Functional Staging of ADP/ATP Carrier Translocation across the Outer Mitochondrial Membrane

Abstract

The ADP/ATP carrier (AAC) is the major representative of the inner membrane carrier proteins of mitochondria that are synthesized without cleavable presequences. The characterization of the import pathway of AAC into mitochondria has mainly depended on an operational staging system. Here, we introduce two approaches for analyzing the import of AAC, blue native electrophoresis and folding-induced translocation arrest, that allow a functional staging of AAC transport across the outer membrane. (i) Blue native electrophoresis permits a direct monitoring of the receptor stage of AAC and its chase into mitochondria. Binding to this stage requires the receptor protein Tom70 but not Tom37 or Tom20. (ii) A fusion protein between AAC and dihydrofolate reductase can be selectively arrested in the general import pore complex of the outer membrane by ligand induced folding of the passenger protein. Cross-linking demonstrates that the arrested preprotein is in close contact not only with several receptors and Tim10 but also with the channel protein Tom40, providing the first direct evidence that cleavable preproteins and carrier preproteins interact with the same outer membrane channel. The staging system presented here permits a molecular dissection of AAC transport across the outer mitochondrial membrane, relates it to functional units of the translocases, and indicates a coordinated and successive cooperation of distinct translocase subcomplexes during transfer of the preprotein. The ADP/ATP carrier (AAC) is the major representative of the inner membrane carrier proteins of mitochondria that are synthesized without cleavable presequences. The characterization of the import pathway of AAC into mitochondria has mainly depended on an operational staging system. Here, we introduce two approaches for analyzing the import of AAC, blue native electrophoresis and folding-induced translocation arrest, that allow a functional staging of AAC transport across the outer membrane. (i) Blue native electrophoresis permits a direct monitoring of the receptor stage of AAC and its chase into mitochondria. Binding to this stage requires the receptor protein Tom70 but not Tom37 or Tom20. (ii) A fusion protein between AAC and dihydrofolate reductase can be selectively arrested in the general import pore complex of the outer membrane by ligand induced folding of the passenger protein. Cross-linking demonstrates that the arrested preprotein is in close contact not only with several receptors and Tim10 but also with the channel protein Tom40, providing the first direct evidence that cleavable preproteins and carrier preproteins interact with the same outer membrane channel. The staging system presented here permits a molecular dissection of AAC transport across the outer mitochondrial membrane, relates it to functional units of the translocases, and indicates a coordinated and successive cooperation of distinct translocase subcomplexes during transfer of the preprotein. Although many mitochondrial preproteins are synthesized with cleavable amino-terminal targeting sequences (presequences), several groups of mitochondrial proteins, in particular membrane proteins, are synthesized without presequences (1Ryan K.R. Jensen R.E. Cell. 1995; 83: 517-519Abstract Full Text PDF PubMed Scopus (117) Google Scholar, 2Schatz G. Dobberstein B. Science. 1996; 271: 1519-1526Crossref PubMed Scopus (913) Google Scholar, 3Neupert W. Annu. Rev. Biochem. 