A synthetic presequence reversibly inhibits protein import into yeast mitochondria

We show that a synthetic peptide corresponding to the N-terminal 22 residues of the cytochrome c oxidase subunit IV presequence blocked import of pre-subunit IV into yeast mitochondria. The 22-residue peptide pL4-(1-22) did not alter the electrical potential across the mitochondrial inner membrane (the delta psi). Inhibition of import was reversible and could be overcome by the addition of increased amounts of precursor. Two other peptides, pL4-(1-16) and pL4-(1-23), which correspond to, respectively, the N-terminal 16 and 23 residues of the same presequence, also blocked import of pre-subunit IV. However, pL4-(1-16) was a much weaker inhibitor of import, while the inhibitory effect of pL4-(1-23) was due to its ability to completely collapse the delta psi. pL4-(1-22) seems to be a general inhibitor of mitochondrial import, in that it also blocked uptake of several other proteins. These included the precursors of the yeast proteins cytochrome c oxidase subunit Va, the F1-ATPase beta subunit, mitochondrial malate dehydrogenase, and the ATP/ADP carrier. In addition, uptake of two non-yeast precursor proteins (human ornithine transcarbamylase and a cytochrome oxidase subunit IV-dihydrofolate reductase fusion), was also blocked by the peptide. Subsequent studies revealed that pL4-(1-22) did not block the initial recognition or binding of proteins to mitochondria. Rather, our results suggest that the peptide acts at a subsequent translocation step which is common to the import pathways of many different precursor proteins.     

A truncated analog of a pre-light-harvesting chlorophyll a/b protein II transit peptide inhibits protein import into chloroplasts

It is unclear how transit peptides target nuclear-encoded precursor proteins to the chloroplast. This study establishes the feasibility of using synthetic peptides as competitive inhibitors of chloroplast protein import and as probes for the function of domains within transit peptides. We show that peptide pL(1-20), MAASTMALSSPAFAGKAVNY, an analog of the NH2 terminus of a pre-light harvesting chlorophyll a/b protein II from Arabidopsis, inhibits the import of several Arabidopsis and pea precursor proteins into pea chloroplasts. Inhibition occurs at a step between the initial binding of precursors to the chloroplast and the first proteolytic cleavage event and is not due to interference with ATP availability or chloroplast integrity. Presumably this reflects specific binding of the peptide to the import machinery in the chloroplast envelope. Our data are consistent with the suggestion (Karlin-Neumann, G. A., and Tobin, E. M. (1986) EMBO J. 5, 9-13) that two conserved blocks of amino acids near the NH2-terminus of transit peptides (spanned by peptide pL(1-20] participate in protein targeting. Computer analysis also shows peptide pL(1-20) lacks the amphiphilic properties characteristic of pre-sequences of many nuclear-encoded mitochondrial proteins. This shows a difference in the mechanisms for targeting proteins to chloroplasts and mitochondria.     

Early events in the transport of proteins into mitochondria. Import competition by a mitochondrial presequence.

