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VDAC and the bacterial porin PorB of Neisseria gonorrhoeae share mitochondrial import pathways

2002; Springer Nature; Volume: 21; Issue: 8 Linguagem: Inglês

10.1093/emboj/21.8.1916

1460-2075

Anne Müller,

DNA Repair Mechanisms

Article15 April 2002free access VDAC and the bacterial porin PorB of Neisseria gonorrhoeae share mitochondrial import pathways Anne Müller Anne Müller Max Planck Institute for Infection Biology, Department of Molecular Biology, Schumannstrasse 21/22, D-10117 Berlin, Germany Search for more papers by this author Joachim Rassow Joachim Rassow University of Hohenheim, Department of Microbiology, Garbenstrasse 30, D-70593 Stuttgart-Hohenheim, Germany Search for more papers by this author Jan Grimm Jan Grimm Max Delbrück Centrum für Molekulare Medizin, Abteilung Zellbiologie, Robert-Rössle-Strasse 10, D-13092 Berlin, Germany Search for more papers by this author Nikolaus Machuy Nikolaus Machuy Max Planck Institute for Infection Biology, Department of Molecular Biology, Schumannstrasse 21/22, D-10117 Berlin, Germany Search for more papers by this author Thomas F. Meyer Corresponding Author Thomas F. Meyer Max Planck Institute for Infection Biology, Department of Molecular Biology, Schumannstrasse 21/22, D-10117 Berlin, Germany Search for more papers by this author Thomas Rudel Thomas Rudel Max Planck Institute for Infection Biology, Department of Molecular Biology, Schumannstrasse 21/22, D-10117 Berlin, Germany Search for more papers by this author Anne Müller Anne Müller Max Planck Institute for Infection Biology, Department of Molecular Biology, Schumannstrasse 21/22, D-10117 Berlin, Germany Search for more papers by this author Joachim Rassow Joachim Rassow University of Hohenheim, Department of Microbiology, Garbenstrasse 30, D-70593 Stuttgart-Hohenheim, Germany Search for more papers by this author Jan Grimm Jan Grimm Max Delbrück Centrum für Molekulare Medizin, Abteilung Zellbiologie, Robert-Rössle-Strasse 10, D-13092 Berlin, Germany Search for more papers by this author Nikolaus Machuy Nikolaus Machuy Max Planck Institute for Infection Biology, Department of Molecular Biology, Schumannstrasse 21/22, D-10117 Berlin, Germany Search for more papers by this author Thomas F. Meyer Corresponding Author Thomas F. Meyer Max Planck Institute for Infection Biology, Department of Molecular Biology, Schumannstrasse 21/22, D-10117 Berlin, Germany Search for more papers by this author Thomas Rudel Thomas Rudel Max Planck Institute for Infection Biology, Department of Molecular Biology, Schumannstrasse 21/22, D-10117 Berlin, Germany Search for more papers by this author Author Information Anne Müller1, Joachim Rassow2, Jan Grimm3, Nikolaus Machuy1, Thomas F. Meyer 1 and Thomas Rudel1 1Max Planck Institute for Infection Biology, Department of Molecular Biology, Schumannstrasse 21/22, D-10117 Berlin, Germany 2University of Hohenheim, Department of Microbiology, Garbenstrasse 30, D-70593 Stuttgart-Hohenheim, Germany 3Max Delbrück Centrum für Molekulare Medizin, Abteilung Zellbiologie, Robert-Rössle-Strasse 10, D-13092 Berlin, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:1916-1929https://doi.org/10.1093/emboj/21.8.1916 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The human pathogen Neisseria gonorrhoeae induces host cell apoptosis during infection by delivering the outer membrane protein PorB to the host cell's mitochondria. PorB is a pore-forming β-barrel protein sharing several features with the mitochondrial voltage-dependent anion channel (VDAC), which is involved in the regulation of apoptosis. Here we show that PorB of pathogenic Neisseria species produced by host cells is efficiently targeted to mitochondria. Imported PorB resides in the mitochondrial outer membrane and forms multimers with similar sizes as in the outer bacterial membrane. The mitochondria completely lose their membrane potential, a characteristic previously observed in cells infected with gonococci or treated with purified PorB. Closely related bacterial porins of non-pathogenic Neisseria mucosa or Escherichia coli remain in the cytosol. Import of PorB into mitochondria in vivo is independent of a linear signal sequence. Insertion of PorB into the mitochondrial outer membrane in vitro depends on the activity of Tom5, Tom20 and Tom40, but is independent of Tom70. Our data show that human VDAC and bacterial PorB are imported into mitochondria by a similar mechanism. Introduction Many bacterial and viral pathogens manipulate induction of host cell apoptosis to their own advantage. Some pathogens, such as Chlamydia species and many viruses, prevent apoptosis and benefit from prolonged host cell survival because it enables them to replicate and produce viable progeny (Fan et al., 1998; Meinl et al., 1998; Tschopp et al., 1998; Rajalingam et al., 2001). Others, like the enteropathogenic bacterial species Salmonella, Shigella and Yersinia, escape