1997; 66: 863-917Crossref PubMed Scopus (970) Google Scholar, 4Pfanner N. Craig E.A. Hönlinger A. Annu. Rev. Cell Dev. Biol. 1997; 13: 25-51Crossref PubMed Scopus (146) Google Scholar). Instead, these noncleavable preproteins possess internal targeting information. An example is the ADP/ATP carrier (AAC), 1The abbreviations AACADP/ATP carrierΔψmembrane potentialDHFRdihydrofolate reductaseEGSethylene glycol bis(succinimidylsuccinate)GIPgeneral import porePAGEpolyacrylamide gel electrophoresisTomtranslocase of outer mitochondrial membraneTimtranslocase of inner mitochondrial membraneMOPS4-morpholinepropanesulfonic acid 1The abbreviations AACADP/ATP carrierΔψmembrane potentialDHFRdihydrofolate reductaseEGSethylene glycol bis(succinimidylsuccinate)GIPgeneral import porePAGEpolyacrylamide gel electrophoresisTomtranslocase of outer mitochondrial membraneTimtranslocase of inner mitochondrial membraneMOPS4-morpholinepropanesulfonic acidwhich is a major representative of the large class of carrier proteins of the inner membrane. The current information shows that the import route of carrier proteins into mitochondria is largely distinct from that of cleavable preproteins. ADP/ATP carrier membrane potential dihydrofolate reductase ethylene glycol bis(succinimidylsuccinate) general import pore polyacrylamide gel electrophoresis translocase of outer mitochondrial membrane translocase of inner mitochondrial membrane 4-morpholinepropanesulfonic acid ADP/ATP carrier membrane potential dihydrofolate reductase ethylene glycol bis(succinimidylsuccinate) general import pore polyacrylamide gel electrophoresis translocase of outer mitochondrial membrane translocase of inner mitochondrial membrane 4-morpholinepropanesulfonic acid Presequence-carrying cleavable preproteins are typically recognized by the receptor Tom20, the 20-kDa subunit of the translocase of the outer membrane (5Söllner T. Griffiths G. Pfaller R. Pfanner N. Neupert W. Cell. 1989; 59: 1061-1070Abstract Full Text PDF PubMed Scopus (247) Google Scholar, 6Moczko M. Gärtner F. Pfanner N. FEBS Lett. 1993; 326: 251-254Crossref PubMed Scopus (45) Google Scholar, 7Moczko M. Ehmann B. Gärtner F. Hönlinger A. Schäfer E. Pfanner N. J. Biol. Chem. 1994; 269: 9045-9051Abstract Full Text PDF PubMed Google Scholar, 8Ramage L. Junne T. Hahne K. Lithgow T. Schatz G. EMBO J. 1993; 12: 4115-4123Crossref PubMed Scopus (179) Google Scholar). The preproteins are subsequently transferred to the general import pore (GIP) complex containing the second receptor Tom22 (9Kiebler M. Keil P. Schneider H. van der Klei I.J. Pfanner N. Neupert W. Cell. 1993; 74: 483-492Abstract Full Text PDF PubMed Scopus (154) Google Scholar, 10Lithgow T. Junne T. Suda K. Gratzer S. Schatz G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11973-11977Crossref PubMed Scopus (142) Google Scholar, 11Hönlinger A. Kübrich M. Moczko M. Gärtner F. Mallet L. Bussereau F. Eckerskorn C. Lottspeich F. Dietmeier K. Jacquet M. Pfanner N. Cell. Biol. 1995; PubMed Scopus Google Scholar, A. Neupert W. R. EMBO J. 1995; PubMed Scopus Google the transfer protein K. Hönlinger A. U. Eckerskorn C. Lottspeich F. Kübrich M. Pfanner N. 1997; PubMed Scopus Google and the import channel J. A. Schatz G. 1989; PubMed Scopus Google Scholar, K. K. Dietmeier K. F. R. Pfanner N. PubMed Scopus Google Scholar). a membrane potential the cleavable preproteins the translocase of the inner membrane the complex and are into the by the protein system (1Ryan K.R. Jensen R.E. Cell. 1995; 83: 517-519Abstract Full Text PDF PubMed Scopus (117) Google Scholar, 2Schatz G. Dobberstein B. Science. 1996; 271: 1519-1526Crossref PubMed Scopus (913) Google Scholar, 3Neupert W. Annu. Rev. Biochem. 1997; 66: 863-917Crossref PubMed Scopus (970) Google Scholar, N. M. Biol. 1997; Full Text Full Text PDF PubMed Google Scholar). the the mitochondrial the presequences. the import of the inner membrane the mitochondrial transport N. Biol. Full Text Full Text PDF PubMed Google Scholar). The carrier preproteins are the receptor Tom70 A. Griffiths G. H. H. Schatz G. EMBO J. PubMed Scopus Google Scholar, T. Pfaller R. Griffiths G. Pfanner N. Neupert W. Cell. Full Text PDF PubMed Scopus Google in cooperation with Tom37 S. Lithgow T. R.E. E. F. Junne T. Schatz G. M. J. Cell Biol. 1995; PubMed Scopus Google can also be in the of T. M. Dietmeier K. Pfaller R. M. Neupert W. Pfanner N. J. Cell Biol. PubMed Scopus Google Scholar, J. Dietmeier K. Pfanner N. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The transport of carrier preproteins and cleavable preproteins of the complex K. Hönlinger A. U. Eckerskorn C. Lottspeich F. Kübrich M. Pfanner N. 1997; PubMed Scopus Google Scholar). the outer membrane, the carrier import route from that of cleavable preproteins M. J. W. Pfanner N. Hönlinger A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). preproteins are by the complex in the S. W. K. E. L. Junne T. Schatz G. K. EMBO J. PubMed Scopus Google Scholar, A. M. C. Lottspeich F. Neupert W. M. EMBO J. PubMed Scopus Google and are transferred to the carrier translocase of the inner membrane, of the membrane protein and the proteins and C. B. Neupert W. M. 1996; PubMed Scopus Google Scholar, C. M. H. Neupert W. M. PubMed Scopus Google Scholar, J. M. Jensen R.E. J. Cell Biol. 1997; Scopus Google Scholar, E. K. K. Schatz G. Science. PubMed Scopus (247) Google Scholar). into the inner membrane requires a and is by to the of the carrier protein M. Neupert W. J. Biol. Chem. Full Text PDF PubMed Google Scholar, A. Hönlinger A. A. Pfanner N. F. Biochem. J. PubMed Scopus Google Scholar). on the import pathway of carrier preproteins of a system of import that by the of AAC translocation in mitochondria M. J. W. Pfanner N. Hönlinger A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, N. Neupert W. J. Biol. Chem. Full Text PDF PubMed Google Scholar, N. M. Neupert W. Cell. Full Text PDF PubMed Scopus Google Scholar). of preproteins by of the and of the in the in The also stage AAC to stage AAC to the mitochondrial stage AAC largely a of the inner membrane stage AAC into the inner stage AAC and operational staging system the import of the and The of and to import is from is that the only of a carrier import pathway and that the this we on the transport route of AAC across the outer membrane that several is of the receptors and are in the of carrier preproteins the mitochondrial the not be it is the receptor Tom22 into direct contact with carrier preproteins or of an in the of Tom22 to mitochondria an on carrier import (9Kiebler M. Keil P. Schneider H. van der Klei I.J. Pfanner N. Neupert W. Cell. 1993; 74: 483-492Abstract Full Text PDF PubMed Scopus (154) Google and a of the mitochondrial of Tom22 carrier import A. Neupert W. R. EMBO J. 1995; PubMed Scopus Google Scholar). the in with receptors an of carrier preproteins with Tom70 and Tom20, but not with Tom22 J. Dietmeier K. Pfanner N. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). Although it is that the carrier preproteins are the same outer membrane channel cleavable direct evidence for a contact between and a carrier preprotein has not to the to a carrier preprotein in the outer membrane import this we two approaches for a functional characterization of the carrier Blue native electrophoresis a direct monitoring of the receptor that only but not Tom37 or Tom20, for the A fusion of AAC with dihydrofolate reductase a protein that be selectively and arrested in the complex by folding of the arrested preprotein the first evidence for a close of AAC with Tom22 and the operational staging with the functional staging of AAC permits a molecular dissection of carrier transport across the outer mitochondrial membrane. in this are in from in and G. Schatz G. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. B. Neupert W. Cell. Full Text PDF PubMed Scopus (179) Google in and in preproteins by in and in in the of T. J. Pfanner N. Cell Biol. Scopus Google Scholar, A. 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Chem. 1994; 269: 9045-9051Abstract Full Text PDF PubMed Google in a the in import of mitochondria in in the of and also to a of The mitochondrial membrane potential by the of and to import preproteins of import with mitochondria of to with on for The by the of and for an and in mitochondrial in the and by or blue the of a stage AAC AAC in and mitochondria in import and containing and for to the of preprotein to the mitochondria. the of a stage AAC AAC in containing to mitochondria of a membrane potential by the of the of AAC mitochondria and of to 1995; PubMed Scopus Google to import for of the membrane potential by of and of mitochondrial in an of with or without an system and in the of or for a to The of J. Biol. Chem. Full Text PDF PubMed Google a by with and the and containing and The of by the and and The with and with to with and for the AAC or and for the The on and in a with the with The the AAC and the acid and Blue native polyacrylamide gel electrophoresis H. G. Biochem. PubMed Scopus Google Scholar, H. G. Biochem. 