Role of the intermembrane-space domain of the preprotein receptor Tom22 in protein import into mitochondria

Tom22 is an essential component of the protein translocation complex (Tom complex) of the mitochondrial outer membrane. The N-terminal domain of Tom22 functions as a preprotein receptor in cooperation with Tom20. The role of the C-terminal domain of Tom22, which is exposed to the intermembrane space (IMS), in its own assembly into the Tom complex and in the import of other preproteins was investigated. The C-terminal domain of Tom22 is not essential for the targeting and assembly of this protein, as constructs lacking part or all of the IMS domain became imported into mitochondria and assembled into the Tom complex. Mutant strains of Neurospora expressing the truncated Tom22 proteins were generated by a novel procedure. These mutants displayed wild-type growth rates, in contrast to cells lacking Tom22, which are not viable. The import of proteins into the outer membrane and the IMS of isolated mutant mitochondria was not affected. Some but not all preproteins destined for the matrix and inner membrane were imported less efficiently. The reduced import was not due to impaired interaction of presequences with their specific binding site on the trans side of the outer membrane. Rather, the IMS domain of Tom22 appears to slightly enhance the efficiency of the transfer of these preproteins to the import machinery of the inner membrane.     

Dynamics of the TOM complex of mitochondria during binding and translocation of preproteins

Translocation of preproteins across the mitochondrial outer membrane is mediated by the TOM complex. This complex consists of receptor components for the initial contact with preproteins at the mitochondrial surface and membrane-embedded proteins which promote transport and form the translocation pore. In order to understand the interplay between the translocating preprotein and the constituents of the TOM complex, we analyzed the dynamics of the TOM complex of Neurospora crassa and Saccharomyces cerevisiae mitochondria by following the structural alterations of the essential pore component Tom40 during the translocation of preproteins. Tom40 exists in a homo-oligomeric assembly and dynamically interacts with Tom6. The Tom40 assembly is influenced by a block of negatively charged amino acid residues in the cytosolic domain of Tom22, indicating a cross-talk between preprotein receptors and the translocation pore. Preprotein binding to specific sites on either side of the outer membrane (cis and trans sites) induces distinct structural alterations of Tom40. To a large extent, these changes are mediated by interaction with the mitochondrial targeting sequence. We propose that such targeting sequence-induced adaptations are a critical feature of translocases in order to facilitate the movement of preproteins across cellular membranes.     

Preproteins of chloroplast envelope inner membrane contain targeting information for receptor-dependent import into fungal mitochondria

The amino-terminal transit sequences of two preproteins destined for the chloroplast inner envelope membrane show similarities to mitochondrial presequences in the prevalence of positive charges and the potential formation of an amphipathic alpha-helix. We studied if these preproteins could be imported into mitochondria and found a low, yet significant import into isolated plant mitochondria. The plant mitochondria were previously shown not to import precursors of chloroplast stromal or thylakoidal proteins. To analyze the specificity of import into mitochondria we used the established import systems of fungal mitochondria. The envelope preproteins were efficiently imported into Saccharomyces cerevisiae or Neurospora crassa mitochondria. Their import showed the characteristics of specific mitochondrial protein uptake, including a requirement for the main receptor MOM19 (mitochondrial outer membrane protein of 19 kDa) and a membrane potential across the inner membrane, and depended on the presence of the chloroplast transit sequence. We conclude that some chloroplast transit sequences contain sufficient information for specific interaction with mitochondrial import receptors (at least from fungal sources).     

Mitochondrial transport of mitoribosomal proteins, YmL8 and YmL20, in Saccharomyces cerevisiae

Two mitochondrial ribosomal (mitoribosomal) proteins, YmL8 and YmL20, of the yeast Saccharomyces cerevisiae and their derivatives were synthesized in vitro and their transport into isolated yeast mitochondria was examined. Of the two proteins, YmL20 possesses an N-terminal presequence of 18 amino acid residues, while YmL8 has no such presequence. Both proteins were found to be transported into isolated mitochondria in an energy-dependent manner. Furthermore, YmL20 protein without its N-terminal presequence was also transported, despite the fact that the presequence alone was capable of transporting a fused passenger protein, Chinese hamster dihydrofolate reductase (DHFR). Therefore, YmL20 protein appears to possess redundant transport signals in its structure. Similarly, YmL8 derivatives lacking either 40 or 86 amino acid residues from the N-terminus and/or 52 amino acid residues from the C-terminus were transported. In addition, the N-terminal segment of this protein was capable of transporting Chinese hamster DHFR into mitochondria, while its C-terminal segment was not. Thus, YmL8 protein also appears to possess two or more transport signals in its structure. Perhaps the presence of many basic amino acid residues in these proteins might, at least partly, contribute to their mitochondrial transport.     

