-over-end. Subsequently, the protein-A sepharose beads were washed three times in buffer A and once with 10 mM Tris-HCl, pH 7.4. Bound proteins were eluted by the addition of SDS-sample buffer and were analyzed by SDS-PAGE.

Assessment of the Mitochondrial Membrane Potential Δψ

The membrane potential Δψ of isolated yeast mitochondria was assessed by measuring the fluorescence quenching of the potential-sensitive dye 3,3′-dipropylthiadicarbocyanine iodide (DiSC₃(5); Molecular Probes, Eugene, OR) as described before (Sims et al., 1974 blue right-pointing triangle

; Gärtner et al., 1995b blue right-pointing triangle

). The measurements were performed using a Perkin Elmer-Cetus (Norwalk, CT) LS 50B luminescence spectrometer at 25°C, with excitation at 622 nm, emission at 670 nm, and slits at 5 nm. The measurements were carried out using a buffer containing 600 mM sorbitol, 1% (wt/vol) BSA, 10 mM MgCl₂, 0.5 mM EDTA, and 20 mM KP_i, pH 7.4. The following reagents were successively added to 3 ml of the buffer and the change in fluorescence was recorded: 3 μl of DiSC₃(5) (in ethanol; final concentration, 2 μM); 20 μl of mitochondria (in SEM buffer; final concentration, 33 μg mitochondrial protein per milliliter); and, finally, 3 μl of valinomycin (in ethanol; final concentration, 1 μM) to dissipate [open triangle]

ψ. The difference in the fluorescence before and after the addition of valinomycin represents a relative assessment of the membrane potential.

Miscellaneous Methods

Standard techniques were used for SDS-PAGE, immunodecoration, and DNA manipulation. For the detection and quantitation of radiolabeled proteins, a storage phosphor imaging system with software (ImageQuant version 1.11, Molecular Dynamics, Sunnyvale, CA) was used.

RESULTS

A Deletion in the Sorting Sequence of Cytochrome b₂ Causes a Higher Δψ-Dependence of Transport into Mitochondria

To modulate the magnitude of the mitochondrial membrane potential Δψ under in vitro protein import conditions into isolated mitochondria, we used different concentrations of the protonophore CCCP (Martin et al., 1991 blue right-pointing triangle

; Nicholls and Ferguson, 1982 blue right-pointing triangle

; Gärtner et al., 1995b blue right-pointing triangle

; Bömer et al., 1998 blue right-pointing triangle

). Oligomycin was included to inhibit the F_oF₁-ATPase to prevent the generation of a membrane potential and the depletion of matrix ATP by a reverse action of the ATPase. The membrane potential was assessed by use of the fluorescent dye DiSC₃(5) that is taken up by mitochondria and thus quenched in a Δψ-dependent manner (Sims et al., 1974 blue right-pointing triangle

; Gärtner et al., 1995b blue right-pointing triangle

). The decrease in fluorescence (which is reversed by a complete dissipation of Δψ by the potassium ionophore valinomycin in the presence of potassium in the medium) is used as an assessment for the magnitude of Δψ (Figure (Figure1A,1A, top). By the addition of increasing concentrations of CCCP, this fluorescence quenching was gradually reduced until a complete dissipation was achieved (Figure (Figure1A 1A [middle and bottom] and B).

Figure 1

The mitochondrial import of two b₂-DHFR preproteins shows a differential dependence on the membrane potential Δψ. (A) The membrane potential [open triangle]

ψ of isolated yeast mitochondria was assessed at 25°C using the potential-sensitive (more ...)

b₂-DHFR fusion proteins are widely used to study mitochondrial protein import since they are efficiently synthesized and radiolabeled in rabbit reticulocyte lysate and their intramitochondrial processing and localization can be unambiguously determined (Hartl et al., 1987 blue right-pointing triangle

; Rassow et al., 1989 blue right-pointing triangle

, 1990

; Koll et al., 1992 blue right-pointing triangle

; Beasley et al., 1993 blue right-pointing triangle

; Glick et al., 1993 blue right-pointing triangle

; Schwarz et al., 1993 blue right-pointing triangle

; Voos et al., 1993 blue right-pointing triangle

; Stuart et al., 1994 blue right-pointing triangle

; Voisine et al., 1999 blue right-pointing triangle

). The 167 amino-terminal amino acid residues of cytochrome b₂ that were used as the basis for the preproteins of this study contain the complete targeting and sorting information of the preprotein as follows: the matrix-targeting sequence (residues 1–31); the sorting sequence (residues 32–80); and 87 residues of the mature protein. Fused to the entire DHFR, the resulting chimeric preprotein b₂-DHFR (Figure (Figure1C) 1C) has been shown to be sorted to the intermembrane space like authentic cytochrome b₂ (Koll et al., 1992 blue right-pointing triangle

; Voos et al., 1993 blue right-pointing triangle

) and will be referred to as the wild-type preprotein in this study. b₂-DHFR was synthesized in rabbit reticulocyte lysates in the presence of [³⁵S]methionine/cysteine and was incubated with isolated yeast mitochondria. Import was determined by monitoring the processing of the preprotein to the intermediate- and mature-sized forms and by protection against externally added protease (Figure (Figure1D, 1D, lane 1). On addition of increasing concentrations of CCCP, the import of b₂-DHFR was gradually inhibited (Figure (Figure1D, 1D, lanes 2–8). In the short import time that is required to be in the kinetically linear import range (Söllner et al., 1991 blue right-pointing triangle

; Alconada et al., 1995 blue right-pointing triangle

), the slow second processing step generated only small amounts of the mature form. Since the Δψ-dependent step of import takes place before the generation of the intermediate-sized form by the matrix-processing peptidase (Schatz, 1996 blue right-pointing triangle

; Neupert, 1997 blue right-pointing triangle

; Pfanner et al., 1997 blue right-pointing triangle

), the sum of protease-protected intermediate- and mature-sized forms was used for the quantitation of import (Figure (Figure11E).

The deletion of a 19-residue segment in the sorting sequence of b₂-DHFR generates the preprotein b₂( [open triangle]

47–65)-DHFR, which is sorted into the matrix space and cleaved to the intermediate-sized form (Koll et al., 1992 blue right-pointing triangle

; Voos et al., 1993 blue right-pointing triangle

; Voisine et al., 1999 blue right-pointing triangle

). b₂(

47–65)-DHFR was efficiently imported into yeast mitochondria in the absence of CCCP (Figure (Figure1D,1D, lane 9) yet was inhibited strongly by the addition of CCCP (Figure (Figure1D,1D, lanes 10–16). A quantitative analysis of the inhibitory effect of CCCP on the import of b₂-DHFR and b₂( [open triangle]

47–65)-DHFR revealed a striking difference (Figure (Figure1E,1E, left panel), indicating that the import of b₂( [open triangle]

47–65)-DHFR was significantly more sensitive to a reduction of the mitochondrial membrane potential than that of b₂-DHFR.

