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The sorting route of cytochrome b2 branches from the general mitochondrial
import pathway at the preprotein translocase of the inner membrane
Bomer, U.; Meijer, M.; Guiard, B.; Dietmeijer, K.; Pfanner, N.; Rassow, J.
DOI
10.1074/jbc.272.48.30439
Publication date
1997
Published in
The Journal of Biological Chemistry
Link to publication
Citation for published version (APA):
Bomer, U., Meijer, M., Guiard, B., Dietmeijer, K., Pfanner, N., & Rassow, J. (1997). The
sorting route of cytochrome b2 branches from the general mitochondrial import pathway at the
preprotein translocase of the inner membrane. The Journal of Biological Chemistry, 272,
30439-30446. https://doi.org/10.1074/jbc.272.48.30439
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The Sorting Route of Cytochrome b
2Branches from the
General Mitochondrial Import Pathway at the Preprotein
Translocase of the Inner Membrane*
(Received for publication, May 29, 1997, and in revised form, September 17, 1997)
Ulf Bo¨ mer‡, Michiel Meijer§, Bernard Guiard¶, Klaus Dietmeier‡, Nikolaus Pfanner‡, and
Joachim Rassow‡i
From the ‡Institut fu¨ r Biochemie und Molekularbiologie, Universita¨t Freiburg, Hermann-Herder-Strasse 7, D-79104 Freiburg, Federal Republic of Germany, the §Institute for Molecular Cell Biology, BioCentrum Amsterdam, 1098 SM Amsterdam, The Netherlands, and the¶Centre de Ge´ne´tique Moleculaire CNRS, Universite´ Pierre et Marie Curie, F-91190 Gif-sur-Yvette, France
Cytochrome b2 is synthesized in the cytosol with a
bipartite presequence. The first part of the presequence targets the protein to mitochondria and mediates trans-location into the mitochondrial matrix compartment; the second part contains the sorting signal that is re-quired for delivery of the protein to the intermembrane space. The localization of the structures that recognize the sorting signal is unclear. Here we show that upon import in vivo, the sorting signal of cytochrome b2
causes an early divergence from the general matrix im-port pathway and thereby prevents translocation of a folded C-terminal domain into mitochondria. By co-im-munoprecipitations we find that translocation interme-diates of cytochrome b2 are associated with Tim23, a
component of the inner membrane protein import ma-chinery. Cytochrome b2constructs with an alteration in
the sorting signal are mistargeted to the matrix of wild-type mitochondria. In mitochondria containing a mu-tant form of Tim23, however, the translocation of the altered sorting signal across the inner membrane is in-hibited, and cytochrome b2 is correctly sorted to the
intermembrane space. We suggest that the sorting sig-nal of cytochrome b2 is recognized within the inner
membrane in close vicinity to Tim23.
Nearly all mitochondrial proteins are synthesized by cytoso-lic ribosomes and posttranslationally imported into the or-ganelle (1–3). Translocation across the outer membrane re-quires transport through a general import pore that is formed by a complex of Tom proteins. Translocation across the inner membrane involves a common step for all preproteins carrying a presequence and is mediated by a complex of the translocase proteins Tim171and Tim23 (Refs. 4 – 6; for the uniform
nomen-clature see Ref. 7). The initial entry of preproteins into the inner membrane requires the membrane potential (Dc). The matrix heat shock protein 70 (mtHsp70) then binds to seg-ments of the preproteins emerging on the matrix side and drives their further translocation in an ATP-dependent reac-tion. In addition, mtHsp70 promotes the unfolding of prepro-tein domains during entry into the mitochondrial membranes. This is thought to occur by generation of an inward-directed force by preprotein-bound mtHsp70 (pulling). mtHsp70 tran-siently binds to the inner membrane, to the peripheral mem-brane protein Tim44 (8 –10), and also directly to the import channel (Tim17-Tim23) (11); therefore, conformational changes of mtHsp70 are directional and can exert a force on the preprotein in transit.
Cytochrome b2 of yeast mitochondria has for many years
served as a paradigm in elucidating the more complex import pathways of proteins that have a final location in the inter-membrane space. In this compartment, mature cytochrome b2
(L(1)-lactate cytochrome c oxidoreductase) is a soluble
tet-rameric protein (12–14). The preprotein contains a bipartite presequence. The first part of the presequence (residues 1–31) mediates translocation across both mitochondrial membranes and is cleaved in the matrix. The second part (residues 32– 80) contains the information that is required for sorting of the protein to the intermembrane space. The inner membrane pep-tidase I, which carries the catalytic site on the intermembrane space side (15), removes the second part of the presequence, yielding the mature protein.
The sorting signal of cytochrome b2resembles the targeting signals of secretory bacterial proteins, suggesting a conserva-tion of targeting mechanisms in mitochondria in line with the endo-symbiont theory of organelle evolution (16). The sequenc-ing project of the yeast genome, however, has recently demon-strated that a possible conservation of the prokaryotic target-ing system does not include a conservation of the Sec machinery of the prokaryotic translocase; homologs of sec genes have not been found in the yeast genome (17). This does not exclude a conservation of mechanistic principles. In a series of studies it has been shown that the sorting signal of cytochrome
b2seems to be imported into the matrix in tandem with the
first part of the presequence (18 –23). These experiments sug-gest that the sorting signal is subsequently recognized by a separate system besides the import machinery and re-exported to the intermembrane space. Other data have suggested that the sorting signal serves as a stop signal in the import
machin-* This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 388, and the Fonds der Chemischen Indus-trie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
iTo whom correspondence should be addressed. Tel.: 49 761 203 5245; Fax: 49 761 203 5261; E-mail: rassow@ruf.uni-freiburg.de.