Mapping of functional domains for HIV-2 gag assembly into virus-like particles

The human immunodeficiency virus type 2 gag precursor protein, pr41, self assembles as virus-like particles (VLP) when the gag gene is expressed in insect cells. To map the functional domains for HIV-2 gag VLP formation, a series of deletion mutants was constructed by removing sequentially the C-terminal region of HIV-2 gag precursor protein and expressing the truncated gag genes in SF9 insect cells by means of recombinant baculoviruses. We found that deletion of up to 143 amino acids at the C-terminus of HIV-2 gag, leaving 376 amino acids at the N-terminus of the protein, did not prevent VLP formation. However, an additional four amino acids deletion from the C-terminus, which represents 372 amino acids at the N-terminus, made gag protein fail to form VLP. There is a proline-rich region at amino acid positions 372 and 377 of HIV-2 gag. To analyze the role of these proline residues, we generated five mutants in which proline was changed sequentially into leucine. Our results showed that replacement of one or two prolines did not stop gag VLP formation, whereas replacement of all three prolines by leucine residues completely abolished VLP assembly. Our data demonstrate that the C-terminal p12 region of HIV-2 gag precursor protein and the zinc finger domain are dispensable for gag VLP assembly, but the presence of at least one of the three proline residues located between amino acid positions 372 and 377 of HIV-2NIH-Z is required.     

The protein import receptor MOM19 of yeast mitochondria

We have identified the protein import receptor MOM19 of Saccharomyces cerevisiae mitochondria. MOM19 is exposed on the outer membrane surface and present in the mitochondrial receptor complex. Antibodies raised against MOM19 strongly inhibited the import of preproteins into isolated yeast mitochondria. Fab fragments prepared from the antibodies showed the same inhibitory effect. By using mutant mitochondria, which lacked the second import receptor MOM72, we found that the import of preproteins via MOM19 did not require the presence of MOM72. We conclude that MOM19 is required for preprotein translocation across the yeast mitochondrial outer membrane and is able to function independently of the receptor MOM72.     

The import route of ADP/ATP carrier into mitochondria separates from the general import pathway of cleavable preproteins at the trans side of the outer membrane

The ADP/ATP carrier (AAC) of the mitochondrial inner membrane is synthesized in the cytosol without a cleavable presequence. The preprotein preferentially binds to the mitochondrial surface receptor Tom70 and joins the import pathway of presequence-carrying preproteins at the cis side of the outer membrane. Little is known about the translocation of the AAC across the outer membrane and where its import route separates from that of cleavable preproteins. Here we have characterized a translocation intermediate of AAC during transfer across the outer membrane. The major portion of the preprotein is exposed to the intermembrane space, while a short segment is still accessible to externally added protease. This intermediate can be quantitatively chased to the fully imported form in the inner membrane. Its accumulation depends on Tom7, but not on the intermembrane space domain of Tom22 in contrast to cleavable preproteins. Moreover, opening of the intermembrane space inhibits the import of AAC, but not that of cleavable preproteins into mitoplasts. We conclude that the import route of AAC diverges from the general import pathway of cleavable preproteins already at the trans side of the outer membrane.     

The preprotein translocase of the mitochondrial inner membrane: function and evolution