For comparison, we imported the following two preproteins that were reported to depend differentially on Δψ: the β-subunit of the F₁-ATPase (F₁β) and a fusion protein between the presequence of F_o-ATPase subunit 9 and DHFR (Su9-DHFR) (Martin et al., 1991 blue right-pointing triangle

). The import of F₁β was strongly inhibited by the addition of CCCP (Figure (Figure1D,1D, lanes 25–32), while the import of Su9-DHFR showed a higher resistance to CCCP (Figure (Figure1D,1D, lanes 17–24). The quantitation indicated that the CCCP sensitivity of the import of b₂( [open triangle]

47–65)-DHFR was comparable to that of F₁β, while that of b₂-DHFR was more related to that of Su9-DHFR (Figure (Figure1E).1E). We conclude that the import of b₂-DHFR and of b₂( [open triangle]

47–65)-DHFR show a differential Δψ-dependence. The difference is roughly related to that observed for the import of Su9-DHFR and F₁β. However, Su9-DHFR and F₁β possess quite different matrix-targeting sequences, whereas b₂-DHFR and b₂( [open triangle]

47–65)-DHFR possess the identical matrix-targeting sequence. In the following chapters, we thus asked if characteristics of the sorting sequence of the b₂-fusion proteins were responsible for the differential Δψ-dependence.

A Differential Δψ-Dependence of b₂-DHFR Preproteins with the Same Intramitochondrial Destination

b₂-DHFR is transported to the intermembrane space, whereas b₂( [open triangle]

47–65)-DHFR is completely imported into the matrix (Koll et al., 1992 blue right-pointing triangle

; Voos et al., 1993 blue right-pointing triangle

) as demonstrated here by the differential protease accessibility after opening of the mitochondrial outer membrane by swelling (i.e., formation of mitoplasts). A major fraction of imported b₂-DHFR was sensitive to proteinase K like the intermembrane space-exposed portions of the marker protein ADP/ATP carrier (Figure (Figure2A,2A, lane 2, columns 3 and 5), while b₂( [open triangle]

47–65)-DHFR was mainly protected against the protease, which is comparable to the matrix marker mitochondrial GrpE (Mge1) (Figure (Figure2A,2A, lane 2, columns 4 and 6). After lysis of the mitochondrial membranes with detergent, both b₂-DHFR fusion proteins as well as the marker proteins were fully accessible to and degraded by proteinase K (Gärtner et al., 1995a blue right-pointing triangle

; data not shown), excluding an endogenous protease resistance of the proteins as explanation for the different protease protection.

Figure 2

Lowering of the membrane potential does not change the intramitochondrial sorting of the b₂-DHFR preproteins. (A) pb₂( [open triangle]

47–65)-DHFR is transported into the matrix. Radiolabeled pb₂-DHFR and pb₂( [open triangle]

47–65)-DHFR were synthesized (more ...)

We asked whether a lowering of the membrane potential altered the intramitochondrial sorting of the b₂-fusion proteins. The preproteins were imported at different concentrations of CCCP, and half of each sample was subjected to swelling. Both the i- and m-form of b₂-DHFR were largely degraded by added proteinase K (Figure (Figure2B,2B, lower panel, lanes 1–4), whereas i-b₂( [open triangle]

47–65)-DHFR was mainly protected against the protease (Figure (Figure2B,2B, lower panel, lanes 5–8). A quantitative analysis demonstrated that the intramitochondrial locations of b₂-DHFR and b₂( [open triangle]

47–65)-DHFR were not affected by the addition of CCCP to the import reaction (Figure (Figure2C).2C). (A second processing to i* that was observed for a small amount of b₂( [open triangle]

47–65)-DHFR [Figure 2B] has been reported for a number of matrix-targeted preproteins and is apparently mediated by the matrix-localized mitochondrial intermediate peptidase [Isaya et al., 1991 blue right-pointing triangle

; Kalousek et al., 1993 blue right-pointing triangle

; Schwarz et al., 1993 blue right-pointing triangle

]. In all experiments, the Δψ-dependence of formation of the i*-form correlated with that of formation of the i-form, and thus our quantitations included both forms.)

Does the transport of b₂-fusion proteins into the matrix require a higher membrane potential than the transport into the intermembrane space? An exchange of two basic amino acid residues (lysine 48 and arginine 49) of the sorting sequence by neutral amino acids impaired the sorting function and caused transport of the mutant preprotein into the matrix (Schwarz et al., 1993 blue right-pointing triangle

; Bömer et al., 1997 blue right-pointing triangle

). We synthesized b₂(K48I,R49C)-DHFR and imported it into mitochondria. Figure Figure3A3A demonstrates that the imported fusion protein was protected against protease added to mitoplasts like the matrix marker Mge1 (columns 1 and 3) and thus transported into the matrix space. Then the CCCP sensitivity for the import of b₂(K48I,R49C)-DHFR was determined (Figure (Figure3B).3B). The import of b₂(K48I,R49C)-DHFR was significantly more resistant to CCCP than that of b₂( [open triangle]

47–65)-DHFR yet was similar to that of b₂-DHFR (Figure (Figure3C).3C). Thus, b₂(K48I,R49C)-DHFR and b₂-DHFR show a similar Δψ-dependence, although their sorting pathways are different. In contrast, b₂( [open triangle]

47–65)-DHFR and b₂(K48I,R49C)-DHFR are both translocated into the matrix but have a strikingly different Δψ-dependence.

Figure 3

A matrix-targeted b₂-DHFR construct with a similar Δψ-dependence as an intermembrane space-targeted construct. (A) The fusion protein pb₂(K48I, R49C)-DHFR was imported into mitochondria, and the intramitochondrial localization was (more ...)

These results lead to two related conclusions. First, lowering of Δψ during import does not alter the intramitochondrial sorting of b₂-fusion proteins. Second, a differential Δψ-dependence of import of b₂-fusion proteins cannot be explained by a different intramitochondrial destination (matrix or intermembrane space). Taken together, these results indicate that the sorting pathway of b₂-fusion proteins is not a critical determinant for the Δψ-dependence of import.

The Content of Charged Residues in the Sorting Sequence Is Not Critical for the Δψ-Dependence of Protein Import

The entire presequence of b₂-DHFR contains a net positive charge of 10 (i.e., +7 for the matrix-targeting sequence and +3 for the sorting sequence) (Guiard, 1985 blue right-pointing triangle

). In b₂( [open triangle]

47–65)-DHFR, a segment containing four positively charged residues has been deleted, leading to a net charge of the sorting sequence of −1 (Figure (Figure4A)4A) (Koll et al., 1992 blue right-pointing triangle

). The two classic preproteins that have been shown to differentially depend on a Δψ for import, Su9-DHFR and F₁β (see Figure Figure1,1, D and E) (Martin et al., 1991 blue right-pointing triangle

), differ in the net charge of their presequences from +12 to +6 (Figure (Figure4A,4A, right panel). It was thus conceivable that a difference in the net positive charge of b₂-fusion proteins was responsible for the differential Δψ-dependence.