1994; PubMed Scopus Google Scholar, F. U. H. B. M. J. Pfanner N. EMBO J. 1997; PubMed Scopus Google Scholar, J. H. Hönlinger A. Pfanner N. Cell. Biol. PubMed Scopus Google Scholar). mitochondrial of in of J. M. J. Biochem. 1995; PubMed Scopus Google Scholar). a of Blue to the The on a or polyacrylamide gel a of gel J. H. Hönlinger A. Pfanner N. Cell. Biol. PubMed Scopus Google Scholar). the gel first in by transfer the E. A Scholar). the of the between a to the and a of the and into The by and to the S. and to the of the by on for of and R. R.E. K. in Scholar, C. to and molecular Scholar). containing AAC or a and with ethylene glycol bis(succinimidylsuccinate) A. Gärtner F. Hönlinger A. Kübrich M. Pfanner N. 1995; PubMed Scopus Google Scholar). mitochondria in to in and to A. Gärtner F. Hönlinger A. Kübrich M. Pfanner N. 1995; PubMed Scopus Google Scholar). on a gel H. G. Biochem. PubMed Scopus Google to an from Tom37 by the to T. H. C. R. Cell. Full Text PDF PubMed Scopus (45) Google and into and of mitochondrial proteins, mitochondrial in of or and on for to and into and A. U. A. Eckerskorn C. Lottspeich F. Dietmeier K. Pfanner N. EMBO J. 1996; PubMed Scopus Google Scholar). to and to PubMed Scopus Google Scholar). to E. A and the of proteins, the gel or membrane to to The preprotein of AAC synthesized in in in the of from of AAC stage and mitochondria of and the inner membrane potential The with the mitochondria The mitochondria with the and to blue native electrophoresis and F. U. H. B. M. J. Pfanner N. EMBO J. 1997; PubMed Scopus Google Scholar, J. H. Hönlinger A. Pfanner N. Cell. Biol. PubMed Scopus Google Scholar, H. J. Pfanner N. Chem. 1996; PubMed Scopus Google Scholar). AAC stage and a the of the mitochondria with the that the AAC the mitochondrial of AAC stage the membrane potential a and of native protein by blue native AAC in a in the molecular also of mitochondria with the of a stage N. Neupert W. J. Biol. Chem. Full Text PDF PubMed Google Scholar, N. M. Neupert W. Cell. Full Text PDF PubMed Scopus Google Scholar). in the of and a AAC into the inner membrane and to the stage blue native the in AAC by and into a from the in AAC that by A. Hönlinger A. A. Pfanner N. F. Biochem. J. PubMed Scopus Google Scholar, H. J. Pfanner N. Chem. 1996; PubMed Scopus Google Scholar). the import and by blue native electrophoresis translocation be to import and is only and be by blue native electrophoresis N. Neupert W. J. Biol. Chem. Full Text PDF PubMed Google AAC stage and mitochondria The into the and the preprotein stage the to the the the preprotein to the molecular of the stage AAC also stage but in the of a and to in the chase of AAC from stage to stage AAC first stage the mitochondria and into two the by of the in the second the a by the in the second and of an system. the stage to the stage A of AAC the stage the second a the first in the of and a the second in the of a an system in the also the stage to the stage that also this stage a the of in and not to AAC import and mitochondrial that blue native electrophoresis permits a direct monitoring of the transport of AAC from stage to stage and to the stage and translocation are into mitochondria. receptor for the of AAC stage we mitochondria from T. M. Dietmeier K. Pfaller R. M. Neupert W. Pfanner N. J. Cell Biol. PubMed Scopus Google or M. Ehmann B. Gärtner F. Hönlinger A. Schäfer E. Pfanner N. J. Biol. 