Growing mitochondria acquire most of their proteins by the uptake of mitochondrial preproteins from the cytosol. To mediate this protein import, both mitochondrial membranes contain independent protein transport systems: the Tom machinery in the outer membrane and the Tim machinery in the inner membrane. Transport of proteins across the inner membrane and sorting to the different inner mitochondrial compartments is mediated by several protein complexes which have been identified in the past years. A complex containing the integral membrane proteins Tim17 and Tim23 constitutes the import channel for preproteins containing amino-terminal hydrophilic presequences. This complex is associated with Tim44 which serves as an adaptor protein for the binding of mtHsp70 to the membrane. mtHsp70, a 70 kDa heat shock protein of the mitochondrial matrix, drives the ATP-dependent import reaction of the processed preprotein after cleavage of the presequence. Preproteins containing internal targeting information are imported by a separate import machinery, which consists of the intermembrane-space proteins Tim9, Tim10, and Tim12, and the inner membrane proteins Tim22 and Tim54. The proteins Tim17, Tim22, and Tim23 have in common a similar topology in the membrane and a homologous amino acid sequence. Moreover, they show a sequence similarity to OEP16, a channel-forming amino acid transporter in the outer envelope of chloroplasts, and to LivH, a component of a prokaryotic amino acid permease, defining a new PRAT-family of preprotein and amino acid transporters.     

Charged amino acids at the carboxyl-terminal portions determine the intracellular locations of two isoforms of cytochrome b5

Outer mitochondrial membrane cytochrome b5 (OMb), which is an isoform of cytochrome b5 (cyt b5) in the endoplasmic reticulum, is a typical tail-anchored protein of the outer mitochondrial membrane. We cloned cDNA containing the complete amino acid sequence of OMb and found that the protein has no typical structural feature common to the mitochondrial targeting signal at the amino terminus. To identify the region responsible for the mitochondrial targeting of OMb, various mutated proteins were expressed in cultured mammalian cells, and the subcellular localization of the expressed proteins was analyzed. The deletion of more than 11 amino acid residues from the carboxyl-terminal end of OMb abolished the targeting of the protein to the mitochondria. When the carboxyl-terminal 10 amino acids of OMb were fused to the cyt b5 that was previously deleted in the corresponding 10 residues, the fused protein localized in the mitochondria, thereby indicating that the carboxyl-terminal 10 amino acid residues of OMb have sufficient information to transport OMb to the mitochondria. The replacement of either of the two positively charged residues within the carboxyl-terminal 10 amino acids by alanine resulted in the transport of the mutant proteins to the endoplasmic reticulum. The mutant cyt b5, in which the acidic amino acid in its carboxyl-terminal end was replaced by basic amino acid, could be transported to the mitochondria. It would thus seem that charged amino acids in the carboxyl-terminal portion of these proteins determine their locations in the cell.     

An import signal in the cytosolic domain of the Neurospora mitochondrial outer membrane protein TOM22

TOM22 is an integral component of the preprotein translocase of the mitochondrial outer membrane (TOM complex). The protein is anchored to the lipid bilayer by a central trans-membrane segment, thereby exposing the amino-terminal domain to the cytosol and the carboxyl-terminal portion to the intermembrane space. Here, we describe the sequence requirements for the targeting and correct insertion of Neurospora TOM22 into the outer membrane. The orientation of the protein is not influenced by the charges flanking its trans-membrane segment, in contrast to observations regarding proteins of other membranes. In vitro import studies utilizing TOM22 preproteins harboring deletions or mutations in the cytosolic domain revealed that the combination of the trans-membrane segment and intermembrane space domain of TOM22 is not sufficient to direct import into the outer membrane. In contrast, a short segment of the cytosolic domain was found to be essential for the import and assembly of TOM22. This sequence, a novel internal import signal for the outer membrane, carries a net positive charge. A mutant TOM22 in which the charge of the import signal was altered to -1 was imported less efficiently than the wild-type protein. Our data indicate that TOM22 contains physically separate import and membrane anchor sequences.     

Biogenesis of Tim23 and Tim17, integral components of the TIM machinery for matrix-targeted preproteins

We analysed the import pathway of Tim23 and of Tim17, components of the mitochondrial import machinery for matrix-targeted preproteins. Tim23 contains two independent import signals. One is located within the first 62 amino acid residues of the hydrophilic domain that, in the assembled protein, is exposed to the intermembrane space. This signal mediates translocation of Tim23 across the outer membrane independently of the membrane potential, DeltaPsi. A second import signal is located in the C-terminal membrane-integrated portion of Tim23. It mediates translocation across the outer membrane and insertion into the inner membrane in a strictly DeltaPsi-dependent fashion. Structurally, Tim17 is related to Tim23 but lacks a hydrophilic domain. It contains an import signal in the C-terminal half and its import requires DeltaPsi. The DeltaPsi-dependent import signals of Tim23 and Tim17 are located at corresponding sites in these two homologous proteins. They exhibit features reminiscent of the positively charged N-terminal presequences of matrix-targeted precursors. Import of Tim23 and its insertion into the inner membrane requires Tim22 but not functional Tim23. Thus, biogenesis of the Tim23.17 complex depends on the Tim22 complex, which is the translocase identified as mediating the import of carrier proteins.     