Figure 4

Substitution of charged residues in the sorting sequence does not alter the [open triangle]

ψ dependence of import. (A) b₂-fusion proteins employed. The net charges (amino acid side chains) of the matrix-targeting sequence and the sorting sequence (more ...)

To test this, three or four positively charged residues in the sorting sequence were replaced by uncharged amino acid residues, leading to the fusion proteins b₂(QIC)-DHFR and b₂(QIC-Q)-DHFR with net charges of the sorting sequence of 0 or −1, respectively (Figure (Figure4A).4A). These preproteins were imported into mitochondria at different concentrations of CCCP (Figure (Figure4B,4B, lanes 1–16). Surprisingly, the import of the preproteins revealed a similar sensitivity to a decrease of the membrane potential as that of the wild-type presequence of b₂-DHFR but was clearly different from that of b₂( [open triangle]

47–65)-DHFR (Figure (Figure4B,4B, left panel and middle panel). In particular, b₂(QIC-Q)-DHFR contains the identical charged residues as b₂( [open triangle]

47–65)-DHFR throughout the entire preprotein but shows a much lower Δψ-dependence (Figure (Figure4B, 4B, middle panel). Moreover, the replacement of two negatively charged residues of the sorting sequence by uncharged ones generated the preprotein b₂(E43Q,D45N)-DHFR with a higher net charge than the wild-type sorting sequence (i.e., +5) (Figure (Figure4A).4A). The import of b₂(E43Q,D45N)-DHFR again revealed a Δψ-dependence that was similar to b₂-DHFR (Figure (Figure4B,4B, lanes 17–24, right panel). These results demonstrate that the Δψ-dependence of import is independent of the net charge of the b₂ sorting sequence.

The wild-type b₂-DHFR and the constructs b₂(K48I,R49C)-DHFR, b₂(QIC)-DHFR, and b₂(QIC-Q)-DHFR, which share a similar Δψ-dependence (Figures (Figures3C3C and and4B),4B), all harbor an uncharged segment (residues 58–65) that is lacking in b₂( [open triangle]

47–65)-DHFR. We asked whether a lack of this segment was responsible for the high Δψ-dependence of b₂( [open triangle]

47–65)-DHFR. Thus, we constructed the fusion protein b₂( [open triangle]

47–57)-DHFR that only lacked the segment containing the four positively charged residues (Figure (Figure5A).5A). The preprotein was imported into mitochondria in the presence of different concentrations of CCCP (Figure (Figure5B).5B). The import of b₂( [open triangle]

47–57)-DHFR showed a high sensitivity toward the lowering of the membrane potential that was close to that of b₂( [open triangle]

47–65)-DHFR (Figure (Figure5C).5C). (Although the difference of the Δψ-dependence of b₂( [open triangle]

47–57)-DHFR compared with b₂( [open triangle]

47–65)-DHFR was statistically significant, the difference was rather small when compared with the Δψ-dependence of b₂-DHFR [Figure 5C].) This result suggests that a lack of this uncharged segment is not the main determinant for the strong difference in Δψ-dependence of import observed between the wild-type preprotein and b₂( [open triangle]

47–65)-DHFR.

Figure 5

Effect of the uncharged stretch within the sorting sequence. (A) The fusion protein b₂( [open triangle]

47–57)-DHFR lacks the positive charges of the sorting sequence like b₂( [open triangle]

47–65)-DHFR but maintains most of the uncharged stretch (more ...)

The Differential Δψ-Dependence Is Not Attributable to the Dependence on mtHsp70 or Tim23

We asked whether the differential Δψ-dependence of b₂-fusion proteins could be explained by the requirement for the second import driving force, mtHsp70, or the dependence on the function of Tim23. We used the preproteins b₂-DHFR, b₂( [open triangle]

47–65)-DHFR, and b₂(QIC)-DHFR to address this problem. In the yeast mutant ssc1–3, mtHsp70 carries an amino acid exchange in the ATPase domain that strongly inhibits its function (Gambill et al., 1993 blue right-pointing triangle

; Voos et al., 1993 blue right-pointing triangle

, 1996

). The isolated mitochondria were preincubated at 37°C to induce the mutant phenotype. The import of b₂( [open triangle]

47–65)-DHFR and b₂(QIC)-DHFR was strongly inhibited in the mutant mitochondria (Figure (Figure6A,6A, middle panel and lower panel, lanes 3 and 4), while the import of b₂-DHFR was only partially reduced (Figure (Figure6A, 6A, upper panel, lanes 3 and 4). Therefore, the requirement for mtHps70 does not correlate with the Δψ-dependence, since b₂-DHFR and b₂(QIC)-DHFR have a similar low Δψ-dependence while b₂( [open triangle]

47–65)-DHFR has a strong Δψ-dependence. The dependence on Tim23 function was analyzed with tim23–2 mutant mitochondria in which the oligomerization of the TIM23 translocase is destabilized and, thus, the transport efficiency of preproteins is reduced (Dekker et al., 1997 blue right-pointing triangle

; Bömer et al., 1997 blue right-pointing triangle

). All three b₂-fusion proteins were inhibited in import into tim23–2 mitochondria by ~60–70% compared with wild-type mitochondria (Figure (Figure6B).6B). In particular, no significant difference of b₂( [open triangle]

47–65)-DHFR compared with b₂-DHFR or b₂(QIC)-DHFR was recognizable, indicating that the differential Δψ-dependence cannot be attributed to a different requirement for Tim23.

A b₂-DHFR Construct That Is Less Dependent on Δψ Than the Wild-Type Version

The results obtained so far have suggested a novel characteristic of the membrane potential-driven import of preproteins, which depends on properties of the sorting sequence in a charge-independent manner. We wondered whether further independent evidence for this novel role of Δψ could be obtained. Beasley et al. (1993) blue right-pointing triangle

had selected a number of mutations in the preprotein of cytochrome b₂. We asked whether the mutation of an uncharged residue to another uncharged residue in the sorting sequence of cytochrome b₂ would influence the Δψ-dependence of import. The residues in the segment deleted in b₂( [open triangle]

47–65)-DHFR seemed to be of particular importance to us.

Finally, we found a single mutation, a substitution of alanine 63 by proline, that generated a preprotein with a remarkable Δψ-dependence. b₂(A63P)-DHFR was targeted to the matrix space since it remained protease-protected in mitoplasts like the matrix marker Mge1 (Figure (Figure7A, 7A, lane 2, columns 3 and 5) (Beasley et al., 1993 blue right-pointing triangle

). When the sensitivity of import to CCCP was analyzed, b₂(A63P)-DHFR showed a higher resistance than any preprotein tested before (Figure (Figure7B,7B, lanes 1–8, quantitation). CCCP concentrations up to 30 μM that lead to a clear reduction of Δψ (see Figure Figure1B)1B) did not inhibit the import of b₂(A63P)-DHFR but actually led to a slight stimulation (Figure (Figure7B,7B, panel on the right). The import of b₂(A63P)-DHFR was only inhibited at higher concentrations of CCCP. Therefore, b₂(A63P)-DHFR requires less Δψ for import than the wild-type presequence of b₂-DHFR, proving that uncharged residues in the b₂ sorting sequence can play a critical role in the membrane potential-dependence of import.