1The abbreviations used are: Timx, component of the protein
trans-locase of the mitochondrial inner membrane of molecular weight x; Tomx, component of the protein translocase of the mitochondrial outer membrane of molecular weight x; Tricine, N-[2-hydroxy-1,1-bis(hy-droxymethyl)ethyl]glycine; DHFR, dihydrofolate reductase; Fe/S pro-tein, Rieske iron/sulfur protein of respiratory chain complex III; HB, heme-binding domain of cytochrome b2; MOPS,
4-morpholinopropane-sulfonic acid; mtHsp70, mitochondrial heat shock protein of 70 kDa; pb2-DHFR, precursor of cytochrome b2-DHFR fusion protein; PAGE,
polyacrylamide gel electrophoresis; SP, spheroplasts; PCR, polymerase chain reaction.
© 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org
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ery and does not proceed into the matrix (24 –28). In this concept, the sorting signal leaves the import machinery in the inner membrane laterally, without involvement of a separate reexport system.
In a previous study we used different constructs of cyto-chrome b2as a molecular ruler to localize the functional sorting
signal within the mitochondrion (29). By studying in vitro import into isolated mitochondria, we found that the sorting signal functions already during insertion into the inner mem-brane. mtHsp70 could exert an unfolding (pulling) activity only on folded domains that were located close to the cytochrome b2
presequence. A folded domain located at the C terminus of a cytochrome b2fusion protein was not unfolded and therefore
not translocated across the outer membrane, suggesting that the sorting signal directed an early divergence of the preprotein from the mtHsp70-driven matrix import pathway. We now asked whether these in vitro findings were of relevance for protein sorting in vivo. We indeed observed that the sorting signal of cytochrome b2strongly restricts the unfolding activity of the mitochondrial import machinery in intact cells. It pre-vents import of a folded C-terminal domain and finally leads to retrograde translocation and accumulation of the mature sized protein in the cytosol. To further localize the submitochondrial site of sorting of cytochrome b2, we characterized a mutant form of the channel protein Tim23 and found that the tim23-2 mutation suppressed the mis-sorting of a cytochrome b2
con-struct containing an altered sorting signal. Our results indicate a branching site of import pathways in the inner membrane Tim machinery, separating the import pathway of cytochrome
b2from the general transport channel of matrix targeted pro-teins. We suggest that the sorting signal of cytochrome b2 is
recognized in close vicinity to Tim23.
MATERIALS AND METHODS
Saccharomyces cerevisiae Strains and Construction of Plasmids—For
expression in yeast, the DNA constructs were cut from pGEM4 plas-mids (29, 30) and transferred to the yeast expression plasmid pYE DP 1/8 –10 (31). The pGEM4 plasmids were cut by HindIII at the 39-end of the inserts and filled by Klenow fragment, and a BglII linker was inserted. The inserts were subsequently released from the plasmids by cutting with EcoRI and BglII and inserted between the EcoRI and
BamHI sites of the vector pYE DP 1/8 –10. The plasmids were
propa-gated in the yeast strain 334 (32). This strain has no glucose repression of GAL expression and cannot utilize galactose.
The cytochrome b2* constructs were made by
oligonucleotide-di-rected mutagenesis. Two subsequent PCRs were performed on pb2-(1–
84)-DHFR in pGEM4 (30). In the first reaction, a 59-primer correspond-ing to the SP6 promotor was combined with the 39-mutagenesis primer. The resulting PCR product was used as 59-primer in a second PCR with a 39-primer corresponding to the T7 promotor. An EcoRI-MscI fragment of the PCR product was inserted between the EcoRI and MscI sites of the corresponding cytochrome b2constructs to introduce the mutations.
The presequences of the resulting constructs were sequenced. All DNA manipulations were carried out according to standard procedures (33–34).
Expression and Localization of Cytochrome b2Constructs in S.