the attack of the host's immune system by efficiently killing key cells involved in the immune response (Zychlinsky et al., 1992; L.M.Chen et al., 1996; Y.Chen et al., 1996; Monack et al., 1996, 1997; Ruckdeschel et al., 1997). Only recently, mitochondria emerged as targets for bacterial and viral manipulation of apoptotic pathways (Müller and Rudel, 2001). These organelles irreversibly trigger cell death upon receiving an apoptotic signal by increasing the permeability of both mitochondrial membranes (Green and Reed, 1998; Kroemer and Reed, 2000). This leads to a breakdown of the inner membrane potential and leakiness for intermembrane space proteins such as cytochrome c, AIF and others (Kroemer and Reed, 2000). One of the bacterial pathogens acting on mitochondria to modulate host cell apoptosis is Neisseria gonorrhoeae (Müller et al., 2000). The genus Neisseria comprises the human pathogenic species N.gonorrhoeae and N.meningitidis, which cause gonorrhoea and meningitis, respectively, and several commensal, non-infectious species such as N.mucosa. The first contact takes place at epithelial surfaces, where tight association of the pathogen to the cells is brought about by means of pili (Swanson et al., 1987) and Opa proteins, which interact with specific receptors on the cell surface (Makino et al., 1991; Chen and Gotschlich, 1996; Gray-Owen et al., 1997; Dehio et al., 1998). Attachment to epithelia initiates the transfer of the outer membrane porin PorB to host cell membranes (Weel and van Putten, 1991; Müller et al., 1999). Both our group and others (Massari et al., 2000; Müller et al., 2000) have demonstrated that PorB is subsequently targeted to mitochondria. This occurs in infected epithelial cells (Müller et al., 2000), but also in cultured lymphocytes that have been treated with purified porin in vitro (Massari et al., 2000). The outcome of PorB targeting to these organelles is a matter of debate and seems to depend on the Neisseria species, the purification procedure and/or the cell type. In cultured epithelial cells infected with N.gonorrhoeae, PorB triggers apoptosis by inducing the release of cytochrome c from mitochondria, a process that is accompanied by a complete breakdown of the membrane potential, matrix swelling, and the activation of caspases (Müller et al., 1999, 2000). PorB resembles the mitochondrial porin or voltage-dependent anion channel (VDAC) with respect to several features. Both proteins are ATP-regulated trimeric β-barrel proteins, forming voltage-gated pores of similar size (Rudel et al., 1996). Interestingly, the VDAC along with other components appears to constitute the permeability transition pore complex (PTPC; Zoratti and Szabo, 1995), which has been implicated in many forms of cell death (Kroemer and Reed, 2000). The fact that both porins play a role in apoptosis regulation invites speculations regarding a common ancestor (Rudel et al., 1996; Frade and Michaelidis, 1997; Kroemer, 1997). This hypothesis is addressed in this study with respect to possible similarities in the targeting to mitochondria. Nearly all mitochondrial proteins are encoded by the nuclear genome. They are synthesized in the cytosol and post-translationally imported into the mitochondria (Pfanner, 2000). Targeting and import are mediated by distinct segments of the newly synthesized proteins. Many preproteins contain a positively charged presequence at the N-terminus. Other mitochondrial proteins including the ANT (Brix et al., 1999), the cytochrome c haem lyase (Diekert et al., 1999), the BCS1 protein (Fölsch et al., 1996) and Tom70 (McBride et al., 1992) contain internal or C-terminal targeting signals. Preproteins bind to the import receptors Tom20 or Tom70 at the mitochondrial surface and are subsequently inserted into a general import pore, GIP, which is essentially formed by Tom40. In addition to these components, the TOM complex (translocase of the outer membrane) includes the import receptor Tom22 and the small subunits Tom5, 6 and 7. The import pathway of VDAC into mitochondria has recently been elucidated in more detail. Whereas there is no doubt that import of VDAC requires the surface receptor Tom20, the involvement of the GIP complex is a subject of debate (Pfaller and Neupert, 1987; Schleiff et al., 1997, 1999; Krimmer et al., 2001). As for other β-barrel proteins of the mitochondrial outer membrane, the targeting signal of VDAC is enigmatic (Krimmer et al., 2001). In this study we compared the import mechanisms of the porins human VDAC and bacterial PorB both in vitro and in whole cells. We found that PorB is specifically inserted into the mitochondrial outer membrane. Although bacteria and mitochondria are related in evolution, their requirements for the insertion of membrane proteins are different. To reach the outer membrane of N.gonorrhoeae, PorB initially has to traverse the bacterial inner membrane and react with the bacterial Sec machinery. For import into