Figure 7

An alteration in the hydrophobic segment of the sorting sequence lowers the Δψ-dependence. (A) The hybrid protein b₂(A63P)-DHFR was imported into isolated yeast wild-type mitochondria, and the intramitochondrial localization was determined (more ...)

In Figure Figure7C,7C, we compared the b₂-fusion proteins used in this study. First, all preproteins required a membrane potential for import since a complete dissipation of Δψ by the addition of valinomycin (in the presence of potassium in the import buffer) blocked the import of each protein (Figure (Figure7C,7C, even-numbered columns). Second, roughly three classes of preproteins can be distinguished when an intermediate level of Δψ is generated by the addition of CCCP (shown are the import results at 30 μM CCCP) (Figure (Figure7C,7C, odd-numbered columns): (1) A series of constructs with a different number of charged residues in the sorting sequence (Figure (Figure7C,7C, columns 5, 7, 9, and 11) show a Δψ-dependence that is roughly similar to that of b₂-DHFR with the wild-type presequence (Figure (Figure7C,7C, column 3); (2) b₂( [open triangle]

47–65)-DHFR and b₂( [open triangle]

47–57)-DHFR reveal a much stronger Δψ-dependence (Figure (Figure7C,7C, columns 13 and 15); while (3) b₂(A63P)-DHFR still can be efficiently imported at a low Δψ (Figure (Figure7C,7C, column 1).

Early Function of the Sorting Sequence for Translocation of the Matrix-targeting Sequence

The experiments described so far have analyzed the influence of the sorting sequence on the entire import process, since the processed forms of the fusion proteins protected against externally added proteinase K were quantified. We asked whether the sorting sequence already had affected the early import stage of translocation of the matrix-targeting sequence or whether the sorting sequence functioned only in a later stage by promoting the translocation of carboxy-terminal parts of the presequence and the mature protein parts. In the latter case, the translocation of the matrix-targeting sequence of b₂( [open triangle]

47–65)-DHFR should have a lower Δψ-dependence than the translocation of the entire fusion protein. Therefore, we analyzed the efficiency of processing of b₂( [open triangle]

47–65)-DHFR, i.e., the removal of the matrix-targeting sequence by the matrix-processing peptidase, without treating the mitochondria with proteinase K (Figure (Figure8A,8A, lanes 1–8). A direct comparison with the protease protection of the processed protein, however, did not reveal a difference (Figure (Figure8,8, A, lanes 9–16, and B, left panel). The Δψ-dependence of the formation of i-b₂( [open triangle]

47–65)-DHFR was indistinguishable from that of translocation of the entire protein to a protease-protected location (Figure (Figure8B,8B, left panel). With both b₂(K48I,R49C)-DHFR and b₂(A63P)-DHFR, which are imported into the matrix like b₂( [open triangle]

47–65)-DHFR, the translocation of the matrix-targeting sequence to the matrix-processing peptidase showed a lower Δψ-dependence than that of b₂( [open triangle]

47–65)-DHFR (Figure (Figure8B);8B); however, the Δψ-dependence was not lower than that of the complete import of the respective protein (Figure (Figure8B,8B, middle panel and right panel). These results suggested that the sorting sequence influenced the Δψ-dependence at a very early import stage.

Figure 8

The sorting sequence influences the Δψ-dependence of translocation of the matrix-targeting sequence. (A and B) The preproteins of b₂( [open triangle]

47–65)-DHFR, b₂(K48I, R49C)-DHFR, and b₂(A63P)-DHFR were imported into mitochondria (more ...)

To obtain independent evidence, we probed the accessibility of the preprotein to matrix Hsp70. mtHsp70 can bind to the matrix-targeting sequence as soon as it emerges on the matrix side of the inner membrane import channel, even when the processing site has not yet been exposed to the matrix (Ungermann et al., 1994 blue right-pointing triangle

). Thereby, the very first stage of translocation of the matrix-targeting signal into the matrix can be analyzed. b₂( [open triangle]

47–65)-DHFR was imported at different concentrations of CCCP. Mitochondria were lysed with nonionic detergent, and the association of the fusion protein with mtHsp70 was determined by coimmunoprecipitation. In the absence of CCCP, ~2.5% of processed b₂( [open triangle]

47–65)-DHFR was recovered together with mtHsp70 (Figure (Figure8C,8C, column 2); this represents a typical yield for the coimmunoprecipitation (including several washing steps) of accumulated substrate with mtHsp70 (Ungermann et al., 1994 blue right-pointing triangle

; Voisine et al., 1999 blue right-pointing triangle

). In the presence of CCCP, the processing of b₂( [open triangle]

47–65)-DHFR (Figure (Figure8C,8C, upper panel, columns 4 and 6) as well as the coprecipitation of i-b₂( [open triangle]

47–65)-DHFR decreased (Figure (Figure8C,8C, lower panel, columns 4 and 6). The amount of the precursor form of b₂( [open triangle]

47–65)-DHFR associated with mitochondria increased in the presence of CCCP (Figure (Figure8C,8C, upper panel, columns 3 and 5 versus column 1); however, coprecipitation of the precursor form with antimtHsp70 remained at a very low level under all conditions and was not increased at low Δψ (Figure (Figure8C,8C, lower panel, columns 1, 3 and 5), indicating that the precursor form was not accessible for binding to mtHsp70. Together with the protease sensitivity of the precursor form (Figure (Figure8A),8A), this result shows that the precursor form is located on the mitochondrial surface and has not yet entered the matrix space. The coprecipitation of b₂( [open triangle]

47–65)-DHFR with anti-mtHsp70 thus confirms the result obtained with the processing assay that the initial translocation of the amino-terminal portion of the preprotein across the inner membrane shows a strong sensitivity to CCCP like the translocation of the complete protein. We conclude that the deletion in the sorting sequence of b₂( [open triangle]

47–65)-DHFR already affects the Δψ-dependence of translocation of the matrix-targeting sequence across the inner membrane.