Studies with a synthetic presequence peptide, F1 beta 1-20, corresponding to the NH2-terminal 20 amino acids of the F1-ATPase beta-subunit precursor (pF1 beta) show that although this peptide binds avidly to phospholipid bi-layers it does not efficiently compete for import of full-length precursor into mitochondria, Ki approximately 100 microM (Hoyt, D.W., Cyr, D.M., Gierasch, L.M., and Douglas, M.G. (1991) J. Biol. Chem. 266, 21693-21699). Herein we report that longer F1 beta presequence peptides F1 beta 1-32 + 2, F1 beta 1-32SQ + 2, and F1 beta 21-51 + 3 compete for mitochondrial import at 1000-, 250-, and 25-fold lower concentrations, respectively, than F1 beta 1-20. A longer peptide, F1 beta 1-51 + 3, was no more effective as an import competitor than F1 beta 1-32 + 2. Both minimal length and amphiphilic character appear required in order for F1 beta peptides to block mitochondrial import. Import competition by longer F1 beta peptides seems to occur at a step common to all precursors since they blocked import of precursors to F1-ATPase alpha- and beta-subunits and the ADP/ATP carrier protein. Dissipation of membrane potential (delta psi) across the inner mitochondrial membrane is observed in the presence of F1 beta-peptides, but this mechanism alone does not account for the observed import inhibition. F1 beta 1-32 + 2 and 21-51 + 3 block import of pF1 beta 100% at peptide concentrations which dissipate delta psi less than 25%. In contrast, experiments with valinomycin demonstrate that when mitochondrial delta psi is reduced 25% import of pF1 beta is inhibited only 25%. Therefore, at least 75% of maximal import inhibition observed in the presence of F1 beta 1-32 + 2 and F1 beta 21-51 + 3 does not result from dissipation of delta psi. Import inhibition by F1 beta-peptides is reversible and can be overcome by increasing the amount of full-length precursor in import reactions. F1 beta presequence peptides and full-length precursor are therefore likely to compete for a common import step. Presequence dependent binding of pF1 beta to trypsin-sensitive elements on the outer mitochondrial membrane is insensitive to inhibitory concentrations of F1 beta presequence peptide. We conclude that import inhibition by F1 beta presequence peptides is competitive and occurs at a site beyond initial interaction of precursor proteins with mitochondria.     

A synthetic signal peptide blocks import of precursor proteins destined for the mitochondrial inner membrane or matrix

A peptide corresponding to amino acids 1-27 of preornithine carbamyltransferase (pOCT) has been chemically synthesized. When added to energized mitochondria in vitro, 20 microM of the peptide, designated pO(1-27), resulted in a collapse of the electrochemical potential across the mitochondrial inner membrane. This effect on transmembrane potential was not observed, however, when pO(1-27) was added to energized mitochondria under conditions that support in vitro import of precursor proteins (i.e. in the presence of reticulocyte lysate). The latter finding, therefore, made possible an examination of the ability of pO(1-27) to block import of homologous and heterologous proteins into the organelle. At 5-10 microM, pO(1-27) prevented import of pOCT in vitro; inhibition was overcome by increasing the concentration of pOCT. In contrast, pO(16-27), a peptide corresponding to amino acids 16-27 of pOCT and exhibiting a charge:mass ratio similar to pO(1-27) had no such inhibitory effect. pO(1-27) blocked import of other unrelated precursor proteins destined either for the mitochondrial matrix (pre-malate dehydrogenase and a hybrid protein containing the signal sequence of pre-carbamyl phosphate synthetase) or for the mitochondrial inner membrane (pre-thermogenin).     

Import of chemically synthesized signal peptides into rat liver mitochondria

The signal peptides of pre-aldehyde dehydrogenase (22-mer) and pre-ornithine transcarbamylase (27-mer) were chemically synthesized and their imports into rat liver mitochondria were studied. Both signal peptides were imported rapidly (within 2 min) in the absence of a membrane potential, exogenous ATP, or rabbit reticulocyte lysate. Signal peptides also were imported into mitochondria treated with a low concentration of trypsin which removed the outer membrane proteins. It was concluded that the chemically synthesized signal peptide could be imported differently than the precursor proteins. The imported signal peptide were found to be associated with both outer and inner membranes. Pulse-chase experiments showed that the import was unidirectional and that the signal peptides associated with inner membranes increased during the chase time. The signal peptides inhibited import of precursor proteins to different extents. Association of signal peptides with inner membrane near or at translocator sites might result in inhibition of precursor import.     

Cytosolic and mitochondrial surface factor-independent import of a synthetic peptide into mitochondria.