mitochondria, PorB may insert spontaneously or it may interact with the TOM complex, although this has no bacterial counterpart. Endogenous mitochondrial homologues of bacterial proteins usually contain additional targeting signals (Hartl and Neupert, 1990). However, PorB is apparently devoid of such sequences. Moreover, the outer membranes of bacteria and mitochondria have a completely different lipid composition. Surprisingly, we found that PorB follows a similar import pathway as VDAC, including Tom20-dependent targeting and an involvement of the GIP. Productive targeting and insertion of PorB into the outer membrane were found to be strictly dependent on a cooperative effect of discontinuous parts of PorB that are distributed in the sequence. Truncated parts that could act as autonomous modules were not observed. These characteristics are different both from other Tom20-dependent precursor proteins and from Tom70-dependent preproteins, thus defining a unique and possibly primordial system of mitochondrial protein targeting. Results Endogenously expressed neisserial porin targets mitochondria Porin genes from N.gonorrhoeae, N.meningitidis, the commensal strain N.mucosa as well as from Escherichia coli were amplified from genomic DNA, cloned into the mammalian expression vector pCMV-Tag-1 and transiently transfected into HeLa cells. Co-staining of transfected cells with the potential-sensitive dye Mitotracker and a specific antibody against the FLAG tag revealed a complete loss of mitochondrial inner membrane potential in cells transfected with porins from the two pathogenic Neisseria species N.gonorrhoeae and N.meningitidis, whereas cells expressing either porin from N.mucosa or E.coli did not differ from neighbouring non-transfected cells with respect to their Mitotracker staining (Figure 1A). Whereas the two latter porins display a diffuse staining pattern, which probably reflects a cytosolic localization, the porins of pathogenic Neisseria are distributed in a granular compartment resembling mitochondria. Counter staining with antibodies against mitochondrial antigens such as cytochrome c (Figure 1A), cytochrome c oxidase and Hsp60 (not shown) did show an almost complete overlap, thereby demonstrating that endogenously synthesized porin targets these organelles just like its bacterial counterpart. This could be confirmed by isolating mitochondria from transiently transfected HeLa cells and subsequent detection of porin in lysates and the mitochondrial preparation by western blotting (Figure 1B). Interestingly, the mitochondria of porin-transfected cells take on the peculiar swollen shape also seen in infected cells (Müller et al., 2000). However, although the targeting to mitochondria of endogenously made porin is more efficient than during an infection, where only up to 50% of all porin in the cell is found in this compartment, most of the mitochondria of porin-transfected cells, in contrast to infected cells, retain cytochrome c (Müller et al., 2000). Furthermore, porin-transfected cells do not undergo apoptosis in the time frame of the experiment. Whereas transfection of eukaryotic cells with porin is, therefore, not suitable for investigating the mechanism of apoptosis induction by neisserial porin at the mitochondrial level, it does represent an ideal model for studying the targeting process. Figure 1.Transient transfection of epithelial cells with bacterial porins reveals mitochondrial localization of porins from pathogenic but not commensal species. (A) HeLa cells were transfected with constructs encoding the indicated FLAG-tagged bacterial porins. After an expression time of 24 h, the cells were stained with Mitotracker, fixed, and co-stained with specific antibodies against cytochrome c (Anti- cyto c) and the FLAG tag. Confocal images of single colours and an overlay of all three colours are shown. PorBNgo (P.IA), porB gene isolated from N.gonorrhoeae strain VPI; PorBNgo (P.IB), porB gene isolated from N.gonorrhoeae strain MS11; PorBNme, porB gene from N.meningitidis; OmpC, ompC gene from E.coli; PorBNmu, porB gene from N.mucosa. (B) Cell lysates and mitochondria were isolated from transiently transfected cells according to the protocol detailed in Materials and methods, separated by SDS–PAGE and blotted onto PVDF membrane. The blots were probed with specific antibodies against the FLAG tag and PorBIA. Detection was performed using the ECL system (Amersham) according to the manufacturer's instructions. Download figure Download PowerPoint Porin is an integral protein of the mitochondrial outer membrane and forms multimers In order to elucidate further the conformation of porin in the cell, cross-linking experiments were performed on isolated mitochondria from transfected cells (Figure 2A). Surprisingly, porin in transfected cells adopts the same multimeric conformation as in the bacterial outer membrane (Figure 2A) and in mitochondria from infected cells (not shown). The sizes of the detected