The specific ligand methotrexate (MTX) stabilizes the DHFR moiety of the cytochrome b₂ fusion proteins and, thus, arrests the importing protein after the first step of translocation in a processed state, with the folded DHFR still outside the mitochondria (Eilers and Schatz, 1986 blue right-pointing triangle

; Rassow et al., 1989 blue right-pointing triangle

). The preproteins of b₂( [open triangle]

47–65)-DHFR, b₂(K48I,R49C)-DHFR, and b₂(A63P)-DHFR were imported in the presence of MTX and different concentrations of CCCP (Figure (Figure8D).8D). The efficiency of translocation arrest was demonstrated by the accessibility of the intermediates to proteinase K (not shown). A quantification of the processing efficiency revealed that the Δψ-dependence of the constructs significantly differed (Figure (Figure8E),8E), as was observed for the translocation of the entire proteins; i.e., b₂(A63P)-DHFR showed the lowest Δψ-dependence, and b₂( [open triangle]

47–65)-DHFR showed the highest Δψ-dependence (compare Figure Figure8E8E to to7C).7C). We conclude that the sorting sequence of cytochrome b₂ contributes to the Δψ-dependence of import at an early stage when the major portion of the mature protein is still outside the mitochondrion.

DISCUSSION

The membrane potential Δψ is essential for the transport of preproteins into or across the mitochondrial inner membrane. We report that the sorting sequence of a cleavable preprotein strongly influences the requirement for Δψ. All cytochrome b₂ fusion proteins used here contain the identical matrix-targeting sequence and the identical mature protein part, and differences were only introduced in the sorting sequence in the form of deletions or of amino acid substitutions. All b₂-fusion proteins were efficiently imported into fully energized mitochondria (i.e., at a high Δψ) and were blocked fully in transport across the inner membrane upon a complete dissipation of Δψ. However, significant differences in import efficiency became apparent when the magnitude of the membrane potential was gradually lowered by the protonophore CCCP. Since the sorting sequence determines the intramitochondrial sorting of b₂-fusion proteins to the intermembrane space or matrix, an obvious assumption was that a differential Δψ-dependence would be related to the sorting pathway of the preproteins. However, we found that the Δψ-dependence was independent of the intramitochondrial destination, and, in particular, matrix-targeted b₂-fusion proteins with both a high and a low Δψ-dependence were found.

It has to be emphasized that CCCP selectively inhibits the Δψ-dependent step of protein import and does not unspecifically impair the import competence of preproteins or mitochondrial function for the following reasons. In the presence of high concentrations of CCCP, preproteins still specifically interact with the TOM machinery of the outer membrane (Hines and Schatz, 1993 blue right-pointing triangle

; Haucke et al., 1995 blue right-pointing triangle

; Ryan et al., 1999 blue right-pointing triangle

). The import block by CCCP can be reversed by the removal of CCCP, and, thus, arrested preproteins can be imported completely (Hines and Schatz, 1993 blue right-pointing triangle

; Haucke et al., 1995 blue right-pointing triangle

; Ryan et al., 1999 blue right-pointing triangle

). The induction of a potassium diffusion potential (by valinomycin in the presence of low potassium in the medium) abolishes the dissipation of Δψ by CCCP and allows import of preproteins, even in the presence of high concentrations of CCCP (Pfanner and Neupert, 1985 blue right-pointing triangle

; Martin et al., 1991 blue right-pointing triangle

The differential Δψ-dependence of the b₂-fusion proteins was not attributable to a differential dependence on the function of mtHsp70. The intramitochondrial sorting pathway of b₂-fusion proteins is critical for the requirement for mtHsp70, since matrix-targeted preproteins, but not intermembrane space-targeted preproteins, strongly depend on the chaperone (Voos et al., 1993 blue right-pointing triangle

; Stuart et al., 1994 blue right-pointing triangle

; Gärtner et al., 1995a blue right-pointing triangle

), while the Δψ-dependence is independent of the sorting pathway. Moreover, preproteins with a low and a high Δψ-dependence showed the same requirement for Tim23 function. In fact, the modulatory effect of the sorting sequence on the Δψ-dependence of protein import was much stronger than the effect of Δψ on Tim23 dimerization (Figure (Figure7C)7C) (Bauer et al., 1996 blue right-pointing triangle

), excluding the fact that the effect of the sorting sequence on the Δψ-dependence was mediated by Tim23.

b₂(

47–65)-DHFR that strongly depends on Δψ lacks four positively charged residues compared with the wild-type sorting sequence of b₂-DHFR with a lower Δψ-dependence, raising the possibility that the membrane potential exerted an electrophoretic effect not only on the matrix-targeting sequence, but also on the sorting sequence. Thus, we constructed a series of b₂-fusion proteins in which positively or negatively charged residues of the sorting sequence were replaced by neutral residues. Surprisingly, however, no differences in the responses to the membrane potential were observed, although the difference in net charge of the sorting sequence was up to 6 among different fusion proteins. These results indicate that the sorting sequence of cytochrome b₂ influences the requirement for a Δψ in a novel manner that is independent of the net charge of the sorting sequence.

As the deleted segment of b₂( [open triangle]

47–65)-DHFR not only contained charged residues but also an uncharged stretch, we reinserted this segment in a further construct, but the Δψ-dependence did not change substantially. Since neither the charge nor the length of the hydrophobic segment of the sorting sequence seems to be crucial, it is unlikely that the simple physicochemical properties of this segment are critical for the differential Δψ-dependence, raising the possibility that more complex structural properties of the sorting sequence are important. Evidence for this hypothesis was obtained by constructing a b₂-fusion protein that showed a lower Δψ-dependence than the wild-type presequence. The only modification was the replacement of an alanine (residue 63) by a proline, thereby breaking a predicted α-helix in the hydrophobic segment of the sorting sequence. This observation is puzzling in view of the typical model that the precursor polypeptide is translocated as an extended chain across both mitochondrial membranes, since this residue then would not even be in contact with the inner membrane. As an extended chain, 50 residues are sufficient to span both mitochondrial membranes (Rassow et al., 1990 blue right-pointing triangle

; Ungermann et al., 1994 blue right-pointing triangle

; Matouschek et al., 1997 blue right-pointing triangle

; Bömer et al., 1998 blue right-pointing triangle

); the first processing step occurs after residue 31, while residues of the mature part (beyond 80) are still on the outside of the outer membrane. Residue 63, therefore, would have to be positioned at the inner side of the outer membrane, making it difficult to explain the profound effect on the Δψ-dependence of the first processing event because the translocation of preproteins across the outer membrane does not require a Δψ (Schatz, 1996 blue right-pointing triangle

; Neupert, 1997 blue right-pointing triangle

; Pfanner et al., 1997 blue right-pointing triangle

). The observation, however, fits well with studies on the mechanism of insertion and sorting of cytochrome b₂ at the inner membrane that indicated the formation of a loop in the inner membrane that was formed mainly by the sorting sequence (Gruhler et al., 1995 blue right-pointing triangle

; Gärtner et al., 1995a blue right-pointing triangle

; Kanamori et al., 1997 blue right-pointing triangle

). It is tempting to speculate that insertion of a helix-breaking residue increases the conformational flexibility of the sorting sequence, thereby facilitating insertion of the sorting sequence into the inner membrane and substituting in part for Δψ as the driving force.