cer-evisiae—Yeast cells were grown at 30 °C in selective synthetic complete
medium containing 3% glycerol to an A600of 0.8 –1.2. Galactose was
added to a final concentration of 2% to induce the expression of the cytochrome b2fusion proteins. After 4 h of induction,;20 mg of cells
were collected by centrifugation and converted to spheroplasts by zy-molyase treatment as described previously (12). The suspension was divided into 5 aliquots, and the spheroplasts were reisolated by centrif-ugation (1 min, 16,0003 g). Two aliquots were resuspended in 350ml of cold permeabilization buffer (0.1Mpotassium acetate, 0.2Msorbitol, 2 mMMgCl2, supplemented with 0.02 volumes of 1MHEPES-KOH, pH
7.2) to open the plasma membrane (“perm. SP”) (35). The permeabilized spheroplasts were reisolated by centrifugation (10 min, 16,0003 g). Pellet and supernatant were separated and the proteins released to the supernatant were precipitated by 10% trichloroacetic acid in the pres-ence of 0.05% sodium deoxycholate. One aliquot of the suspensions of spheroplasts was resuspended in 350ml of EM buffer (1 mMEDTA, 10
mMMOPS-KOH, pH 7.2) to open the plasma membrane and the outer mitochondrial membrane (“swelling”). The spheroplasts of an addi-tional aliquot were resuspended in EM buffer containing 1% Triton X-100 to lyse all organelles. The latter two samples and one of the samples with permeabilized spheroplasts were treated with proteinase K (50mg/ml) to digest the cytochrome b2fusion proteins not protected by
membranes. The fifth aliquot of spheroplasts was taken as a control and analyzed directly (“total SP”). Samples were resuspended in 100ml of sample buffer, and portions of 40ml were analyzed by SDS-PAGE and immunoblotting with antibodies directed against DHFR. Antibodies directed against the mitochondrial outer membrane protein Tom20 were used to control the fractionation procedure.
To compare the growth rate of different yeast strains (as shown in Fig. 2), the cells were first grown in liquid selective minimal medium at 28 °C containing 2% glucose. The cultures were diluted with Ringer’s solution and spotted on selective minimal medium plates containing 2% glucose or 2% glucose and 2% galactose. After 5 days at 28 °C, cells were scraped from the plates and again diluted with Ringer’s solution and spotted onto plates. Plates were incubated for 5 days at 28 °C.
Import of Proteins into Isolated Mitochondria—Yeast cells were
grown in YPG medium (1% Bacto-yeast extract, 2% Bacto-peptone, 3% glycerol), and mitochondria were prepared according to published pro-cedures (12). We used the strains PK82 (36), MB3 (WT, Ref. 37), and MB3– 46 (tim23-2; Ref. 38). Radiolabeled preproteins were synthesized in rabbit reticulocyte lysate in the presence of [35S]methionine/[35
S]cys-teine after in vitro transcription by SP6 RNA polymerase (Amersham Corp., Stratagene) or by coupled transcription/translation with T7 po-lymerase (Stratagene). Isolated yeast mitochondria (15–25 mg) were incubated with reticulocyte lysate containing the radiolabeled prepro-tein (5–10% (v/v)) in import buffer (3% bovine serum albumin (w/v), 250
Msucrose, 60 mMKCl, 5 mMMgCl2, 5 mMsodium malate, 2 mMATP, 20
mMpotassium phosphate, 10 mMMOPS-KOH, pH 7.2) at 25 °C. Reac-tions were stopped by addition of 1mMvalinomycin and cooling on ice. Samples with a dissipated membrane potential received valinomycin prior to incubation with preprotein. For generation of mitoplasts, im-port reactions were diluted with 9 volumes of EM buffer and left on ice for 30 min. Control mitochondria were diluted with isotonic SEM buffer (EM buffer supplemented with 250 mMsucrose). Proteinase K treat-ment, reisolation of mitochondria, and separation by SDS-PAGE or Tricine-SDS-PAGE have been described previously (39, 40). Autoradio-graphs were obtained and quantified using a storage PhosphorImaging system (Molecular Dynamics).
Co-immunoprecipitations—Specific antibodies were pre-bound to
protein A-Sepharose (10ml wet volume, Pharmacia Biotech Inc.) for 1 h in 480ml of lysis buffer (1% digitonin (Merck, 1 3 recrystallized from ethanol), 10% glycerol (w/v), 50 mMNaCl, 2 mMEDTA, 30 mM HEPES-KOH, pH 7.4). Import reactions were performed for 20 min with 25mg of mitochondrial protein per lane and 20% (v/v) reticulocyte lysate. After reisolation and washing with SEM buffer, mitochondria were resuspended in lysis buffer supplemented with protease inhibitors (2 mg/ml antipain, 5 mg/ml aprotinin, 0.25 mg/ml chymostatin, 1.25 mg/ml leupeptin, 0.5mg/ml pepstatin A, 2 mMphenylmethylsulfonyl fluoride)
and shaken end-over-end for 10 min at 8 °C. Unsolubilized material was removed by ultracentrifugation (30 min at 100,0003 g). The superna-tants were incubated for 1 h at 8 °C by end-over-end shaking with antibodies prebound to protein A-Sepharose. After three washing cycles with lysis buffer, the protein A-Sepharose pellets were heated in sample buffer and applied to SDS-PAGE.
RESULTS
Localization of Different Cytochrome b2Constructs after Im-port in Vivo—Ga¨rtner et al. (29) used three building blocks to
monitor the sorting and unfolding pathway of cytochrome b2in
isolated mitochondria (Fig. 1A) as follows: the bipartite prese-quence; the;100-residue non-covalent heme binding domain (HB) of cytochrome b2that forms a tightly folded domain and
strictly depends on the function of mtHsp70 for unfolding and translocation across the mitochondrial membranes (26, 41); and the cytosolic enzyme dihydrofolate reductase (DHFR) as passenger protein, the unfolding of DHFR requires only a low energy input and thus does not depend on functional mtHsp70 (29, 41). In the in vitro experiments, a heme binding domain located at the C terminus (pb2-DHFR-HB) was not imported but got stuck in the mitochondrial import sites. When the intramitochondrial sorting signal was inactivated by deletion
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of a 19-residue segment, the resulting preprotein pb2
D-DHFR-HB was completely imported (29).