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The of the stage mitochondria indicates that Tom22 is not for of the receptor stage of Gratzer S. Lithgow T. R.E. E. F. Junne T. Schatz G. M. J. Cell Biol. 1995; PubMed Scopus Google Tom37 a subunit of the a and and an of Tom37 in the import of Tom37 is to to it is on the mitochondrial S. Lithgow T. R.E. E. F. Junne T. Schatz G. M. J. Cell Biol. 1995; PubMed Scopus Google Scholar). information on the of Tom37 with the outer membrane and its for import of AAC is of the of a that a to the of Tom37 to to of a Tom37 and this to of (i) Tom37 of mitochondria with to the mitochondria to proteins and membrane proteins S. H. J. Cell Biol. PubMed Scopus Google Tom37 from the a an protein that is only with the inner membrane, Tom70 and Tom40, proteins in the membrane N. Craig E.A. Hönlinger A. Annu. Rev. Cell Dev. Biol. 1997; 13: 25-51Crossref PubMed Scopus (146) Google Scholar). Tom37 is the first protein that a membrane protein. (ii) Blue native electrophoresis of mitochondria by in the second the into distinct subcomplexes in Tom70 and the complex containing Tom40, and the proteins K. Hönlinger A. U. Eckerskorn C. Lottspeich F. Kübrich M. Pfanner N. 1997; PubMed Scopus Google Scholar, J. H. Hönlinger A. Pfanner N. Cell. Biol. PubMed Scopus Google Tom37 in a in blue native electrophoresis and distinct from the subcomplexes the that Tom37 with Tom70 in of AAC S. Lithgow T. R.E. E. F. Junne T. Schatz G. M. J. Cell Biol. 1995; PubMed Scopus Google we a with a of the The are S. Lithgow T. R.E. E. F. Junne T. Schatz G. M. J. Cell Biol. 1995; PubMed Scopus Google Scholar). The of Tom37 by with the mitochondria for of the stage of AAC by blue native electrophoresis these that Tom37 is not an membrane protein but is with the outer membrane an with Tom37 is not for the of AAC to Tom70 but a stage of AAC that the of the stage of AAC requires Tom37 and Tom20, and also are not for the of the AAC preprotein to mitochondria. Although the that the stage of AAC is with the M. J. W. Pfanner N. Hönlinger A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google R. J. Pfanner N. Neupert W. J. Cell Biol. PubMed Scopus Google Scholar, M. Pfaller R. T. Griffiths G. H. Pfanner N. Neupert W. PubMed Scopus Google stage on blue native electrophoresis a molecular and the and the complex J. H. Hönlinger A. Pfanner N. Cell. Biol. PubMed Scopus Google this demonstrates that the stage is not with of the to by native The of a stage is only the of the inner membrane, that is by of the that these the major of an AAC is transferred into the that the with the complex is to an AAC that be arrested in the we a fusion protein between AAC and the synthesized in with mitochondria and to a in the of a in the of a of to the of a stage to the inner membrane in a we blue native in the of a a that AAC a of The ligand the folding of and import of cleavable preproteins that passenger protein M. Schatz G. PubMed Scopus Google Scholar, J. B. U. Neupert W. J. Cell Biol. 1989; PubMed Scopus Google Scholar). the of The the import of AAC not by of the of and a a stage but a molecular the AAC stage the of and in a distinct molecular of The of this of a that the not the inner membrane to of and A of in and mitochondria a in the mitochondria that Tom70 AAC T. Pfaller R. Griffiths G. Pfanner N. Neupert W. Cell. 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M. 1996; PubMed Scopus Google and only of The fusion protein across the outer membrane in the of and of several that recognized by Tom70 a major Tom22 and the also to Tim10 that into the demonstrates that the arrested preprotein the complex and the first direct evidence that AAC in is in close to Tom22 and two approaches to the translocation of the ADP/ATP carrier across the outer mitochondrial membrane. the operational staging of AAC import also and the import to the of the translocase the of blue native the contact of AAC with the mitochondrial the import can be The of this receptor stage selectively requires the receptor Tom70 proteins, Tom20, and large to the and to import Tom37 has to a receptor subunit with Tom70 S. Lithgow T. R.E. E. F. Junne T. Schatz G. M. J. 