Aminotransferase Variants as Probes for the Role of the N-terminal Region of a Mature Protein in Mitochondrial Precursor Import and Processing

Abstract

Of the two homologous isozymes of aspartate aminotransferase that are also nearly identical in their folded structures, only the mitochondrial form (mAAT) is synthesized as a precursor (pmAAT). After its in vitro synthesis in rabbit reticulocyte lysate, it can also be efficiently imported into isolated rat liver mitochondria, where it is processed to its native form by removal of the N-terminal presequence. The homologous cytosolic isoenzyme (cAAT) is not imported into mitochondria, even after fusion of the mitochondrial presequence from pmAAT to its N-terminal end. Substitution of the 30-residue N-terminal peptide of the mature portion of pmAAT with the corresponding sequence from the homologous, import-incompetent cytosolic isozyme (pcmAAT) does not prevent import but reduces substantially its processing in the matrix. A detectable amount of the pcmAAT chimera is found associated with the inner mitochondrial membrane. Single and double substitution mutants of Trp-5 and Trp-6 at the N-terminal end of the mature protein are imported into mitochondria with efficiency similar to that of wild type. However, replacement of Trp-5 with proline, or of both tryptophans with either alanine (W5A/W6A mutant) or valine and alanine (W5V/W6A mutant), allows import but interferes with the correct processing of the imported protein despite the presence of an intact cleavage site for the processing peptidase. Similar cleavage results were obtained using newly synthesized proteins and mitochondrial matrix extracts. These results indicate that translocation and processing for a precursor are independent events and that sequences C-terminal to the cleavage site are indeed important for the correct maturation of pmAAT in the matrix, probably because of their contribution to the conformation and flexibility of the peptide region surrounding the cleavage site required for efficient processing. The same region from the mature component of the protein may play a role in the commitment of the passenger protein to complete its translocation into the matrix. Of the two homologous isozymes of aspartate aminotransferase that are also nearly identical in their folded structures, only the mitochondrial form (mAAT) is synthesized as a precursor (pmAAT). After its in vitro synthesis in rabbit reticulocyte lysate, it can also be efficiently imported into isolated rat liver mitochondria, where it is processed to its native form by removal of the N-terminal presequence. The homologous cytosolic isoenzyme (cAAT) is not imported into mitochondria, even after fusion of the mitochondrial presequence from pmAAT to its N-terminal end. Substitution of the 30-residue N-terminal peptide of the mature portion of pmAAT with the corresponding sequence from the homologous, import-incompetent cytosolic isozyme (pcmAAT) does not prevent import but reduces substantially its processing in the matrix. A detectable amount of the pcmAAT chimera is found associated with the inner mitochondrial membrane. Single and double substitution mutants of Trp-5 and Trp-6 at the N-terminal end of the mature protein are imported into mitochondria with efficiency similar to that of wild type. However, replacement of Trp-5 with proline, or of both tryptophans with either alanine (W5A/W6A mutant) or valine and alanine (W5V/W6A mutant), allows import but interferes with the correct processing of the imported protein despite the presence of an intact cleavage site for the processing peptidase. Similar cleavage results were obtained using newly synthesized proteins and mitochondrial matrix extracts. These results indicate that translocation and processing for a precursor are independent events and that sequences C-terminal to the cleavage site are indeed important for the correct maturation of pmAAT in the matrix, probably because of their contribution to the conformation and flexibility of the peptide region surrounding the cleavage site required for efficient processing. The same region from the mature component of the protein may play a role in the commitment of the passenger protein to complete its translocation into the matrix. Over 90% of mitochondrial proteins are encoded by the nuclear DNA, synthesized in the cytoplasm as precursors with an N-terminal targeting sequence, and imported into the organelle. For proteins destined to the mitochondrial matrix such as mitochondrial aspartate aminotransferase (mAAT), 1The abbreviations used are: mAAT, mitochondrial aspartate aminotransferase; cAAT, cytosolic aspartate aminotransferase; pmAAT, precursor to aspartate aminotransferase; pcAAT, precursor chimera to cytosolic aspartate aminotransferase; pcmAAT, chimeric precursor; Δψ, membrane potential; mt-hsp70, mitochondrial hsp70; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride; RRL, rabbit reticulocyte lysate; TPCK,l-1-tosylamido-2-phenylethyl chloromethylketone; HEPPS, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid. the translocation process involves the initial binding of the precursor form of the protein to the surface of mitochondria, its transport across both mitochondrial membranes mediated by the translocation apparatus, and the final processing and folding of the protein in the matrix (1Pfanner N. Douglas M.G. Endo T. Hoogenrad N.J. Jensen R. Meijer M. Neupert W. Schatz G. Schmitz U.K. Shore G.S. Trends Biochem. Sci. 