We now expressed the hybrid proteins (Fig. 1A) from a ga-lactose-inducible vector in intact S. cerevisiae cells. To avoid indirect effects of the induction by galactose, a yeast strain with gal1 mutation was employed that cannot utilize galactose as carbon source and lacks a glucose repression of GAL expres-sion (32). After a 4-h induction, cells were isolated and con-verted to permeabilized spheroplasts (Fig. 1B). We first ana-lyzed two hybrid proteins with a complete presequence and DHFR at the C terminus; the heme binding domain was either directly adjacent to the presequence (pb2-HB-DHFR) or not
included (pb2-DHFR). Both proteins were found in the
organel-lar pellet of permeabilized spheroplasts (Fig. 1B, lanes 2 and 8) and protected against proteinase K (Fig. 1B, lanes 6 and 12) unless the membranes were disrupted with detergent (Fig. 1B,
lanes 5 and 11). In a hypotonic buffer (to swell mitochondria
and open the intermembrane space), both proteins became accessible to added protease (Fig. 1B, lanes 4 and 10). pb2 -HB-DHFR was efficiently processed to the mature sized form (Fig. 1B, lane 1), whereas pb2-DHFR was observed in both the
in-termediate sized form and the mature sized form (Fig. 1B, lane
7). A slower processing of pb2-DHFR has also observed in in vitro import experiments (29) and may be attributed to a lack of
most sequences of mature cytochrome b2. No precursor sized
forms of the hybrid proteins were observed. These results
in-dicate an efficient import of the proteins into mitochondria and a correct sorting to the intermembrane space in vivo.
We then studied the localization of the construct b2
-DHFR-HB (Fig. 1B, lanes 13–18). This protein was efficiently processed to the mature sized form; only minute amounts of precursor or intermediate sized forms were observed (Fig. 1B,
lane 13). However, the processed protein was mainly found in
the cytosolic fraction (Fig. 1B, lane 15) and not protected against proteinase K in permeabilized spheroplasts (Fig. 1B,
lane 18). A placement of the heme binding domain at the C
terminus of a hybrid protein thus reveals a striking effect on its cellular sorting and prevents a complete import into mitochon-dria. We will address below (see Fig. 3) the mechanism how a processed protein can be found in the cytosolic fraction.
When the cytochrome b2sorting signal was inactivated, the resulting hybrid protein b2D-DHFR-HB was found in the or-ganellar fraction (Fig. 1B, lanes 20 and 24). It remained pro-tease-protected also in swollen mitochondria (Fig. 1B, lane 22), demonstrating it was transported across the inner membrane. These results indicate that pb2-HB-DHFR, pb2-DHFR, and pb2D-DHFR-HB were correctly sorted into mitochondria in vivo
comparable to the situation in vitro. Mis-sorting of the hybrid protein depends on both the presence of the heme binding domain at the C terminus and an intact sorting signal (pb2
-DHFR-HB). To determine the effect on the viability of yeast cells, we expressed the hybrid proteins permanently by
grow-FIG. 1. Import of cytochrome b2hybrid proteins in vivo. A, hybrid proteins used in this study. pb2-DHFR-HB contains the first 84 amino
acid residues of the cytochrome b2precursor protein, fused to the entire mouse dihydrofolate reductase (DHFR) and the complete heme binding
domain of cytochrome b2(HB, amino acid residues 81–184). The bipartite cytochrome b2presequence is composed of an N-terminal matrix targeting
sequence (residues 1–31) and a second part (residues 32– 80) containing the intermembrane space sorting signal. The construct pb2-DHFR contains
the complete presequence of cytochrome b2; the heme-binding domain is not included. In pb2-HB-DHFR, the first 220 amino acid residues of the
cytochrome b2precursor, including the complete heme-binding domain, are fused to DHFR. In pb2D-DHFR-HB, the heme-binding domain of
cytochrome b2is separated from the cytochrome b2presequence by the DHFR domain; the intermembrane space sorting signal is inactivated by
deletion of amino acids 47– 65. The construct pb2-DHFR-HB contains the complete presequence of cytochrome b2. B, localization of b2hybrid
proteins after expression in S. cerevisiae. Spheroplasts (SP) were formed by treatment of the cells with zymolyase. To obtain permeabilized spheroplasts (perm. SP), the cells were subsequently subjected to a hypotonic shock. Mitochondria were pelleted by centrifugation (Pel., Sup.). The localization of the hybrid proteins was determined by treatment with proteinase K (Prot. K) of intact mitochondria or after swelling of the organelles to open the intermembrane space. The proteins were precipitated by trichloroacetic acid, separated by SDS-PAGE, and analyzed by immunoblotting, using an antiserum against DHFR (lanes 1–24). To control the efficiency of the fractionation of the b2D-DHFR-HB cells, the
mitochondrial outer membrane protein Tom20 was used as a marker protein (lanes 25–30). For quantification, the total amount of the respective protein in the spheroplasts (total SP) was set to 100% (control).