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Biochem. 1997; 66: 863-917Crossref PubMed Scopus (970) Google Scholar, 4Pfanner N. Craig E.A. Hönlinger A. Annu. Rev. Cell Dev. Biol. 1997; 13: 25-51Crossref PubMed Scopus (146) Google Scholar, T. Schatz G. Biochem. Sci. 1995; Full Text PDF PubMed Scopus Google only Tom70 is for the first of in mitochondria AAC can this and and Tom22 receptors in this the of AAC and import is T. M. Dietmeier K. Pfaller R. M. Neupert W. Pfanner N. J. Cell Biol. PubMed Scopus Google The receptor Tom70 in a native AAC can this be The of AAC, a is not a but is in with PubMed Scopus Google Scholar, R. Neupert W. J. Biochem. PubMed Scopus Google Scholar, N. K. Suda K. M. Schatz G. Lithgow T. 1995; PubMed Scopus Google Scholar, T. S. Schatz G. K. EMBO J. 1997; PubMed Scopus Google Scholar, T. S. C. R. Schatz G. K. EMBO J. PubMed Scopus Google Scholar). of a preprotein from the mitochondrial import that preproteins to has to N. K. Suda K. M. Schatz G. Lithgow T. 1995; PubMed Scopus Google Scholar, T. S. 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AAC stage is largely to these the operational staging system in providing direct evidence for an of AAC with the import channel of AAC to import stage and stage to the major Tom70 stage and stage Although this of AAC to be the first for a direct contact of the two proteins, the the of the to a transport of AAC in the we a fusion protein between AAC and the that selectively arrested across the outer membrane by the with the ligand The fusion protein the carrier import targeting of a stage and membrane import and in the of The of in the complex is of the or of the inner membrane potential the first that selectively the channel in a a of preproteins in the channel requires the in the channel of the inner membrane translocation contact F. U. H. B. M. J. Pfanner N. EMBO J. 1997; PubMed Scopus Google Scholar, M. Neupert W. Cell. Full Text PDF PubMed Scopus Google Scholar, M. S. W. Schatz G. EMBO J. 1995; PubMed Scopus Google Scholar). Cross-linking demonstrates that the is in close to several proteins Tom20, and to providing the first direct evidence that Tom22 and are in the of AAC in that the channel is for import of cleavable preproteins and carrier preproteins and the of a direct of Tom22 in the import of carrier preproteins. Although carrier preproteins synthesized in not interact with the of Tom22 J. Dietmeier K. Pfanner N. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google from a carrier preprotein to to this and N. that of carrier preproteins can interact with to the that Tom22 is not in the first of a carrier preprotein. the by the carrier preproteins are during the translocation and can for to that the of arrested to a large of proteins and to the protein Tim10 to an of the import Although the of the preprotein the mitochondrial is by the the translocation across the outer membrane a coordinated cooperation of several subcomplexes in the membrane and on the and the receptor subcomplexes of Tom70 and Tom20, the complex containing Tom22 and the import channel Tom40, and the complex in the of the on the and characterization of import K. Hönlinger A. U. Eckerskorn C. Lottspeich F. Kübrich M. Pfanner N. 1997; PubMed Scopus Google Scholar, A. Griffiths G. H. H. Schatz G. EMBO J. PubMed Scopus Google Scholar, T. Pfaller R. Griffiths G. Pfanner N. Neupert W. Cell. 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EMBO J. 1997; PubMed Scopus Google we a in the import of carrier preproteins is by a successive of functional units of the import in AAC to to the receptor an the are and Tom70 the complex and the AAC to the import channel in a Tom20, and on and on the The complex with AAC the carrier translocase of the inner membrane, on the and the is the of of AAC into the inner membrane by the carrier translocase and the to the A of this inner membrane on the of a and translocation in the inner membrane carrier translocase it has with the outer membrane K. A M. T. J. and S. for and