1996; 21: 51-52Abstract Full G. 1996; to in the form of in the matrix and a membrane across the inner membrane (1Pfanner N. Douglas M.G. Endo T. Hoogenrad N.J. Jensen R. Meijer M. Neupert W. Schatz G. Schmitz U.K. Shore G.S. Trends Biochem. Sci. 1996; 21: 51-52Abstract Full G. 1996; a conformation of the imported protein to be also for efficient translocation M. Schatz G. Full M. Full The to be to the of mitochondrial Neupert W. Trends Biochem. Sci. Full that the of the as it into the matrix Schatz G. Neupert W. binding the of the process by as a in with of the protein import in the inner membrane M. Neupert W. W. N. 1996; and mitochondrial W. N. in the matrix, the Neupert W. Trends Biochem. Sci. Full Schatz G. and mitochondrial Schatz G. Sci. N. M. the final folding of at proteins into their the translocation the presequence of matrix precursors is to the found mitochondrial precursors are processed in a by the mitochondrial processing M. Neupert W. G. M. Neupert W. Full M. Sci. an cleavage by mitochondrial Sci. the of the mitochondrial processing G. M. Neupert W. Full of processing in the presence of such as and G. M. Neupert W. Full G. M. W. Schatz G. Full are for the precursor of mitochondrial proteins M. Neupert W. the of are in sequence such as the presence of and as as the to form in G. are to be important for the targeting of the Schatz G. Full the for the cleavage of the presequence mitochondria at the with the of the mature protein are cleavage and of of sequences from G. Sci. The contribution of sequences C-terminal to the cleavage site N-terminal end of mature to the import and correct processing of mitochondrial precursors is of sequences of the processing site of mitochondrial were to import correct processing M. Shore of mature sequences from the maturation site found to prevent processing of the mitochondria Douglas M.G. Full using either T. T. Full Full or chimeric proteins M. Full Full M. M. Neupert W. Full indicate that the in C-terminal to the processing site may a role in the of processing. The of the sequences at the processing of mitochondrial precursors to the that the conformation of the presequence peptide and the flexibility in the region surrounding the cleavage site may be important for the and maturation of precursors in the matrix T. T. Full T. T. T. Full Full M. Full Full it is not the of the imported protein or its with in the matrix also to the of the aminotransferase in in two homologous sequence in the (cAAT) and the in the mitochondrial matrix are encoded in the nuclear and synthesized in the only the mitochondrial form is for translocation into is synthesized as a precursor with a at its N-terminal end (pmAAT). N-terminal also of the of the two The presequence of pmAAT from rat liver is and two of in from the maturation site M. Biochem. pmAAT to the of mitochondrial precursors the cleavage G. M. Full Full where is and is for processing of imported pmAAT to in a and is a the of the of at the N-terminal end of the mature portion of the processing of the imported protein as as the translocation of the protein to the matrix The for rat liver pmAAT in M. Full used as the for The region of pmAAT from by and into the The to the by the native pmAAT, the in and are by at either or both of were by using from to in in the in the of the were to the for for for for for and for and for for and and for and for were used to and the were by using the from The the pcmAAT from two isolated pmAAT M. Full and M. Full using as M. Full Full were by in vitro using the from and either for the or for the The obtained were in rabbit reticulocyte for at using of and as the as M. Full M. Full were by and to a final of were isolated from rat liver by a N. Sci. in a The import as M. Full M. Full precursor protein in were with of isolated mitochondria in for at After mitochondria were by at for at The and with an of the protein that does not to The mitochondria were by in of by and in an of or mitochondrial either with protein to with for that or the same after mitochondria with protein that is were by at an to a The of the mature in the mitochondria to that of the precursor mature in the complete import used to the of precursor protein of the imported protein by mitochondria with for at import of the pmAAT as to the of for pmAAT import and the of with the import with and of the associated with mitochondria as precursor not After by mitochondrial were with for to the protein associated with mitochondria but not by a of and phenylmethylsulfonyl to mitochondria of the membrane by were to by with of After for were by for at and in the same the and and were to the the amount of protein that into the matrix, were for or with of and by of with The of the by the of the matrix into the using a for into their and membrane were in and in a The and the in a for at to the matrix from the membrane matrix were from rat liver mitochondria by in by of the by of and as in the presence of and and and not the processing a final of of pmAAT, of mitochondrial matrix of of and of were at for or were by the of an of and were by and The amount of processing from the of the with a and is as the of precursor that to mature to the amount of protein with the using the U.K. in polyacrylamide in After and the the of the using a as M. Full Full M. Full were synthesized at the in the of of the of and and were obtained from and from the N-terminal end of mature isolated from rat liver R. T. Biochem. and the complete of rat precursor pmAAT