Role of the negative charges in the cytosolic domain of TOM22 in the import of precursor proteins into mitochondria

TOM22 is an essential mitochondrial outer membrane protein required for the import of precursor proteins into the organelles. The amino-terminal 84 amino acids of TOM22 extend into the cytosol and include 19 negatively and 6 positively charged residues. This region of the protein is thought to interact with positively charged presequences on mitochondrial preproteins, presumably via electrostatic interactions. We constructed a series of mutant derivatives of TOM22 in which 2 to 15 of the negatively charged residues in the cytosolic domain were changed to their corresponding amido forms. The mutant constructs were transformed into a sheltered Neurospora crassa heterokaryon bearing a tom22::hygromycin R disruption in one nucleus. All constructs restored viability to the disruption-carrying nucleus and gave rise to homokaryotic strains containing mutant tom22 alleles. Isolated mitochondria from three representative mutant strains, including the mutant carrying 15 neutralized residues (strain 861), imported precursor proteins at efficiencies comparable to those for wild-type organelles. Precursor binding studies with mitochondrial outer membrane vesicles from several of the mutant strains, including strain 861, revealed only slight differences from binding to wild-type vesicles. Deletion mutants lacking portions of the negatively charged region of TOM22 can also restore viability to the disruption-containing nucleus, but mutants lacking the entire region cannot. Taken together, these data suggest that an abundance of negative charges in the cytosolic domain of TOM22 is not essential for the binding or import of mitochondrial precursor proteins; however, other features in the domain are required.     

The essential yeast protein MIM44 (encoded by MPI1) is involved in an early step of preprotein translocation across the mitochondrial inner membrane

The essential yeast gene MPI1 encodes a mitochondrial membrane protein that is possibly involved in protein import into the organelle (A. C. Maarse, J. Blom, L. A. Grivell, and M. Meijer, EMBO J. 11:3619-3628, 1992). For this report, we determined the submitochondrial location of the MPI1 gene product and investigated whether it plays a direct role in the translocation of preproteins. By fractionation of mitochondria, the mature protein of 44 kDa was localized to the mitochondrial inner membrane and therefore termed MIM44. Import of the precursor of MIM44 required a membrane potential across the inner membrane and involved proteolytic processing of the precursor. A preprotein in transit across the mitochondrial membranes was cross-linked to MIM44, whereas preproteins arrested on the mitochondrial surface or fully imported proteins were not cross-linked. When preproteins were arrested at two distinct stages of translocation across the inner membrane, only preproteins at an early stage of translocation could be cross-linked to MIM44. Moreover, solubilized MIM44 was found to interact with in vitro-synthesized preproteins. We conclude that MIM44 is a component of the mitochondrial inner membrane import machinery and interacts with preproteins in an early step of translocation.     

Analysis of the sorting signals directing NADH-cytochrome b5 reductase to two locations within yeast mitochondria

Mitochondrial NADH-cytochrome b5 reductase (Mcr1p) is encoded by a single nuclear gene and imported into two different submitochondrial compartments: the outer membrane and the intermembrane space. We now show that the amino-terminal 47 amino acids suffice to target the Mcr1 protein to both destinations. The first 12 residues of this sequence function as a weak matrix-targeting signal; the remaining residues are mostly hydrophobic and serve as an intramitochondrial sorting signal for the outer membrane and the intermembrane space. A double point mutation within the hydrophobic region of the targeting sequence virtually abolishes the ability of the precursor to be inserted into the outer membrane but increases the efficiency of transport into the intermembrane space. Import of Mcr1p into the intermembrane space requires an electrochemical potential across the inner membrane, as well as ATP in the matrix, and is strongly impaired in mitochondria lacking Tom7p or Tim11p, two components of the translocation machineries in the outer and inner mitochondrial membranes, respectively. These results indicate that intramitochondrial sorting of the Mcr1 protein is mediated by specific interactions between the bipartite targeting sequence and components of both mitochondrial translocation systems.     