We chemically synthesized a peptide, 11 beta-45, which was composed of 45 amino acid residues including the whole extension peptide and some of the mature portion of bovine cytochrome P-450(11 beta) precursor. 11 beta-45 was imported into mitochondria in vitro depending on the mitochondrial membrane potential, but its import did not require extramitochondrial ATP. Although cytosolic protein factors in the high speed supernatant of reticulocyte lysate are known to stimulate the import of various precursor proteins into mitochondria, the import of 11 beta-45 was not stimulated by cytosolic factors in reticulocyte lysate. The import of the peptide did not require mitochondrial surface protein components because its import was not affected by trypsin treatment of mitochondria. On the other hand, trypsin treatment of mitoplasts resulted in a great reduction in the import of the peptide, indicating that 11 beta-45 interacts during the import process with some protein components located inside mitochondria. These observations indicated that the peptide 11 beta-45 was imported via the potential-dependent pathway as in the case of precursor proteins, but skipped the interactions with cytosolic factors and mitochondrial surface components normally required for the import of precursor proteins.     

Interaction of peptides corresponding to mitochondrial presequences with membranes.

The transport of the F1-ATPase beta-subunit precursor into mitochondria is dependent upon a presequence at its amino terminus. Within the mitochondrial membrane translocation site the potential amphiphilic character of the presequence region may be necessary to stabilize binding to the mitochondrial inner membrane. To better understand its role in protein import, the interaction of the F1 beta-presequence with lipid membranes was measured using circular dichroism and surface tensiometry. These studies reveal that a 20-residue peptide containing the F1 beta-presequence binds to phospholipid vesicles (Kd = 4.5-6.0 x 10(-8)M and adopts a predominantly alpha-helical structure. Although the presequence peptide binds avidly to lipids, it does not appear to penetrate deeply into the bilayer to perturb a reporter probe in the membrane interior. Compared with the effect of the peptides with demonstrated membrane insertion and lytic properties, the F1-beta-presequence appears to displace phospholipid head groups but not insert deeply into the bilayer. High concentrations (greater than 50 microM) of presequence peptides are required to noticibly perturb import of the full length F1 alpha- or F1 beta-subunit precursors. Thus, the F1 beta-presequence alone is not sufficient to efficiently compete for import but may require a protein context or a minimal length to assist insertion into the transport site. These observations are discussed in light of the different requirements for import of various presequence containing precursors into mitochondria.     

A multisubunit complex of outer and inner mitochondrial membrane protein translocases stabilized in vivo by translocation intermediates.

Translocation of nuclear encoded preproteins into the mitochondrial matrix requires the coordinated action of two translocases: one (Tom) located in the outer mitochondrial membrane and the other (Tim) located in the inner membrane. These translocases reversibly cooperate during protein import. We have previously constructed a chimeric precursor (pPGPrA) consisting of an authentic mitochondrial precursor at the N terminus (Delta(1)-pyrroline-5-carboxylate dehydrogenase, pPut) linked, through glutathione S-transferase, to protein A. When pPGPrA is expressed in yeast, it becomes irreversibly arrested during translocation across the outer and inner mitochondrial membranes. Consequently, the two membranes of mitochondria become progressively "zippered" together, forming long stretches in which they are in close contact (Schülke, N., Sepuri, N. B. V., and Pain, D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 7314-7319). We now demonstrate that trapped PGPrA intermediates hold the import channels stably together and inhibit mitochondrial protein import and cell growth. Using IgG-Sepharose affinity chromatography of solubilized zippered membranes, we have isolated a multisubunit complex that contains all Tom and Tim components known to be essential for import of matrix-targeted proteins, namely Tom40, Tom22, Tim17, Tim23, Tim44, and matrix-localized Hsp70. Further characterization of this complex may shed light on structural features of the complete mitochondrial import machinery.     