M. Biochem. indicate that processing of pmAAT in by cleavage and of the precursor The sequence of the cleavage site and its region is identical in from Full R. N. Full but from found in mitochondrial matrix proteins Sci. the contribution of sequences from the cleavage site to both efficient import and correct processing of rat liver pmAAT, fusion protein and substitution mutants of the fusion pcAAT, the presequence of pmAAT in to the of the homologous cytosolic substantially from in its N-terminal M. Full The pcmAAT, both the mitochondrial presequence and from the of the cytosolic to the for of the mitochondrial M. Full Full a of and double substitution mutants targeting the initial of the N-terminal of the mature form of the is to be for protein folding and of either or both of the found in and of the N-terminal peptide and from the cleavage were with a of or even mutants the wild cleavage of and double substitution mutants at and in the of the processing of mature protein associated with mitochondria to the amount of precursor mature protein in the complete import in of imported protein that is to in the presence of of imported protein mature or of mature protein associated with mitochondria to the amount of precursor mature protein in the complete import of imported protein that is to in the presence of of imported protein mature or in a of import of pmAAT and pcmAAT are in T. M. Full synthesized pmAAT efficiently imported into isolated rat liver mitochondria and processed to a The amount of protein with mitochondria to be as it by of the processed mature protein intact The of the mature protein in the mitochondria with to that of the precursor mature in the complete import used to the of precursor protein imported from to with in the presence of the mitochondrial membranes and allows of the to the that of the imported protein folded for folding the by folded M. and of R. The precursor form pmAAT is also to in to a conformation similar to the of the mature protein M. Full T. M. Full only the presequence peptide is to by of M. Full after at and in the presequence peptide M. T. N. Sci. the a protein of the pmAAT presequence to the of the cytosolic can even in M. Full not only not imported into isolated mitochondria, it not even to the not for pcmAAT, a chimera in only the N-terminal of into the N-terminal sequence of pmAAT with isolated mitochondria, a amount of protein to mitochondria A of to with the complete of the wild protein associated with that pcmAAT to a but of it The amount of associated with mitochondria but of the imported protein in of mitochondria with in the presence of that only of the imported pcmAAT for the wild and in the folded precursor only the presequence is to M. Full the amount of chimera as a mature to the of the presequence to The results indicate that C-terminal to the peptide by the mitochondrial processing in pmAAT, in the N-terminal region of the mature be important for processing. However, the sequence in pcmAAT from to the in the for found at both the and of maturation of mitochondrial precursors Sci. it is not of the in the sequence from the processing site in pcmAAT is for the of processing. to a of mutants the wild cleavage site sequence, but with or double in and of the N-terminal peptide of mature that to and C-terminal from the processing site in pmAAT the of the of mature is as to sequence with the cytosolic isozyme of two at the N-terminal M. N. R. Full These two are because are in mitochondrial for sequences are the in the cytosolic either or but not M. Biochem. M. N. R. Full are also to to the of the of the native The of in by the in the region of the The of the proteins by in vitro of the corresponding were identical to that of the wild The amount of protein synthesized also to the wild not as mature or were in the of mutants are also in the of wild pmAAT and probably from of at proteins were in with isolated rat liver mitochondria after of were imported with the same efficiency as from the amount of protein that associated with the mitochondrial However, not of were processed import into of in results in the of a amount of precursor associated with mitochondria and to by However, the amount of protein for pcmAAT and and of the protein associated with The in the precursor and processed is identical to that found with the precursor and mature of the wild protein that the processing of the probably at the cleavage of the double and can be into mitochondria, a processing in for the the associated with mitochondria is as in precursor and mature results were obtained for the not that precursors may be processed at an site the presequence. the of the cleavage not the of the with that of an that obtained and by wild pmAAT with of M. T. N. Sci. that precursor by after at the processing of the and mutants probably in the of is important to that the mutants to be in an folded of the of mitochondria with in the presence of to the of the pmAAT mutants either or in or were both imported into isolated mitochondria and processed to a with similar to that of wild processed protein for are in to the of of the efficiency of the processing from the of protein associated with mitochondria to that of wild precursor of mutants after translocation into the matrix, a of mutants and efficiently The results in and indicate that a of pcmAAT and mitochondria as after import in and of the associated with by of the membrane by mitochondria and the inner membrane intact but the of the The precursor