Effect of truncated forms of the steroidogenic acute regulatory protein on intramitochondrial cholesterol transfer

It has been proposed that the steroidogenic acute regulatory (StAR) protein controls hormone-stimulated steroid production by mediating cholesterol transfer to the mitochondrial inner membrane. This study was conducted to determine the effect of wild-type StAR and several modified forms of StAR on intramitochondrial cholesterol transfer. Forty-seven N-terminal or 28 C-terminal amino acids of the StAR protein were removed, and COS-1 cells were transfected with pCMV vector only, wild-type StAR, N-47, or the C-28 constructs. Lysates from the transfected COS-1 cells were then incubated with mitochondria from MA-10 mouse Leydig tumor cells that were preloaded with [3H]cholesterol. After incubation, mitochondria were collected and fractionated on sucrose gradients into outer membranes, inner membranes, and membrane contact sites, and [3H]cholesterol content was determined in each membrane fraction. Incubation of MA-10 mitochondria with wild-type StAR containing cell lysate resulted in a significant 34.9% increase in [3H]cholesterol content in contact sites and a significant 32.8% increase in inner mitochondrial membranes. Incubations with cell lysate containing N-47 StAR protein also resulted in a 16.4% increase in [3H]cholesterol in contact sites and a significant 26.1% increase in the inner membrane fraction. In contrast, incubation with the C-28 StAR protein had no effect on cholesterol transfer. The cholesterol-transferring activity of the N-47 truncation, in contrast to that of the C-28 mutant, was corroborated when COS-1 cells were cotransfected with F2 vector (containing cytochrome P450 side-chain cleavage enzyme, ferridoxin, and ferridoxin reductase) and either pCMV empty vector or the complementary DNAs of wild-type StAR, N-47 StAR, or C-28 StAR. Pregnenolone production was significantly increased in both wild-type and N-47-transfected cells, whereas that in C-28-transfected cells was similar to the control value. Finally, immunolocalization studies with confocal image and electron microscopy were performed to determine the cellular location of StAR and its truncated forms in transfected COS-1 cells. The results showed that wild-type and most of the C-28 StAR protein were imported into the mitochondria, whereas most of N-47 protein remained in the cytosol. These studies demonstrate a direct effect of StAR protein on cholesterol transfer to the inner mitochondrial membrane, that StAR need not enter the mitochondria to produce this transfer, and the importance of the C-terminus of StAR in this process.     

Targeting of NH2-terminal-processed microsomal protein to mitochondria: a novel pathway for the biogenesis of hepatic mitochondrial P450MT2

Cytochrome P4501A1 is a hepatic, microsomal membrane-bound enzyme that is highly induced by various xenobiotic agents. Two NH2-terminal truncated forms of this P450, termed P450MT2a and MT2b, are also found localized in mitochondria from beta-naphthoflavone-induced livers. In this paper, we demonstrate that P4501A1 has a chimeric NH2-terminal signal that facilitates the targeting of the protein to both the ER and mitochondria. The NH2-terminal 30-amino acid stretch of P4501A1 is thought to provide signals for ER membrane insertion and also stop transfer. The present study provides evidence that a sequence motif immediately COOH-terminal (residues 33-44) to the transmembrane domain functions as a mitochondrial targeting signal under both in vivo and in vitro conditions, and that the positively charged residues at positions 34 and 39 are critical for mitochondrial targeting. Results suggest that 25% of P4501A1 nascent chains, which escape ER membrane insertion, are processed by a liver cytosolic endoprotease. We postulate that the NH2-terminal proteolytic cleavage activates a cryptic mitochondrial targeting signal. Immunofluorescence microscopy showed that a portion of transiently expressed P4501A1 is colocalized with the mitochondrial-specific marker protein cytochrome oxidase subunit I. The mitochondrial-associated MT2a and MT2b are localized within the inner membrane compartment, as tested by resistance to limited proteolysis in both intact mitochondria and mitoplasts. Our results therefore describe a novel mechanism whereby proteins with chimeric signal sequence are targeted to the ER as well as to the mitochondria.     