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ing yeast on galactose-containing medium (Fig. 2, right panel). While pb2D-DHFR-HB and pb2-DHFR did not affect cellular
growth, cells expressing pb2-DHFR-HB grew poorly. The
mis-sorting of pb2-DHFR-HB is thus accompanied by an inhibitory effect on cellular viability.
Chase of Cytochrome b2Hybrid Proteins after Accumulation in Translocation Sites—The results obtained with b2
-DHFR-HB in vivo (this study) and in vitro (29) are on the one hand comparable; under both conditions, the hybrid protein was not completely imported into mitochondria, indicating that the presence of an intact sorting signal prevents import of the heme binding domain located at the C terminus. On the other hand, the processing and the final location of b2-DHFR-HB
were different between the in vivo and the in vitro situation; in
vivo the hybrid protein was processed to the mature form and
found in the cytosolic fraction; in vitro processing was mainly to the intermediate sized form that got stuck in mitochondrial import sites (29). This difference can be attributed to the slow kinetics of the second processing step. Since the in vitro exper-iments have to be performed within a time range of;1 h due to the limited stability of the isolated mitochondria, the time range used for the in vivo experiments (4 h) is not possible.
For further characterization of the b2-DHFR-HB transport
intermediates, we again used isolated mitochondria and im-ported the preprotein from reticulocyte lysate. To complete the processing of b2-DHFR-HB in vitro, a short term import
incu-bation was followed by a reisolation of the mitochondria and a second incubation (chase). pb2-DHFR-HB and, as control, pb2 -DHFR were synthesized in rabbit reticulocyte lysates in the presence of [35S]methionine/[35S]cysteine and incubated with
isolated energized yeast mitochondria for 10 min. The mito-chondria were reisolated and incubated again (chases, without further addition of preproteins) (Fig. 3, A and B). b2-DHFR was
processed to the intermediate and mature sized forms (Fig. 3A,
upper panel, columns 5 and 7) and transported to a
protease-protected location (Fig. 3A, upper panel, columns 6 and 8). No b2-DHFR was found in the supernatant after the chase reaction
(Fig. 3A, lower panel). b2-DHFR-HB was also processed to the
intermediate and mature sized forms (Fig. 3A, columns 1 and
3), but none of the forms was protected against proteinase K
added to the mitochondria after the chase reaction (Fig. 3A,
columns 2 and 4). Moreover, a major portion of the mature
protein was found in the supernatant after the chase (Fig. 3A,
lower panel, column 3). Fig. 3B shows the time-dependent
formation of mature b2-DHFR-HB during the chase reaction
and the release into the supernatant. When the mitochondrial membrane potential was dissipated before the import reaction, no processing to intermediate or mature sized forms was ob-served (not shown), demonstrating that the processing oc-curred in mitochondria. We conclude that b2-DHFR-HB was
transported partially into mitochondria such that the prese-quence could be cleaved by the matrix processing peptidase and the inner membrane peptidase I (the folded heme binding domain remained outside mitochondria as shown previously (29)). The intermediate sized form remained stuck in the mi-tochondrial membranes (accessible to externally added prote-ase), whereas the mature sized form was mainly released to the supernatant.
FIG. 2. Growth of yeast strains expressing different b2-DHFR
constructs. Yeast cells containing the empty vector pYE DP 1/8 –10 (2)
or the vector with an inserted cytochrome b2construct (b2-DHFR-HB,
b2D-DHFR-HB, b2-DHFR, see Fig. 1A) were grown on selective minimal
medium plates containing 2% glucose (Glucose) or 2% glucose and 2% galactose (Galactose) for 5 days at 28 °C. Expression of the hybrid proteins is induced in the presence of galactose.
FIG. 3. Import of b2-DHFR hybrid proteins into isolated
mito-chondria. A, b2-DHFR-HB is released into the supernatant after
proc-essing inside mitochondria. Mitochondria were isolated from a yeast wild-type strain. The hybrid proteins pb2-DHFR-HB and pb2-DHFR
(see Fig. 1A) were synthesized in reticulocyte lysate in the presence of [35S]methionine and incubated with isolated mitochondria for 10 min at
12 °C for accumulation in import sites of the mitochondrial membranes (Import). After reisolation, the mitochondria were resuspended in im-port buffer and incubated for 15 min at 30 °C (Chase). Samples were divided into halves, and one-half was treated with proteinase K (Prot.
K). After reisolation of the mitochondria by centrifugation, the
mito-chondrial pellets (Pel.) and the supernatants (Sup.) were analyzed by SDS-PAGE and autoradiography. i, processing intermediate; m, mature protein. B, time course of the release of b2-DHFR-HB from
mitochon-dria. The experiment was performed as in A, varying the time of chase. The total amount of m-b2-DHFR-HB (pellet1 supernatant) present
after 60 min of chase was set to 100% (control).