Nascent polypeptide-associated complex stimulates protein import into yeast mitochondria

To identify yeast cytosolic proteins that mediate targeting of precursor proteins to mitochondria, we developed an in vitro import system consisting of purified yeast mitochondria and a radiolabeled mitochondrial precursor protein whose C terminus was still attached to the ribosome. In this system, the N terminus of the nascent chain was translocated across both mitochondrial membranes, generating a translocation intermediate spanning both membranes. The nascent chain could then be completely chased into the mitochondrial matrix after release from the ribosome. Generation of this import intermediate was dependent on a mitochondrial membrane potential, mitochondrial surface proteins, and was stimulated by proteins that could be released from the ribosomes by high salt. The major salt-released stimulatory factor was yeast nascent polypeptide-associated complex (NAC). Purified NAC fully restored import of salt-washed ribosome-bound nascent chains by enhancing productive binding of the chains to mitochondria. We propose that ribosome-associated NAC facilitates recognition of nascent precursor chains by the mitochondrial import machinery.     

Functions of outer membrane receptors in mitochondrial protein import

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins and are imported into mitochondria. The targeting signals for mitochondria are encoded in the presequences or in the mature parts of the precursor proteins, and are decoded by the receptor sites in the translocator complex in the mitochondrial outer membrane. The recently determined NMR structure of the general import receptor Tom20 in a complex with a presequence peptide reveals that, although the amphiphilicity and positive charges of the presequence is essential for the import ability of the presequence, Tom20 recognizes only the amphiphilicity, but not the positive charges. This leads to a new model that different features associated with the mitochondrial targeting sequence of the precursor protein can be recognized by the mitochondrial protein import system in different steps during the import.     

N-terminal domain of the dual-targeted pea glutathione reductase signal peptide controls organellar targeting efficiency

Import of nuclear-encoded proteins into mitochondria and chloroplasts is generally organelle specific and its specificity depends on the N-terminal signal peptide. Yet, a group of proteins known as dual-targeted proteins have a targeting peptide capable of leading the mature protein to both organelles. We have investigated the domain structure of the dual-targeted pea glutathione reductase (GR) signal peptide by using N-terminal truncations. A mutant of the GR precursor (pGR) starting with the second methionine residue of the targeting peptide, pGRdelta2-4, directed import into both organelles, negating the possibility that dual import was controlled by the nature of the N terminus. The deletion of the 30 N-terminal residues (pGRdelta2-30) inhibited import efficiency into chloroplasts substantially and almost completely into mitochondria, whereas the removal of only 16 N-terminal amino acid residues (pGRdelta2-16) resulted in the strongly stimulated mitochondrial import without significantly affecting chloroplast import. Furthermore, N-terminal truncations of the signal peptide (pGRdelta2-16 and pGRdelta2-30) greatly stimulated the mitochondrial processing activity measured with the isolated processing peptidase. These results suggest a domain structure for the dual-targeting peptide of pGR and the existence of domains controlling organellar import efficiency therein.     

Critical region in the extension peptide for the import of cytochrome P-450(SCC) precursor into mitochondria

In order to establish the role of the extension peptide of the precursor of P-450(SCC), a mitochondrial inner membrane protein, in the import into the organella, three deletion mutants of the precursor, in which the deletions were in the mature portion, were constructed. These mutant precursors were imported into mitochondria in vitro as efficiently as the original precursor, indicating that the extension peptide contains sufficient information for the import of the precursor into mitochondria. To investigate which portion of the extension peptide contains the mitochondrial targeting signal, various lengths of the amino-terminal portion of the extension peptide of P-450(SCC) precursor were fused to the mature portion of adrenodoxin. The fusion proteins consisting of 44 and 19 amino-terminal amino acids and mature adrenodoxin were imported into mitochondria, whereas those containing 14, 7, and 2 amino-terminal amino acid residues were not. The importance of the amino-terminal portion of the extension peptide was confirmed by the deletion from the amino-terminal end of a fusion protein consisting of the amino-terminal 44 amino acid residues of P-450(SCC) precursor and mature adrenodoxin, SCC44RAd. The amino-terminal deletions abolished the import of the fusion proteins into mitochondria. Substitution of all of the three basic amino acids, Arg(4), Arg(9), and Lys(14) in the extension peptide of SCC44RAd to Ser or Thr inhibited the binding of the fusion protein to mitochondria as well as its import.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein import into yeast mitochondria: the inner membrane import site protein ISP45 is the MPI1 gene product.