Mature carnitine palmitoyltransferase I retains the N-terminus of the nascent protein in rat liver

Carnitine palmitoyltransferase I was isolated from octylglucoside extracts of rat liver mitochondrial outer membranes. This native enzyme was digested proteolytically with V8 protease. Five major peptides were obtained all of which were found in the amino acid sequence predicted from the full-length cDNA sequence of the protein. One peptide was found to correspond to the extreme N-terminus of the deduced amino acid sequence. Therefore, the mature protein retains the N-terminus of the nascent protein after import into the mitochondrial membrane. Knowledge of the identity of the N-terminus of the mature protein allows a reappraisal of the role of the two main. N-terminal hydrophobic domains of the protein and of the possible topology of the protein within the membrane.     

cis and trans sites of the TOM complex of mitochondria in unfolding and initial translocation of preproteins

Translocation of preproteins across the mitochondrial outer membrane is mediated by the TOM complex. Our previous studies led to the concept of two preprotein binding sites acting in series, the surface-exposed cis site and the trans site exposed to the intermembrane space. We report here that preproteins are bound to the cis site in a labile fashion even at low ionic strength, whereas intermediates arrested at the trans site remained firmly bound at higher salt concentration. The stability of the trans site intermediate results from interactions of both the presequence and unfolded parts of the mature part of the preprotein with the TOM complex. Binding to the trans site proceeded at rates comparable with those of unfolding of the mature domain and appeared to be kinetically limited by the unfolding reaction. Efficient binding to the trans site and unfolding were observed with both outer membrane vesicles and intact mitochondria whose membrane potential, DeltaPsi, was dissipated. Upon re-establishing DeltaPsi, trans site-bound preprotein resumed translocation into the matrix. The rates of unfolding and binding to the trans site were the same as those for translocation into intact energized mitochondria. We conclude that preprotein unfolding in intact mitochondria can take place without the involvement of the translocation machinery of the inner membrane and, in particular, the matrix Hsp70 chaperone. Further, preprotein unfolding at the outer membrane can be a rate-limiting step for formation of the trans site intermediate and for the entire translocation reaction.     

N-terminal hydrophobic sorting signals of preproteins confer mitochondrial hsp70 independence for import into mitochondria

The requirement of mitochondrial hsp70 (mt-hsp70) for the import of a series of preproteins containing hydrophobic sorting signals into isolated yeast mitochondria was investigated. Here we demonstrate that the presence of such a sorting signal in proximity to the N-terminal matrix-targeting sequence of a preprotein can secure a translocating polypeptide chain in the import channel in a manner that does not require mt-hsp70 activity. Trapping the translocating chain in this fashion leads to efficient processing by the mitochondrial processing peptidase and to complete translocation across the outer mitochondrial membrane into the intermembrane space. These mt-hsp70-independent effects appear to be exerted at the level of the inner membrane through an interaction of the hydrophobic core of the sorting signal with component(s) of the translocase of the inner membrane. Hydrophobic sorting signals of inner membrane proteins inserted into the membrane from the matrix, as well as those of intermembrane space proteins, are capable of causing this mt-hsp70-independent stabilization, demonstrating that this phenomenon is not unique to those preproteins normally sorted to the intermembrane space.     

The molecular structure of mitochondrial contact sites. Their role in regulation of energy metabolism and permeability transition

Contact sites between the outer and peripheral inner membrane of mitochondria are involved in protein precursor uptake and energy transfer. Hexokinase and mitochondrial creatine kinase could be attributed by different techniques to the energy transfer contacts. Kinetic analyses suggested a functional interaction between the kinases, outer membrane pore protein, and inner membrane adenylate translocator (ANT). This suggestion was strongly supported by isolation of hexokinase and creatine kinase complexes that were constituted of kinase oligomers, porin and ANT. Phospholipid vesicles carrying reconstituted kinase-porin-ANT complexes enclosed internal ATP in contrast to vesicles containing free porin only. This indicated that unspecific transport through porin was regulated by its interaction with a specific antiporter, ANT. A direct interaction between porin and ANT in the hexokinase complex conferred the reconstituted system with permeability properties reminiscent of the mitochondrial permeability transition (PT) pore. In the creatine kinase complex this interaction between porin and ANT was replaced by contact of both with the kinase octamer. Thus PT-pore-like functions were not observed unless the creatine kinase octamer was dissociated, suggesting that the ANT was locked in the antiporter state by interaction with the octamer. Indeed, reconstituted pure ANT showed PT-pore-like properties concerning Ca2+ sensitivity. However, as cyclophilin was missing, sensitivity against cyclosporin was not observed.