A comparison of these findings to protein export into and across the bacterial plasma membrane reveals both interesting differences and similarities. Several distinct effects of the electrochemical potential were described for bacterial export, including an electrophoretic effect (Driessen and Wickner, 1991 blue right-pointing triangle

; Geller et al., 1993 blue right-pointing triangle

; Andersson and von Heijne, 1994 blue right-pointing triangle

; Cao et al., 1995 blue right-pointing triangle

; Duong et al., 1997 blue right-pointing triangle

; Kiefer et al., 1997 blue right-pointing triangle

; Kiefer and Kuhn, 1999 blue right-pointing triangle

; Schuenemann et al., 1999 blue right-pointing triangle

). Some bacterial preprotein constructs could be transported into the plasma membrane in the absence of any electrochemical gradient, apparently driven by an increase of hydrophobic force (upon removal of charged amino acid residues and the extension of a hydrophobic segment) (Zimmermann et al., 1982 blue right-pointing triangle

; Geller and Wickner, 1985 blue right-pointing triangle

; Lee et al., 1992 blue right-pointing triangle

; Cao et al., 1995 blue right-pointing triangle

; Kiefer and Kuhn, 1999 blue right-pointing triangle

; Schuenemann et al., 1999 blue right-pointing triangle

). In contrast, protein transport at the mitochondrial inner membrane is blocked when the membrane potential is dissipated completely, but, as shown here, it can occur at a low membrane potential when a sequence beyond the matrix-targeting sequence supports transport. Moreover, the model preprotein b₂(A63P)-DHFR, which shows the lowest Δψ-dependence of any preprotein transported at the mitochondrial inner membrane, was generated by lowering the hydrophobic moment in the sorting sequence. Interestingly, the insertion of a proline residue directly after the signal peptide of OmpF or OmpA fusion proteins strongly decreased the requirement for a membrane potential during bacterial export (Lu et al., 1991 blue right-pointing triangle

). It was concluded that the conformational flexibility caused by the inserted proline (helix break) facilitated a loop formation under conditions of low proton motive force (Lu et al., 1991 blue right-pointing triangle

). Although mitochondrial import and bacterial export occur in opposite directions across the evolutionary conserved membrane, the proline-effect suggests that in both cases secondary structure properties such as conformational flexibility facilitate membrane insertion and lower the requirement for a membrane potential.

Finally, we found that the cytochrome b₂ sorting sequence functioned at a very early import stage since it modulated the efficiency of translocation of the matrix-targeting sequence across the inner membrane under conditions of low Δψ. Since the first processing step (i.e., the removal of the matrix-targeting sequence) represents an early event in the import of cytochrome b₂ proteins independently of their final destination (Glick et al., 1992 blue right-pointing triangle

; Koll et al., 1992 blue right-pointing triangle

; Gärtner et al., 1995a blue right-pointing triangle

; Gruhler et al., 1995 blue right-pointing triangle

; Stuart and Neupert, 1996 blue right-pointing triangle

), this provides further evidence that the influence of the sorting sequence on the Δψ-dependence of translocation of the matrix-targeting sequence is independent of the sorting pathway of the preproteins. In summary, we report the unexpected observation that a preprotein region outside the matrix-targeting sequence strongly influences the dependence of mitochondrial protein import on the membrane potential. This modulatory effect of the sorting sequence is independent of the charge, hydrophobicity, and actual sorting function of the sorting sequence but is related to a conformational flexibility of this segment. We propose that an electrophoretic effect of Δψ on the matrix-targeting sequence is complemented by an additional import-driving activity of the sorting sequence. The sorting sequence thus can modulate the effectiveness of Δψ action.

ACKNOWLEDGMENTS

We thank Drs. Elizabeth Craig and Michiel Meijer for yeast strains and Dr. Wolfgang Voos for helpful discussion. This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 388 Freiburg, and the Fonds der Chemischen Industrie.