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Association of Cytochrome b2Fusion Proteins with the Inner Membrane Translocase Protein Tim23—If the cytochrome b2
sorting sequence is arrested in the mitochondrial inner mem-brane, it should be possible to demonstrate interactions of translocation intermediates with distinct components of the inner membrane protein import machinery. By co-immunopre-cipitation, we first tested for association of preproteins with Tim23. The following hybrid proteins were used: b2-DHFR with a fully functional cytochrome b2presequence; b2*-DHFR with a
partial inactivation of the sorting signal by replacing two pos-itively charged residues (lysine 48 and arginine 49) by un-charged residues (Fig. 4A), the resulting hybrid protein is mistargeted into the matrix space (see below, Fig. 6B) (20, 27); and b2D-DHFR-HB with a complete inactivation of the sorting
signal (due to the 19-residue deletion). The hybrid proteins were accumulated in mitochondrial import sites and processed to the intermediate sized form. All three hybrid proteins were found in association with Tim23 (Fig. 4B, lanes 2, 7 and 12), suggesting that cytochrome b2remains in contact with Tim23
irrespective of an alteration (b2*-DHFR) or partial deletion (b2D-DHFR-HB) of the sorting sequence. At least in this stage
of translocation, cytochrome b2 seems to be imported by the
general import channel of matrix targeted proteins. Tim23 is suggested to participate in the constitution of this protein translocation channel (38, 42– 48).
Association of Cytochrome b2 Fusion Proteins with mtHsp70 —In parallel we tested for the interaction of the
fu-sion proteins with mtHsp70 (Fig. 4B, lanes 4, 9, and 14). Of the three constructs tested, only b2*-DHFR and b2D-DHFR-HB
were co-immunoprecipitated in significant amounts. b2-DHFR
was only precipitated in minor amounts that were close to the level observed with preimmune serum (Fig. 4B, lane 4 versus
lane 1). We conclude that in b2-DHFR the intact sorting signal
of cytochrome b2 prevents stable binding of the translocation
intermediate to mtHsp70. With b2*-DHFR and b2D-DHFR-HB,
mtHsp70 can gain access to additional segments of the polypep-tide chains (Fig. 4B, lanes 9 and 14) that are not accessible when the authentic sorting sequence is present (Fig. 4B, lane
4). Tim44 serves as a membrane anchor for mtHsp70 (49 –51).
No significant association with Tim44 was observed with either of the three constructs (Fig. 4B, lanes 3, 8, and 13). This result is in agreement with our recent observation that Tim44 is a component of a separate subcomplex of the import machinery that only transiently interacts with the Tim23zTim17 core complex (11).
Preprotein Sorting in tim23-2 Mutant Mitochondria—The
yeast mutant tim23-2 is impaired in preprotein translocation into the mitochondrial matrix in vivo and in vitro (38, 52). We used tim23-2 mitochondria to characterize preprotein sorting to the intermembrane space in dependence of the function of the inner membrane Tim machinery. We first tested the sta-bility of the Tim23zTim17 core complex (44, 46, 47) by co-immunoprecipitations with antibodies against Tim23. The to-tal amounts of Tim23 and Tim17 were comparable between wild-type and mutant mitochondria; however, the stability of the complex with Tim17 was reduced in the tim23-2 mutant (Fig. 5A). The membrane potential of the mutant mitochondria was intact (not shown). The amounts of Tim44 and mtHsp70 were the same in the mitochondria of both strains (Fig. 5A,
samples 5 and 6).
We next analyzed the import kinetics of two mitochondrial preproteins, cytochrome b2and the iron/sulfur protein of com-plex III of the respiratory chain (Rieske Fe/S protein), which are “classical” preproteins in the analysis of mitochondrial sorting pathways. The Fe/S protein is completely imported into the matrix and then exported across the inner membrane to its functional destination at the intermembrane space side of the inner membrane (53, 54). As shown in Fig. 5B, cytochrome b2
was imported into tim23-2 mitochondria nearly with the same kinetics as into wild-type mitochondria. Processing to the ma-ture protein and translocation to a protease-protected position within the mitochondria were only slightly retarded. The result was different with the Fe/S protein. Processing of the protein was drastically delayed (Fig. 5B). Interestingly, the import reaction was already delayed in the step from the preprotein to the processing intermediate. The step from the intermediate to
FIG. 4. Co-immunoprecipitation of b2-DHFR hybrid proteins with components of the mitochondrial protein import machinery. A,
The hybrid protein pb2*-DHFR is a derivative of pb2-DHFR (Fig. 1A) with two amino acid exchanges in the intermembrane space sorting signal
(K48I and R49C) leading to mistargeting of the protein into the matrix. B, co-immunoprecipitation of b2-DHFR, b2*-DHFR, and b2D-DHFR-HB
with antibodies directed against Tim23, Tim44, mtHsp70, and the ADP/ATP carrier (AAC). Radiolabeled b2-DHFR, b2*-DHFR, and b2D-DHFR-HB
were synthesized in reticulocyte lysate and imported into isolated yeast mitochondria for 20 min at 25 °C. The mitochondria were reisolated and lysed in buffer containing 1% digitonin. After a clarifying spin, aliquots were subjected to immunoprecipitation with antibodies against Tim23, Tim44, mtHsp70, and ADP/ATP carrier or with preimmune antibodies. Precipitated proteins were analyzed by SDS-PAGE. Std., standard, 5% of the material added to the antibodies; p, precursor protein; i, processing intermediate; m, mature protein.