multimers correspond to porin dimers, trimers and a high-molecular-weight band that probably represents a hexamer. This argues in favour of an intrinsic ‘multimerization signal’ in the primary porin sequence and suggests that porin might form a membrane channel in either mitochondrial membrane. Figure 2.Endogenously expressed PorBIA forms multimers and is an integral protein of the mitochondrial outer membrane. (A) HeLa cells were transfected with a construct encoding PorBIA, the mitochondria were isolated, and both purified mitochondria and cultured gonococci were subjected to chemical cross-linking. Samples were separated by SDS–PAGE in the absence or presence of β-mercaptoethanol as indicated. The blot was probed with a specific antibody against PorBIA. (B) Mitochondria from transfected or infected cells as well as cultured gonococci were subjected to sodium carbonate extraction. After 30 min of incubation in 0.1 M sodium carbonate, the membranes were pelleted by ultracentrifugation. Pellets (P) and supernatants (SN) were separated by SDS–PAGE, blotted, and probed with antibodies against the marker proteins Hsp60, VDAC, cytochrome c oxidase (cyto c ox) and cytochrome c (cyto c) (left panel). Immunoblots of PorBIA extracted from mitochondria of transfected (transfected) or infected cells (Ngo infected) or of intact bacteria (bacteria) are shown in the right panel. (C) Mitochondria were isolated from S.cerevisiae expressing human VDAC or gonococcal PorBIA from a plasmid under an inducible promoter according to a protocol detailed in Materials and methods and subjected to carbonate extraction as described in (B). Shown are immunoblots of pellets (P) and supernatants (SN) of mitochondria containing human VDAC (VDAC) or gonococcal PorBIA (PorBIA). Yeast cytochrome c (cyto c) was detected as a soluble marker protein. (D) Purified mitochondria were sonicated and the resulting vesicles were separated by centrifugation over a linear sucrose gradient for 16 h. Fractions of 0.8 ml were harvested, TCA precipitated and separated by SDS–PAGE. After blotting onto PVDF membrane, bound proteins were detected with specific antibodies against VDAC, ANT and PorBIA. (E) HeLa cells were transfected with constructs encoding mitochondrial matrix (MiMat)-targeted RFP and PorBIA or human VDAC, respectively, or with MiMat–RFP alone. After 24 h of expression, cells were fixed and stained with an anti-FLAG antibody followed by an Alexa 488-coupled secondary antibody. Confocal images of single colours and an overlay of both colours are shown. Download figure Download PowerPoint Sodium carbonate extraction of mitochondria was performed to investigate whether porin is an integral membrane protein under these circumstances or only associates with mitochondria loosely. In the latter case, the interaction should be sensitive to the basic pH of sodium carbonate. The fate of marker proteins revealed the principal usefulness of the method (Figure 2B), as soluble proteins such as cytochrome c and the matrix protein Hsp60 were found in the supernatant, whereas integral membrane proteins such as VDAC and cytochrome c oxidase were detected in the pellet of carbonate-extracted mitochondria. The method further revealed that neisserial porin, regardless of whether it was endogenously synthesized or delivered to mitochondria during an infection, is always resistant to carbonate extraction (Figure 2B). Porin in its natural environment, the bacterial outer membrane, served as an additional control (Figure 2B). The mitochondrial porin VDAC is an integral protein of the outer mitochondrial membrane. Owing to the overall similarities between mitochondrial and neisserial porin, it was expected that the latter would also reside in the outer rather than the inner membrane. This was examined with the help of a technique that allows the density gradient separation of both membranes on the basis of their different protein contents. This method requires large amounts of mitochondria to start with, a demand not met by the transfection system. For the purpose of collecting large quantities of mitochondrial material, recombinant yeast strains were generated expressing either neisserial porin or human VDAC under an inducible (glucose-repressed and galactose-induced) promoter. The structural and functional homology between human and yeast VDAC has been demonstrated earlier (Shimizu et al., 1999). Indeed, the correct targeting of both porins to mitochondria could be confirmed in this biological system (Figure 2C). By digesting purified mitochondria from the recombinant strains with proteinase K (PK), it could further be shown that both proteins are integral components (and therefore PK resistant) of the membrane rather than merely associated (data not shown). This was confirmed by sodium carbonate extraction of isolated mitochondria (Figure 2C), which revealed the localization of VDAC and PorBIA in the membrane (pellet) and cytochrome c in the