Protein import across both mitochondrial membranes is mediated by the cooperation of two distinct protein transport systems, one in the outer and the other in the inner membrane. Previously we described a 45 kDa yeast mitochondrial inner membrane protein (ISP45) that can be cross-linked to a partially translocated precursor protein (Scherer et al., 1992). We have now purified ISP45 to homogeneity and identified it as the product of the nuclear MPI1 gene. Identity of ISP45 with the MPI1 gene product was shown by microsequencing of three tryptic ISP45 peptides and by demonstrating that an antibody against an Mpi1p-beta-galactosidase fusion protein specifically recognizes ISP45. Antibodies monospecific for ISP45 inhibited protein import into right-side-out mitochondrial inner membrane vesicles, but not into intact mitochondria. On solubilizing mitochondria, ISP45 was rapidly converted to a 40 kDa proteolytic fragment unless mitochondria were first denatured with trichloroacetic acid. The combined genetic and biochemical evidence identifies ISP45/Mpi1p as a component of the protein import system of the yeast mitochondrial inner membrane.     

The cytosolic factor required for import of precursors of mitochondrial proteins into mitochondria

The mechanism of import of proteins into mitochondria was studied by using the peptide of the presequence of ornithine aminotransferase (the extrapeptide), which was chemically synthesized and is composed of 34 amino acids. When the extrapeptide was incubated with isolated mitochondria in the presence of a rabbit reticulocyte lysate at 25 degrees C, it was imported into the mitochondrial matrix, and the import depended on the inner membrane potential, but not added ATP. The import of several precursors of mitochondrial proteins was competitively inhibited by the presence of excess extrapeptide in the reaction system, indicating that the extrapeptide and mitochondrial proteins were imported by the same machinery. Import of the extrapeptide was significantly stimulated by addition of a rabbit reticulocyte lysate, and a component of the lysate (the cytosolic factor) stimulating import of the extrapeptide was purified about 20,000 times by successive column chromatography on DEAE-cellulose and aminopentyl-Sepharose 4B. The binding of the extrapeptide to liposomes composed of egg lecithin and partially purified receptor of the precursor of mitochondrial protein (Ono, H., and Tuboi, S., (1985) Biochem. Int. 10, 351-357) required the cytosolic factor when the concentration of the peptide was less than 1.5 X 10(-8) M, suggesting that the physiological binding of the precursors of mitochondrial proteins to the receptor is dependent on the cytosolic factor. The extrapeptide and the cytosolic factor were shown to form a complex. From these results, the mechanism of binding of the extrapeptide to the receptor of the mitochondrial outer membrane is suggested to be as follows: the peptide (the precursor of mitochondrial protein) and the cytosolic factor form a complex, and then the complex is recognized by and bound to the receptor.     

Translocation arrest by reversible folding of a precursor protein imported into mitochondria. A means to quantitate translocation contact sites

Passage of precursor proteins through translocation contact sites of mitochondria was investigated by studying the import of a fusion protein consisting of the NH2-terminal 167 amino acids of yeast cytochrome b2 precursor and the complete mouse dihydrofolate reductase. Isolated mitochondria of Neurospora crassa readily imported the fusion protein. In the presence of methotrexate import was halted and a stable intermediate spanning both mitochondrial membranes at translocation contact sites accumulated. The complete dihydrofolate reductase moiety in this intermediate was external to the outer membrane, and the 136 amino acid residues of the cytochrome b2 moiety remaining after cleavage by the matrix processing peptidase spanned both outer and inner membranes. Removal of methotrexate led to import of the intermediate retained at the contact site into the matrix. Thus unfolding at the surface of the outer mitochondrial membrane is a prerequisite for passage through translocation contact sites. The membrane-spanning intermediate was used to estimate the number of translocation sites. Saturation was reached at 70 pmol intermediate per milligram of mitochondrial protein. This amount of translocation intermediates was calculated to occupy approximately 1% of the total surface of the outer membrane. The morphometrically determined area of close contact between outer and inner membranes corresponded to approximately 7% of the total outer membrane surface. Accumulation of the intermediate inhibited the import of other precursor proteins suggesting that different precursor proteins are using common translocation contact sites. We conclude that the machinery for protein translocation into mitochondria is present at contact sites in limited number.     