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the mature protein was not inhibited. In the context of the conservative sorting pathway of the Fe/S protein (53), this implies that it is the import of the Fe/S protein into the matrix that is reduced in the tim23-2 mutant.
We then used two cytochrome b2 hybrid proteins with a
partial inactivation of the sorting signal due to the replacement of the positively charged residues 48 and 49 by uncharged ones, b2*-DHFR (Fig. 4A) and b2*-HB-DHFR. With wild-type
mito-chondria, both hybrid proteins were efficiently processed to the intermediate forms but not or only very poorly to the mature forms (Fig. 6A, lanes 1–3). The intermediate forms were pro-tected against proteinase K added to intact mitochondria (Fig. 6A, lanes 1–3) and to swollen mitochondria (Fig. 6B, columns 3 and 6). The swelling opened the mitochondrial intermembrane space, as evidenced by the decrease of the content of cyto-chrome b2(Fig. 6B, column 2), but left the matrix space intact,
shown with the level of mtHsp70 (Fig. 6B, column 1). Imported ib2*-DHFR (Fig. 6B, column 3) and ib2*-HB-DHFR (Fig. 6B,
column 6) behaved like mtHsp70, indicating that they were
mistargeted into the matrix.
With tim23-2 mitochondria, however, both constructs were processed further to the mature sized forms (Fig. 6A, lanes
5–7). 30 – 40% of the imported proteins were processed to the
mature forms. The mature forms were protected against pro-teinase K in intact mitochondria (Fig. 6A, lanes 5–7) but be-came accessible to the protease after opening of the outer membrane (Fig. 6B, columns 5 and 8). mb2*-DHFR and mb2
*-HB-DHFR thus fractionated like mature cytochrome b2,
indi-cating that they were correctly sorted to the intermembrane space in the mutant mitochondria. The processing to the ma-ture forms was abolished by a dissipation of the inner mem-brane potential (Dc), demonstrating that it strictly depended on the insertion of the preproteins into the inner membrane. We conclude that the defect in Tim23 suppresses the sorting defect caused by the amino acid alteration in the presequence of cytochrome b2.
DISCUSSION
We have characterized the sorting of cytochrome b2in vivo
and in organello and present evidence that its import route
FIG. 5. Characterization of the yeast mutant tim23-2. A, reduced stability of the Tim17zTim23 complex in the mutant tim23-2. Tim17 was synthesized in reticulocyte lysate in the presence of [35
S]methi-onine and imported into wild-type (WT) or tim23-2 mitochondria (Mito.) for 30 min at 25 °C. Non-imported material was removed by treatment with proteinase K (200mg/ml). Mitochondria were lysed with digitonin and a co-immunoprecipitation was performed with antibodies directed against Tim23. Upper panels, co-immunoprecipitations (lanes 1– 4); 5% of total mitochondrial extract applied to immunoprecipitation (lanes 5 and 6). Lower panels, lanes 5 and 6, endogenous proteins analyzed by SDS-PAGE and Western blotting. B, import of mitochondrial prepro-teins. Radiolabeled cytochrome b2(cyt. b2) and Rieske iron/sulfur
pro-tein (Fe/S propro-tein) were imported into isolated wild-type or tim23-2 mitochondria at 25 °C for the times indicated (see “Materials and Meth-ods”). Import reactions were stopped by addition of valinomycin. Half of each sample was treated with proteinase K (50 mg/ml, 1 Prot. K). Mitochondria were reisolated and analyzed by SDS-PAGE and autoradiography.
FIG. 6. Sorting of cytochrome b2hybrid proteins in
mitochon-dria of the mutant tim23-2. A, import into mitochonmitochon-dria.
Radiola-beled b2*-DHFR and b2*-HB-DHFR were imported into wild-type (WT)
or tim23-2 mitochondria at 25 °C for the times indicated. In lanes 4 and
8 the membrane potential was dissipated prior to import by including 1
mMvalinomycin (2Dc). Import reactions were stopped by addition of
valinomycin. All samples were treated with proteinase K (50mg/ml) to remove non-imported proteins. The mitochondria were reisolated and analyzed by SDS-PAGE and autoradiography. B, localization of im-ported cytochrome b2* proteins. Radiolabeled b2*-DHFR and b2
*-HB-DHFR were imported into wild-type or tim23-2 mitochondria for 30 min at 25 °C as in A. Import was stopped, and mitochondria were divided into halves. One-half was diluted with cold SEM buffer and left on ice. The other half was subjected to hypotonic swelling and treated with proteinase K for 30 min. Reisolated mitochondria were washed and proteins were separated by SDS-PAGE and transferred to nitrocellu-lose. Radiolabeled cytochrome b2* proteins were analyzed by
autora-diography. The amount of endogenous marker proteins (mtHsp70, cy-tochrome b2) was determined on the same blots by immunodecoration.
p, precursor protein; i, processing intermediate; m, mature protein.