supernatant. Separation of the two membranes by sucrose density gradient centrifugation revealed that both recombinant proteins appear in the same gradient fractions (Figure 2D), which also contained the yeast VDAC (not shown). In contrast, a typical inner membrane protein, the ANT, is exclusively found in a remote fraction (Figure 2D). There is no overlap of both membranes, as judged by the markers. An additional experimental design directed towards determining the sub-mitochondrial localization of both porins involved co-expressing them with a variant of the red-fluorescing protein (RFP), which is artificially targeted to the mitochondrial matrix by a typical cleavable localization signal (Figure 2E). The shape of mitochondria containing only the RFP did not differ from that of normal mitochondria in non-transfected cells (upper panel); neither did those co-expressing RFP and VDAC (lower panel). These mitochondria revealed a complete overlap of both antigens. In the case of mitochondria containing both RFP and porin, however, the typical swollen morphology was observed (central panel), which can also be triggered by addition of porin to mitochondria in vitro (Müller et al., 2000). Interestingly, hardly any overlap could be observed in these mitochondria; instead, rings of porin seem to surround sphere-shaped matrices. All these data taken together clearly indicate that neisserial porin as well as mitochondrial porin, when expressed by either human or yeast cells, specifically inserts into the mitochondrial outer membrane, where it acquires its typical multimeric conformation and presumably forms functional channels. Targeting of neisserial porin to the mitochondrial outer membrane is independent of inner membrane potential The import of many mitochondrial proteins requires an intact inner membrane potential, which provides the driving force necessary for protein translocation across one or both membranes. In the case of PorB import, the role of the membrane potential for import seems especially intriguing, since breakdown of the membrane potential is one of the earliest features observed both during an infection and upon porin treatment of isolated mitochondria (Müller et al., 2000). In order to evaluate the importance of an intact membrane potential for PorB import, cells were treated with the uncoupler antimycin prior to transfection. Antimycin binds to and irreversibly inactivates complex III of the electron transport chain in the inner membrane. Its action was confirmed by counterstaining cells with the potential-sensitive Mitotracker. Neisserial porin, just like the outer membrane proteins VDAC and Tom20, clearly targets mitochondria independently of an intact membrane potential (Figure 3A). Figure 3.Import of PorBIA does not depend on an intact mitochondrial membrane potential. (A and B) HeLa cells were transfected with constructs encoding the indicated proteins, stained with Mitotracker, fixed and co-stained with antibodies against the FLAG (PorBIA, VDAC) or Myc (Tom20) tags. Where indicated, the cells were treated with the uncoupling agent antimycin at 100 μM prior to transfection. Both single colours and overlays are shown of confocal sections. Note that in the case of MiMat–RFP, no double staining with Mitotracker could be performed, as both dyes emit light at similar wavelengths. Download figure Download PowerPoint On the other hand, protein import of an RFP fused to the N-terminal signal sequence of the cytochrome c oxidase subunit Va localized to the inner mitochondrial membrane (MiMat–RFP) is completely blocked in the presence of antimycin (Figure 3B), demonstrating a clear dependence of translocation across the inner membrane on an intact membrane potential. The primary sequence of neisserial porin does not code for a linear mitochondrial targeting signal Two different strategies were employed to characterize a possible mitochondrial targeting signal: one involved the construction of deletion mutants; the other exploited the fact that the very similar N.mucosa porin is not imported by mitochondria of transfected cells (Figure 1A). Consequently, hybrid molecules were generated by fusing the two genes at the highly conserved membrane-spanning regions, thereby exchanging only the variable loop domains. All constructs were used for transfection experiments in HeLa cells followed by immunostaining and confocal microscopy (Figure 4). Figure 4.The primary sequence of PorBIA does not contain a linear mitochondrial targeting signal. PorBIA mutants carrying deletions in one or several loop domains, as well as hybrid molecules consisting of PorBIA and PorBNmu sequences, were constructed by standard PCR techniques. All proteins were expressed in HeLa cells and analysed with respect to their subcellular distribution as described in Figure 1A. Conserved membrane-spanning regions are schematically depicted in grey, whereas the variable loop domains are depicted in white (PorBIA sequences) or black (PorBNmu sequences). The amino acid positions of the junctions between the conserved membrane-spanning regions and the variable loop domains are indicated below the wild-type PorBNgo and PorBNmu. ΔF308 describes a mutant that lacks the terminal phenylalanine residue. Download figure Download PowerPoint A well-studied prerequisite for the correct assembly of porins into the outer bacterial membrane is the presence of a phenylalanine at the extreme C-terminus of the porin sequence (de Cock et al., 1997). In order to dissect whether a mechanistic parallel can be established between folding of porin into the outer membrane of bacteria and of mitochondria, the terminal phenylalanine was deleted and the resulting construct was expressed in HeLa cells (Figure 4). Since this mutant targeted mitochondria (Figure 4) and induced the breakdown of the mitochondrial membrane potential just like the wild-type protein (not shown), it can be concluded that the terminal phenylalanine of PorB is dispensable for mitochondrial membrane integration. Most targeting sequences identified to date are located at either terminus of the preprotein, which is why the two terminal PorB loops seemed the most likely candidates. However, truncations at either end affecting loop I and loop VIII, respectively, did not lead to a loss of mitochondrial targeting, thereby ruling out the existence of a targeting signal as known from other preproteins. Truncation of loops I–III or IV–VIII, on the other hand, generated mutants that were no longer transported to mitochondria. It must be assumed, however, that these short forms no longer retain the ability to form β-barrel structures, raising doubts about the reliability of a simple deletion approach and stressing the necessity of an alternative strategy. The generation of chimeric molecules provides the opportunity to exchange one loop after the other without interfering with the tertiary structure of the pore. Interestingly, N.mucosa porins engineered to carry either gonococcal loops I and II, loop III, loops IV–VII or loop VIII showed a cytoplasmic localization indistinguishable from the wild type (Figure 4). Consistent with this observation, constructs of gonococcal PorB carrying the respective mucosal sequences did not fail to target mitochondria. In all these cases, the distribution of the porins in the cell follows an all-or-none principle. Partial effects of mitochondrial targeting with low efficiency were not observed. These data strongly argue against the existence of a linear targeting signal and provide evidence in favour of a targeting mechanism that requires the cooperation of discontinuous parts of the sequence. Moreover, it is remarkable that all constructs that co-localized with mitochondria also caused the complete dissipation of the membrane potential that is characteristic for the original PorB, demonstrating not only targeting but functional interactions with the mitochondria. Protein–protein interactions with potential binding partners which could contribute to the ‘trapping’ of PorB in the mitochondrial membranes include VDAC and ANT. Although PorB clearly co-purified with both of these factors when mitochondria were lysed mildly and subjected to non-denaturing purification techniques such as gel filtration or anion-exchange chromatography, no direct interaction was detected by co-immunoprecipitation experiments (data not shown). Gonococcal porin is imported into isolated mitochondria in vitro Isolated mitochondria from Saccharomyces cerevisiae have been used extensively for in vitro import studies. This requires the presence of ATP and an electron source such as NADH. Proteins to be imported are synthesized in vitro using a coupled transcription/translation system and radiolabelled simultaneously with [35S]methionine. Since proteins tend to associate with mitochondria unspecifically, the import reaction is followed by PK digestion. Only the PK-resistant population can truly be classified as ‘imported’. As shown in Figure 5A, in vitro translated gonococcal porin as well as mitochondrial VDAC are readily digested by PK; a 20-fold amount (100 μM) of the PK concentration shown to digest both proteins completely (5 μM) was used in all subsequent assays. One obstacle preventing the in vitro approach, the lack of sulfur-containing amino acids in the PorB sequence, was overcome by the generation of a hybrid containing the N-terminal portion of gonococcal porin and the C-terminal loop VIII of homologous N.mucosa porin (Figure 4). This mutant retains the ability to target mitochondria in transfected cells, but has gained an additional methionine in the last membrane-spanning region. It is, therefore, more readily labelled than the wild type. Figure 5.PorBIA is imported by isolated yeast mitochondria in vitro with lower efficiency but similar kinetics as human VDAC. (A) In vitro translated PorBIA and human VDAC were digested with increasing amounts of PK as indicated for 15 mi