Import into mitochondria of precursors containing hydrophobic passenger proteins: pretreatment of precursors with urea inhibits import

We have studied the import into isolated yeast mitochondria of three hydrophobic passenger proteins attached to the N-terminal cleavable presequence of mitochondrial ATPase subunit 9 from Neurospora crassa. One natural precursor (pN9) contained N. crassa subunit 9; two chimaeric precursors, N9L/Y8-1 and N9L/Y9-2, respectively contained yeast mitochondrial ATPase subunits 8 and 9. In the absence of urea, pN9 and N9L/Y8-1 are imported efficiently but N9L/Y9-2 is not imported. After pretreatment of precursors in 4 M urea, binding of pN9 to mitochondria is marginally affected while its import is substantially inhibited; the binding to mitochondria of chimaeric proteins, N9L/Y8-1 and N9L/Y9-2, is greatly enhanced but no import is observed. This behaviour of import precursors containing hydrophobic passenger proteins is contrasted with that of a hydrophilic chimaeric precursor pCOXIV-DHFR, whose binding and import are enhanced by pretreatment with a high concentration of urea (8 M). The import of N9L/Y8-1 is very sensitive to the presence of low concentrations of urea in the import reaction mixture, and is abolished above 0.5 M urea although precursor binding to mitochondria is increased. By contrast, neither the import nor binding of pCOXIV-DHFR is affected directly by urea up to 0.8 M. These deleterious effects of urea on import of the chimaeric precursors N9L/Y8-1 and N9L/Y9-2 are interpreted in terms of a non-productive binding of these precursors to mitochondria, brought about by exposure of their hydrophobic domains resulting from urea unfolding. The generalization that membrane translocation of mitochondrial import precursors is enhanced by their prior unfolding in urea thus does not apply in the case of these precursors containing hydrophobic passenger proteins.     

Protein import into mitochondria: ATP-dependent protein translocation activity in a submitochondrial fraction enriched in membrane contact sites and specific proteins

To identify the membrane regions through which yeast mitochondria import proteins from the cytoplasm, we have tagged these regions with two different partly translocated precursor proteins. One of these was bound to the mitochondrial surface of ATP-depleted mitochondria and could subsequently be chased into mitochondria upon addition of ATP. The other intermediate was irreversibly stuck across both mitochondrial membranes at protein import sites. Upon subfraction of the mitochondria, both intermediates cofractionated with membrane vesicles whose buoyant density was between that of inner and outer membranes. When these vesicles were prepared from mitochondria containing the chaseable intermediate, they internalized it upon addition of ATP. A non-hydrolyzable ATP analogue was inactive. This vesicle fraction contained closed, right-side-out inner membrane vesicles attached to leaky outer membrane vesicles. The vesicles contained the mitochondrial binding sites for cytoplasmic ribosomes and contained several mitochondrial proteins that were enriched relative to markers of inner or outer membranes. By immunoelectron microscopy, two of these proteins were concentrated at sites where mitochondrial inner and outer membranes are closely apposed. We conclude that these vesicles contain contact sites between the two mitochondrial membranes, that these sites are the entry point for proteins into mitochondria, and that the isolated vesicles are still translocation competent.     