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branches early from the general matrix targeting pathway. A cytochrome b2construct with a C-terminal heme binding
domain, which is able to form a tightly folded structure (26, 29, 41), cannot be completely imported into mitochondria in vivo. It is processed to the intermediate sized form and, after longer import times, to the mature form. The mature form is then released from the mitochondria and found in the cytosol. Due to the long accumulation in mitochondrial import sites, expres-sion of this preprotein causes a growth defect of intact yeast cells. To translocate the heme binding domain across the mi-tochondrial membranes, the domain either has to be placed close to the presequence or the sorting signal in the prese-quence has to be inactivated. In the first case, an interaction of mtHsp70 with the N-terminal portion of the presequence gen-erates a pulling force on the heme binding domain during its entry into/across the outer membrane (29). In the latter case, the preprotein lacking the sorting information is completely imported into the matrix and thus is continuously pulled in by the mtHsp70-driving system. The in vivo experiments shown here strongly support the validity of the in vitro import studies published previously (29) which demonstrated that a C-termi-nal heme-binding domain cannot be imported, indicating that the sorting pathway of cytochrome b2diverges early from the matrix import pathway (and the mtHsp70-dependent unfold-ing machinery).
The in vivo import studies bear an important implication on the role of preprotein folding in the cytosol during mitochon-drial protein import. Since the heme binding domain located at the C terminus prevents mitochondrial import of a cytochrome
b2construct with a high efficiency (more than 80%), it is most
likely that the domain is folded in intact cells as has been observed in the in vitro import system (29). This implies that the 100-residue domain is completely synthesized before trans-location into mitochondria, i.e. its import occurs by a post-translational mechanism. Wienhues et al. (55) used the cytoso-lic enzyme DHFR as passenger protein and a stabilization of its folding by an externally added ligand (aminopterin) to demon-strate a post-translational import mechanism for;75% of the passenger proteins. With the heme binding domain, we present the first case of an authentic mitochondrial domain that is apparently folded in the cytosol. It has been discussed that mitochondrial protein import occurs mainly co-translationally
in vivo (56), and thus preprotein folding should not be relevant.
Moreover, cytosolic chaperone proteins could interfere with and prevent folding of protein domains (57, 58). The results of Wienhues et al. (55) and this study, however, provide strong evidence for a post-translational mechanism of mitochondrial protein import.
It has been discussed for many years whether the sorting signal of cytochrome b2functions during the import reaction by arrest in the inner membrane or whether it requires complete transport into the matrix and becomes functional in a subse-quent re-export step (59, 60). In addition to the in vivo results, we show that a mutation affecting a component of the central core of the preprotein translocase in the inner mitochondrial membrane (Tim23) facilitates the recognition of the cyto-chrome b2 sorting signal. If the cytochrome b2 sorting signal had already left the inner membrane and moved into the ma-trix to engage a separate export machinery, a further move-ment of the polypeptide should not be facilitated by structures that had been left behind. If the sorting function were carried out by an independent export system, it is difficult to explain why a mutation of TIM23 should facilitate a subsequent sort-ing step. The findsort-ings reported here thus favor an early recog-nition of the sorting signal during the entry into the inner membrane. They do not necessarily imply that Tim23 itself
recognizes the sorting signal, but Tim23 seems to mark the location where the sorting signal leaves the general matrix import route and moves into a separate branch directed toward the intermembrane space. The mutant Tim23 protein may delay the movement of the modified sorting signal across the inner membrane and thereby reestablish the translocation ar-rest of cytochrome b2 constructs with a partially inactivated sorting signal. The weakened stability of the Tim23-Tim17 interaction in the tim23-2 mutant mitochondria may facilitate lateral release of the sorting signal from the translocation channel.
An inner membrane localization of the branching site of import pathways is further corroborated by the characteriza-tion of translocacharacteriza-tion intermediates. We find that, after process-ing to the intermediate sized form, cytochrome b2 is stably inserted in the inner membrane and associates with Tim23 without involvement of mtHsp70 (30, 61; data not shown), in contrast to other proteins that require mtHsp70 to prevent release from the import machinery (62). Tokatlidis et al. (63) recently identified Tim11 as an inner membrane protein that is in close contact to the cytochrome b2sorting signal during its
import and sorting. The authors similarly concluded that the branching of import pathways occurs at the inner membrane. However, the precise function of Tim11 in this process is not yet clear (64). It has been proposed that the biogenesis of cytochrome b2involves an interaction of the mature part of the
protein with the Tim machinery (23, 29, 65). The efficient release of the mature sized cytochrome b2 construct into the
cytosol in vivo suggests that such an interaction with the Tim machinery is only transient and of limited stability.
The principle of conservative sorting via the matrix space was originally described for the Rieske Fe/S protein (53). If cytochrome b2 followed a different import pathway, both
pro-teins should reveal differences in the import into mitochondria of the tim23-2 mutant. In fact, the results of our import exper-iments are in agreement with this prediction. Processing of cytochrome b2to the intermediate and to the mature protein
was not affected in tim23-2 mutant mitochondria. In contrast, the Fe/S protein was only poorly processed to the intermediate form in tim23-2 mitochondria. We conclude that the biogenesis of the Fe/S protein requires transport of the preprotein into the mitochondrial matrix that depends on the intact Tim23zTim17 complex. The tim23-2 mutation thus reveals a striking differ-ence between the sorting pathways of cytochrome b2and the
Fe/S protein.
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