and processed after of Similar of mitochondria that imported either wild or a pmAAT that is processed that also of the proteins is with the The results indicate that the proteins the membrane and are associated with of the into its membrane and matrix by of and and that of imported wild or is in the membrane and of the pcmAAT with the membrane and in the matrix the of in the membrane and in the The found in the membrane both precursor and in the of pcmAAT, a amount of an is by of the protein by is by the that of a of the initial of mitochondria and the of the of and also the of the may also be the of the in of precursor and processed after of the results the presence of a amount of imported pcmAAT in the membrane The protein with the matrix a similar and in and a of pmAAT pcmAAT, associated with the inner membrane after translocation into However, does not the efficiency of processing of the precursor in the matrix where it to the processing peptidase. the wild protein and a pmAAT that to be processed A and were found in the matrix The two double mutants (W5A/W6A and that are processed to in the matrix not The of of that in the for the wild and for the These to folded after translocation are found in the matrix. the folded conformation of at of the imported pcmAAT in be the for its binding to the inner membrane. both and pcmAAT are found associated with the membrane the of a of pcmAAT in the inner membrane is not the of the presence of the is to for binding to membranes M. Full M. Biochem. 1996; by the that wild pmAAT, in mitochondria with the processing found in the matrix not These results indicate that translocation and processing of pmAAT are independent the of a of pcmAAT to a in the inner membrane is not the of processing. of wild and pmAAT also by the of precursor to mature form of of in vitro with a matrix as a of processing G. M. Full Full of wild pmAAT in with a matrix for at results in of precursor to mature of is to the of processing using and mitochondrial processing G. T. T. T. Full M. Full Full that the synthesized pmAAT as for processing is by a of to to be in the gel not it is that the of a of precursor into mature results from the of matrix of an folded by be to be at as as that with the processing with and to the by matrix the processing of pmAAT in the presence of and that the is mediated by a in the matrix a mitochondrial processing is G. M. Neupert W. Full G. M. W. Schatz G. Full processing the to its folding into a conformation M. Full the processing site is not to the processing the a of processing is not to the of the presequence the presequence from the folded by after at peptide from the processing site M. Full identical processing for the chimera pcmAAT the N-terminal from the cytosolic isozyme or for However, the double substitution is processed to an in intact mitochondria, also to of similar precursor and mature by the matrix also in the presence of and that it is mediated by the same that the wild A corresponding in to the mature form of pmAAT in the the is in the and is not by with the matrix is to from at an in the mature portion of the The presence of in the of wild pmAAT and but not in that of pcmAAT N-terminal sequence of two in and in the same region of the sequence as in the results obtained for the processing in vitro of pmAAT by matrix with intact mitochondria, the efficiency of processing is with the matrix extracts. the processing of to be processed in intact of the final in the translocation of mitochondrial precursors into the matrix is the removal of the N-terminal presequence peptide that the protein to the organelle. are import of wild rat liver pmAAT into isolated mitochondria precursor is probably processed in a cleavage mediated by the mitochondrial processing peptidase. of the of the of processing to be their to with at the of precursor The complete of the sequences at cleavage G. Sci. to the that of be for the of the cleavage M. Shore T. T. T. Full However, the of the is by the of the presequence or the presequence a of the N-terminal mature sequence Full in However, the of the cleavage region or its to the may be not by the sequences surrounding the peptide but by the conformation of the protein at the of in be by its with mitochondrial results indicate that the folding of the precursor the efficiency of the of the processing of pmAAT in vitro in by a matrix that the is processed to the mature a of the newly synthesized pmAAT can be processed to a form by matrix These results that processing of pmAAT in intact mitochondria probably at the of folding of the imported Similar also for mitochondrial M. Full Full matrix processing in their for cleavage of peptide in of native is not by the of the or the of the peptide but also by the flexibility of the region to the cleavage site and its to a conformation at the site Sci. of the cleavage region can be the in of the of the N-terminal region of in the folded a for the of matrix to process the folded the native folded the N-terminal is of the with of the mature sequence an that with the M.G. Trp-5 and Trp-6 in the surface of the and are to the the processing site be the surface of the in the flexibility or of the from the cleavage site as a of with the processing peptidase. is to that the peptide after at peptide from the pmAAT maturation is by of M. Full that in the folded protein the N-terminal end is the surface of the folded to a conformation of the precursor a of of the region surrounding the cleavage site as