Precursor proteins are transported into mitochondria in the absence of proteolytic cleavage of the additional sequences

Many nuclear-coded mitochondrial proteins are synthesized as larger precursor polypeptides that are proteolytically processed during import into the mitochondrion. This processing appears to be catalyzed by a soluble, metal-dependent protease localized in the mitochondrial matrix. In this report we employ an in vitro system to investigate the role of processing in protein import. Intact Neurospora crassa mitochondria were incubated with radiolabeled precursors in the presence of the chelator o-phenanthroline. Under these conditions, the processing of the precursors of the beta-subunit of F1-ATPase (F1 beta) and subunit 9 of the F0F1-ATPase was strongly inhibited. Protease-mapping studies indicated that import of the precursor proteins into the mitochondria continued in the absence of processing. Upon readdition of divalent metal to the treated mitochondria, the imported precursors were quantitatively converted to their mature forms. This processing of imported precursors occurred in the absence of a mitochondrial membrane potential and was extremely rapid even at 0 degrees C. This suggests that all or part of the polypeptide chain of the imported precursors had been translocated into the matrix location of the processing enzyme. Localization experiments suggested that the precursor to F1 beta is peripherally associated with the mitochondrial membrane while the precursor to subunit 9 appeared to be tightly bound to the membrane. We conclude that proteolytic processing is not necessary for the translocation of precursor proteins across mitochondrial membranes, but rather occurs subsequent to this event. On the basis of these and other results, a hypothetical pathway for the import of F1 beta and subunit 9 is proposed.     

A chimeric mitochondrial precursor protein with internal disulfide bridges blocks import of authentic precursors into mitochondria and allows quantitation of import sites

Bovine pancreatic trypsin inhibitor (which contains three intramolecular disulfide bridges) was chemically coupled to the COOH terminus of a purified artificial mitochondrial precursor protein. When the resulting chimeric precursor was presented to energized isolated yeast mitochondria, its trypsin inhibitor moiety prevented the protein from completely entering the organelle; the protein remained stuck across both mitochondrial membranes, with its NH2 terminus in the matrix and its trypsin inhibitor moiety still exposed on the mitochondrial surface. The incompletely imported protein appeared to "jam" mitochondrial protein import sites since it blocked import of three authentic mitochondrial precursor proteins; it did not collapse the potential across the mitochondrial inner membrane. Quantification of the inhibition indicated that each isolated mitochondrial particle contains between 10(2) and 10(3) protein import sites.     

Adriamycin disrupts phosphatidylserine import into the mitochondria of permeabilized CHO-K1 cells

The action of adriamycin (an inhibitor of precursor protein import into mitochondria) upon phosphatidylserine (PtdSer) import into mitochondria was examined in permeabilized CHO-K1 cells. The decarboxylation of nascent PtdSer to phosphatidylethanolamine was used as an indicator reaction for the lipid translocation process. Adriamycin was without effect upon new PtdSer synthesis but blocked the time- and translocation-dependent decarboxylation of this lipid at the mitochondrial inner membrane of permeabilized cells. The effect of adriamycin was concentration-dependent with an IC50 of 150 microM and was not due to direct inhibition of PtdSer decarboxylase. To determine at which level of PtdSer transport adriamycin was working, the adriamycin-treated permeabilized cells were incubated with 1-acyl-2-[N-(6-[(7-nitrobenz-2-oxa-1,3-diazo-4-yl)] aminocaproyl)]phosphatidyl[1'-14C] serine (NBD-Ptd[1'-14C]Ser), and its decarboxylation was determined. Since the NBD-Ptd[1'-14C]Ser freely partitions into all cell membranes, it can partition into the outer mitochondrial membrane in an ATP-independent fashion. The NBD-Ptd[1'-14C]Ser was readily decarboxylated in an ATP-independent manner in permeabilized cells. Adriamycin inhibited the decarboxylation of NBD-Ptd[1'-14C]Ser, thereby indicating that it can act upon lipid transport processes between the outer and inner mitochondrial membrane.