REFERENCES

Alconada A, Gärtner F, Hönlinger A, Kübrich M, Pfanner N. Mitochondrial receptor complex from Neurospora crassa and Saccharomyces cerevisiae. Methods Enzymol. 1995;260:263–286. [PubMed]
Andersson H, von Heijne G. Membrane protein topology: effects of μ_H⁺ on the translocation of charged residues explain the “positive inside” rule. EMBO J. 1994;13:2267–2272. [PubMed]
Bauer MF, Sirrenberg C, Neupert W, Brunner M. Role of Tim23 as voltage sensor and presequence receptor in protein import into mitochondria. Cell. 1996;87:33–41. [PubMed]
Bauer MF, Hofmann S, Neupert W, Brunner M. Protein translocation into mitochondria: the role of TIM complexes. Trends Cell Biol. 2000;10:25–31. [PubMed]
Beasley EM, Müller S, Schatz G. The signal that sorts yeast cytochrome b₂ to the mitochondrial intermembrane space contains three distinct functional regions. EMBO J. 1993;12:2303–2311. [PubMed]
Bömer U, Meijer M, Guiard B, Dietmeier K, Pfanner N, Rassow J. The sorting route of cytochrome b₂ branches from the general mitochondrial import pathway at the preprotein translocase of the inner membrane. J Biol Chem. 1997;272:30439–30446. [PubMed]
Bömer U, Maarse AC, Martin F, Geissler A, Merlin A, Schönfisch B, Meijer M, Pfanner N, Rassow J. Separation of structural and dynamic functions of the mitochondrial translocase: Tim44 is crucial for the inner membrane import sites in translocation of tightly folded domains, but not of loosely folded preproteins. EMBO J. 1998;17:4226–4237. [PubMed]
Cao G, Kuhn A, Dalbey RE. The translocation of negatively charged residues across the membrane is driven by the electrochemical potential: evidence for an electrophoresis-like membrane transfer mechanism. EMBO J. 1995;14:866–875. [PubMed]
Daum G, Böhni PC, Schatz G. Import of proteins into mitochondria: cytochrome b₂ and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria. J Biol Chem. 1982;257:13028–13033. [PubMed]
Davis AJ, Ryan KR, Jensen RE. Tim23p contains separate and distinct signals for targeting to mitochondria and insertion into the inner membrane. Mol Biol Cell. 1998;9:2577–2593. [PMC free article] [PubMed]
de Kruijff B. Anionic phospholipids and protein translocation. FEBS Lett. 1994;346:78–82. [PubMed]
Dekker PJT, Keil P, Rassow J, Maarse AC, Pfanner N, Meijer M. Identification of MIM23, a putative component of the protein import machinery of the mitochondrial inner membrane. FEBS Lett. 1993;330:66–70. [PubMed]
Dekker PJ, Martin F, Maarse AC, Bömer U, Müller H, Guiard B, Meijer M, Rassow J, Pfanner N. The Tim core complex defines the number of mitochondrial translocation contact sites and can hold arrested preproteins in the absence of matrix Hsp70-Tim44. EMBO J. 1997;16:5408–5419. [PubMed]
Driessen AJ, Wickner W. Proton transfer is rate-limiting for translocation of precursor proteins by the Escherichia coli translocase. Proc Natl Acad Sci USA. 1991;88:2471–2475. [PubMed]
Duong F, Eichler J, Price A, Leonard MR, Wickner W. Biogenesis of the Gram-negative bacterial envelope. Cell. 1997;91:567–573. [PubMed]
Eilers M, Oppliger W, Schatz G. Both ATP and an energized inner membrane are required to import a purified precursor protein into mitochondria. EMBO J. 1987;6:1073–1077. [PubMed]
Eilers M, Schatz G. Binding of a specific ligand inhibits import of a purified precursor protein into mitochondria. Nature. 1986;322:228–32. [PubMed]
Gambill BD, Voos W, Kang PJ, Miao B, Langer T, Craig EA, Pfanner N. A dual role for mitochondrial heat shock protein 70 in membrane translocation of preproteins. J Cell Biol. 1993;123:109–117. [PMC free article] [PubMed]
Gärtner F, Bömer U, Guiard B, Pfanner N. The sorting signal of cytochrome b₂ promotes early divergence from the general mitochondrial import pathway and restricts the unfoldase activity of matrix Hsp70. EMBO J. 1995a;14:6043–6057. [PubMed]
Gärtner F, Voos W, Querol A, Miller BR, Craig EA, Cumsky MG, Pfanner N. Mitochondrial import of subunit Va of cytochrome c oxidase characterized with yeast mutants. J Biol Chem. 1995b;270:3788–3795. [PubMed]
Geller B, Zhu HY, Cheng S, Kuhn A, Dalbey RE. Charged residues render pro-OmpA potential dependent for initiation of membrane translocation. J Biol Chem. 1993;268:9442–9447. [PubMed]
Geller BL, Wickner W. M13 procoat inserts into liposomes in the absence of other membrane proteins. J Biol Chem. 1985;260:13281–13285. [PubMed]
Glick BS, Brandt A, Cunningham K, Müller S, Hallberg RL, Schatz G. Cytochromes c₁ and b₂ are sorted to the intermembrane space of yeast mitochondria by a stop-transfer mechanism. Cell. 1992;69:809–822. [PubMed]
Glick BS, Wachter C, Reid GA, Schatz G. Import of cytochrome b₂ to the mitochondrial intermembrane space: the tightly folded heme-binding domain makes import dependent upon matrix ATP. Protein Sci. 1993;2:1901–1917. [PubMed]
Gruhler A, Ono H, Guiard B, Neupert W, Stuart RA. A novel intermediate on the import pathway of cytochrome b₂ into mitochondria: evidence for conservative sorting. EMBO J. 1995;14:1349–1359. [PubMed]
Guiard B. Structure, expression and regulation of a nuclear gene encoding a mitochondrial protein: the yeast L(+)-lactate cytochrome c oxidoreductase (cytochrome. b₂) EMBO J. 1985;4:3265–3272. [PubMed]
Hartl FU, Ostermann J, Guiard B, Neupert W. Successive translocation into and out of the mitochondrial matrix: targeting of proteins to the intermembrane space by a bipartite signal peptide. Cell. 1987;51:1027–1037. [PubMed]
Haucke V, Lithgow T, Rospert S, Hahne K, Schatz G. The yeast mitochondrial protein import receptor Mas20p binds precursor proteins through electrostatic interaction with the positively charged presequence. J Biol Chem. 1995;270:5565–5570. [PubMed]
Hines V, Schatz G. Precursor binding to yeast mitochondria: a general role for the outer membrane protein Mas70p. J Biol Chem. 1993;268:449–454. [PubMed]
Hurt EC, van Loon APGM. How proteins find mitochondria and intramitochondrial compartments. Trends Biochem Sci. 1986;11:204–207.
Isaya G, Kalousek F, Fenton WA, Rosenberg LE. Cleavage of precursors by the mitochondrial processing peptidase requires a compatible mature protein or an intermediate octapeptide. J Cell Biol. 1991;113:65–76. [PMC free article] [PubMed]
Jensen RE, Johnson AE. Protein translocation: is Hsp70 pulling my chain? Curr Biol. 1999;9:R779–R782. [PubMed]
Kalousek F, Neupert W, Omura T, Schatz G, Schmitz UK. Uniform nomenclature for the mitochondrial peptidases cleaving precursors of mitochondrial proteins. Trends Biochem Sci. 1993;18:249. [PubMed]
Kanamori T, Nishikawa S, Shin I, Schultz PG, Endo T. Probing the environment along the protein import pathways in yeast mitochondria by site-specific photocrosslinking. Proc Natl Acad Sci USA. 1997;94:485–490. [PubMed]
Kang PJ, Ostermann J, Shilling J, Neupert W, Craig EA, Pfanner N. Requirement for hsp70 in the mitochondrial matrix for translocation and folding of precursor proteins. Nature. 1990;348:137–143. [PubMed]
Kerscher O, Sepuri NB, Jensen RE. Tim18p is a new component of the Tim54p-Tim22p translocon in the mitochondrial inner membrane. Mol Biol Cell. 2000;11:103–116. [PMC free article] [PubMed]
Kiefer D, Hu X, Dalbey R, Kuhn A. Negatively charged amino acid residues play an active role in orienting the Sec-independent Pf3 coat protein in the Escherichia coli inner membrane. EMBO J. 1997;16:2197–2204. [PubMed]