for efficient processing. the presequence peptide the presence of both the cleavage site and at the N-terminal region of the presequence to be important for processing T. T. Full Sci. A of flexibility of the presequence peptide also to be Full M. Full Full that the region C-terminal to the processing site is also important for and processing by the liver matrix peptidase. the sequence of the of the mature of pmAAT with processing. the chimeric precursor pcmAAT after import into mitochondria or with a matrix the at to to the cleavage site and by the corresponding sequence form the import-incompetent cytosolic the that are found at of maturation in precursors Sci. in the of the pmAAT processing region substitution be for the of processing. it that at processing but not import of a chimeric protein the presequence of to M. M. Neupert W. Full results the processing of a of pmAAT mutants with in the and indicate that efficient processing is also or at the in can that the processing can in either of as as the wild or The to is the of in the in the is not The processing of by intact mitochondria is for is to that the pcmAAT chimera two at the and A of is that the site of the to at the in the and the is to a in because of the of the peptide at a for peptide the or to the cleavage site are in the A similar may to the matrix processing the in the pcmAAT and precursors in the form or as a of the and at the presence of in may a the conformation of the cleavage region that may its by the or the of the the the even after at in the folded and M. in The for the processing of the mutants at the and are two such mutants are (W5A/W6A and but in both the precursors are not to the mature but to by intact mitochondria, that cleavage at the processing site is of the of to that of a precursor by of folded pmAAT after at M. T. N. Sci. that a cleavage in a region at is processing results from the of the processing and not matrix the presequence of the pcmAAT and that in the matrix and a is with matrix extracts. is that in of a with a such as the conformation of the processing indicate the for of at the and C-terminal of the presequence by to and to M. T. N. Sci. The of alanine in the region the with the mature sequence the of the the maturation of the initial of the mature protein by the processing as for the presequence of the precursor Full may also to the of the peptide region in the site of the the processing of the sequence of the region where the cleavage to the presence of a sequence that the maturation site in pmAAT in that it the in results the of a region of the presequence from the cleavage site can end in the site of the peptidase. A is that region of the presequence a processing of the wild protein by the processing and a binding site in the peptidase. in the conformation of the processing site may to in the of the N-terminal region of the presequence in the site that in the cleavage of the peptide is by the of at from the cleavage site in for by the matrix processing T. T. Full T. T. T. Full it that both the and found in with the site T. T. Full The presence of a region in the of the presequence and in the of two of the presequence in the removal of the found in to the protein Full that presequence flexibility be probably not for correct processing. of the pmAAT mutants that processing to folding after import into the matrix it may be that or of the folded is the for the processing However, such the folding and the processing of the after is processed and as efficiently as the wild only a of imported is to of the pmAAT in are to a mitochondria with similar to the wild a of pcmAAT is in the inner membrane of the N-terminal of pmAAT interferes with of the of its translocation into the matrix. to for protein import into mitochondria, translocation across the inner membrane the of precursors with N. M. Full N. Meijer M. Full Full both the presequence peptide and mature of precursor proteins Neupert W. The presence of the N-terminal sequence from at the of pcmAAT the initial of with of the mature protein the presequence. a of the protein may into the membrane. is with that the N-terminal of the mature protein sequence a site in pmAAT for cytosolic M. Full Full M. in and that does not to cytosolic M. Full Full M. Full Full the N-terminal region of mature for both correct processing and translocation of the precursor into the matrix. is that both the of in the matrix. that with in the translocation and folding of the imported protein may to the correct conformation and of the processing region to and correct processing by the processing peptidase. to C-terminal from the maturation site may its with matrix proteins and its and as for the processing peptidase. into of the translocation and maturation of precursors in the precursor is not into that the presequence peptide is but not for the sequence of the passenger translocation and processing are independent However, of the N-terminal region of the mature protein the efficiency of the for and by processing not be that from of for of peptide in native to in the presequence efficient and correct processing are C-terminal to the cleavage The of the mitochondrial processing probably from a site that can a of at the and However, to the correct of the the the may for both and from the cleavage substitution as the processing The conformation can be by the sequence of the region or by with in the mitochondrial matrix, in the translocation and folding of imported for the pcmAAT chimera used in