Kiefer D, Kuhn A. Hydrophobic forces drive spontaneous membrane insertion of the bacteriophage Pf3 coat protein without topological control. EMBO J. 1999;18:6299–6306. [PubMed]
Koehler CM, Merchant S, Schatz G. How membrane proteins travel across the mitochondrial intermembrane space. Trends Biochem Sci. 1999;24:428–432. [PubMed]
Koll H, Guiard B, Rassow J, Ostermann J, Horwich AL, Neupert W, Hartl FU. Antifolding activity of hsp60 couples protein import into the mitochondrial matrix with export to the intermembrane space. Cell. 1992;68:1163–1175. [PubMed]
Kurz M, Martin H, Rassow J, Pfanner N, Ryan MT. Biogenesis of Tim proteins of the mitochondrial carrier import pathway: differential targeting mechanisms and crossing over with the main import pathway. Mol Biol Cell. 1999;10:2461–2474. [PMC free article] [PubMed]
Lee JI, Kuhn A, Dalbey RE. Distinct domains of an oligotopic membrane protein are Sec-dependent and Sec-independent for membrane insertion. J Biol Chem. 1992;267:938–943. [PubMed]
Lu HM, Yamada H, Mizushima S. A proline residue near the amino terminus of the mature domain of secretory proteins lowers the level of the proton motive force required for translocation. J Biol Chem. 1991;266:9977–9982. [PubMed]
Martin J, Mahlke K, Pfanner N. Role of an energized inner membrane in mitochondrial protein import: Δψ drives the movement of presequences. J Biol Chem. 1991;266:18051–18057. [PubMed]
Matouschek A, Azem A, Ratliff K, Glick BS, Schmid K, Schatz G. Active unfolding of precursor proteins during mitochondrial protein import. EMBO J. 1997;16:6727–6736. [PubMed]
Merlin A, von Ahsen O, Craig EA, Dietmeier K, Pfanner N. A mutant form of mitochondrial GrpE suppresses the sorting defect caused by an alteration in the presequence of cytochrome b₂. J Mol Biol. 1997;273:1–6. [PubMed]
Neupert W. Protein import into mitochondria. Annu Rev Biochem. 1997;66:863–917. [PubMed]
Nicholls GN, Ferguson SJ. Bioenergetics. London, UK: Academic Press; 1982.
Ostermann J, Voos W, Kang PJ, Craig EA, Neupert W, Pfanner N. Precursor proteins in transit through mitochondrial contact sites interact with hsp70 in the matrix. FEBS Lett. 1990;277:281–284. [PubMed]
Pfanner N, Neupert W. Transport of proteins into mitochondria: a potassium diffusion potential is able to drive the import of ADP/ATP carrier. EMBO J. 1985;4:2819–2825. [PubMed]
Pfanner N, Neupert W. Transport of F₁-ATPase subunit β into mitochondria depends on both a membrane potential and nucleoside triphosphates. FEBS Lett. 1986;209:152–156. [PubMed]
Pfanner N, Craig EA, Hönlinger A. Mitochondrial preprotein translocase. Annu Rev Cell Dev Biol. 1997;13:25–51. [PubMed]
Pratje E, Guiard B. One nuclear gene controls the removal of transient pre-sequences from two yeast proteins: one encoded by the nuclear the other by the mitochondrial genome. EMBO J. 1986;5:1313–1317. [PubMed]
Rassow J, Guiard B, Wienhues U, Herzog V, Hartl FU, Neupert W. Translocation arrest by reversible folding of a precursor protein imported into mitochondria: a means to quantitate translocation contact sites. J Cell Biol. 1989;109:1421–1428. [PMC free article] [PubMed]
Rassow J, Hartl FU, Guiard B, Pfanner N, Neupert W. Polypeptides traverse the mitochondrial envelope in an extended state. FEBS Lett. 1990;275:190–194. [PubMed]
Roise D. Interaction of a synthetic mitochondrial presequence with isolated yeast mitochondria: mechanism of binding and kinetics of import. Proc Natl Acad Sci USA. 1992;89:608–612. [PubMed]
Roise D, Schatz G. Mitochondrial presequences. J Biol Chem. 1988;263:4509–4511. [PubMed]
Ryan KR, Jensen RE. Protein translocation across mitochondrial membranes: what a long, strange trip it is. Cell. 1995;83:517–519. [PubMed]
Ryan MT, Müller H, Pfanner N. Functional staging of ADP/ATP carrier translocation across the outer mitochondrial membrane. J Biol Chem. 1999;274:20619–20627. [PubMed]
Schatz G. The protein import system of mitochondria. J Biol Chem. 1996;271:31763–31766. [PubMed]
Schatz G, Dobberstein B. Common principles of protein translocation across membranes. Science. 1996;271:1519–1526. [PubMed]
Scherer PE, Krieg UC, Hwang ST, Vestweber D, Schatz G. A precursor protein partly translocated into yeast mitochondria is bound to a 70 kd mitochondrial stress protein. EMBO J. 1990;9:4315–4322. [PubMed]
Schleyer M, Neupert W. Transport of proteins into mitochondria: translocational intermediates spanning contact sites between outer and inner membranes. Cell. 1985;43:339–350. [PubMed]
Schleyer M, Schmidt B, Neupert W. Requirement of a membrane potential for the posttranslational transfer of proteins into mitochondria. Eur J Biochem. 1982;125:109–116. [PubMed]
Schneider A, Behrens M, Scherer P, Pratje E, Michaelis G, Schatz G. Inner membrane protease I, an enzyme mediating intramitochondrial protein sorting in yeast. EMBO J. 1991;10:247–254. [PubMed]
Schuenemann TA, Delgado-Nixon VM, Dalbey RE. Direct evidence that the proton motive force inhibits membrane translocation of positively charged residues within membrane proteins. J Biol Chem. 1999;274:6855–6864. [PubMed]
Schwarz E, Seytter T, Guiard B, Neupert W. Targeting of cytochrome b₂ into the mitochondrial intermembrane space: specific recognition of the sorting signal. EMBO J. 1993;12:2295–2302. [PubMed]
Sims PJ, Waggoner AS, Wang CH, Hoffman JF. Studies on the mechanism by which cyanine dyes measure membrane potential in red blood cells and phosphatidylcholine vesicles. Biochemistry. 1974;13:3315–3330. [PubMed]
Söllner T, Rassow J, Pfanner N. Analysis of mitochondrial protein import using translocation intermediates and specific antibodies. Methods Cell Biol. 1991;34:345–358. [PubMed]
Stuart RA, Gruhler A, van der Klei I, Guiard B, Koll H, Neupert W. The requirement of matrix ATP for the import of precursor proteins into the mitochondrial matrix and intermembrane space. Eur J Biochem. 1994;220:9–18. [PubMed]
Stuart RA, Neupert W. Topogenesis of inner membrane proteins of mitochondria. Trends Biochem Sci. 1996;21:261–267. [PubMed]
Truscott KN, Pfanner N. Import of carrier proteins into mitochondria. Biol Chem. 1999;380:1151–1156. [PubMed]
Ungermann C, Neupert W, Cyr DM. The role of Hsp70 in conferring unidirectionality on protein translocation into mitochondria. Science. 1994;266:1250–1253. [PubMed]
Voisine C, Craig EA, Zufall N, von Ahsen O, Pfanner N, Voos W. The protein import motor of mitochondria: unfolding and trapping of preproteins are distinct and separable functions of matrix Hsp70. Cell. 1999;97:565–574. [PubMed]
Voos W, Gambill BD, Guiard B, Pfanner N, Craig EA. Presequence and mature part of preproteins strongly influence the dependence of mitochondrial protein import on heat shock protein 70 in the matrix. J Cell Biol. 1993;123:119–126. [PMC free article] [PubMed]
Voos W, von Ahsen O, Müller H, Guiard B, Rassow J, Pfanner N. Differential requirement for the mitochondrial Hsp70-Tim44 complex in unfolding and translocation of preproteins. EMBO J. 1996;15:2668–2677. [PubMed]
Voos W, Martin H, Krimmer T, Pfanner N. Mechanisms of protein translocation into mitochondria. Biochim Biophys Acta. 1999;1422:235–254. [PubMed]
Zimmermann R, Watts C, Wickner W. The biosynthesis of membrane-bound M13 coat protein: energetics and assembly intermediates. J Biol Chem. 1982;257:6529–6536. [PubMed]

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