Characterization of Tic110, a Channel-forming Protein at the Inner Envelope Membrane of Chloroplasts, Unveils a Response to Ca2+ and a Stromal Regulatory Disulfide Bridge

Abstract

Tic110 has been proposed to be a channel-forming protein at the inner envelope of chloroplasts whose function is essential for the import of proteins synthesized in the cytosol. Sequence features and topology determination experiments presently summarized suggest that Tic110 consists of six transmembrane helices. Its topology has been mapped by limited proteolysis experiments in combination with mass spectrometric determinations and cysteine modification analysis. Two hydrophobic transmembrane helices located in the N terminus serve as a signal for the localization of the protein to the membrane as shown previously. The other amphipathic transmembrane helices are located in the region composed of residues 92–959 in the pea sequence. This results in two regions in the intermembrane space localized to form supercomplexes with the TOC machinery and to receive the transit peptide of preproteins. A large region also resides in the stroma for interaction with proteins such as molecular chaperones. In addition to characterizing the topology of Tic110, we show that Ca2+ has a dramatic effect on channel activity in vitro and that the protein has a redox-active disulfide with the potential to interact with stromal thioredoxin. Tic110 has been proposed to be a channel-forming protein at the inner envelope of chloroplasts whose function is essential for the import of proteins synthesized in the cytosol. Sequence features and topology determination experiments presently summarized suggest that Tic110 consists of six transmembrane helices. Its topology has been mapped by limited proteolysis experiments in combination with mass spectrometric determinations and cysteine modification analysis. Two hydrophobic transmembrane helices located in the N terminus serve as a signal for the localization of the protein to the membrane as shown previously. The other amphipathic transmembrane helices are located in the region composed of residues 92–959 in the pea sequence. This results in two regions in the intermembrane space localized to form supercomplexes with the TOC machinery and to receive the transit peptide of preproteins. A large region also resides in the stroma for interaction with proteins such as molecular chaperones. In addition to characterizing the topology of Tic110, we show that Ca2+ has a dramatic effect on channel activity in vitro and that the protein has a redox-active disulfide with the potential to interact with stromal thioredoxin. After the proposed endosymbiotic event that gave rise to chloroplasts and, as a consequence, to the massive transfer of genes from the endosymbiont to the host cell nucleus, two new machineries were developed at the outer and inner envelope of chloroplasts, the TOC and TIC complexes, respectively. The machineries transport plastid proteins, whose synthesis was moved to the cytosol, back to the plastid (for a review, see Refs. 1Jarvis P. New Phytol.. 2008; 179: 257-285Google Scholar and 2Stengel A. Soil J. Bolter B. Biol. Chem... 2007; 388: 765-772Google Scholar). Whereas the channel of the TOC complex is homologous to the Omp85 family of transporters in bacteria (3Bolter B. Soll J. Schulz A. Hinnah S. Wagner R. Proc. Natl. Acad. Sci. U. S. A... 1998; 95: 15831-15836Google Scholar), the TIC complex shows no homology to known transport systems, since the plasma membrane-bacterial transport machineries were relocated to the thylakoid membrane. Moreover, there was an essential requirement for establishing new lines of communication between the organelle and other parts of the cell (4Pesaresi P. Schneider A. Kleine T. Leister D. Curr. Opin. Plant Biol... 2007; 10: 600-606Google Scholar). In chloroplasts of land plants, redox regulation is linked to oxygen and/or light. The regulation of chloroplast-specific processes by redox in accord with the status of the organelle applies not only for transcription and translation but also for translocation. Redox regulation at the translocation level has been proposed for the TIC machinery on the basis of protein composition and dynamics (5Chigri F. Hormann F. Stamp A. Stammers D.K. Bolter B. Soll J. Vothknecht U.C. Proc. Natl. Acad. Sci. U. S. A... 2006; 103: 16051-16056Google Scholar, 6Kuchler M. Decker S. Hormann F. Soll J. Heins L. EMBO J.. 2002; 21: 6136-6145Google Scholar, 7Hormann F. Kuchler M. Sveshnikov D. Oppermann U. Li Y. Soll J. J. Biol. Chem... 2004; 279: 34756-34762Google Scholar, 8Stengel A. Benz P. Balsera M. Soll J. Bolter B. J. Biol. Chem... 2008; 283: 6656-6667Google Scholar) as well as specific in vitro import experiments (9Hirohashi T. Hase T. Nakai M. Plant Physiol... 2001; 125: 2154-2163Google Scholar). However, despite advances in our biochemical understanding of the import process, little is presently known regarding details of the role of redox or of the structure of the TIC complex, particularly with respect to its channel subunits. Tic110 is essential for protein import into plastids (10Schnell D.J. Kessler F. Blobel G. Science.. 1994; 266: Scholar, J. Biol. Chem... 1994; Scholar, T. M. Li M. J. Kessler F. D.J. Plant Scholar). has been proposed to be a protein and of the of the TIC complex L. A. R. Wagner R. Kuchler M. Hormann F. Sveshnikov D. Soll J. EMBO J.. 2002; 21: Scholar). of Tic110 The region from to is in the of translocation between the TOC and the TIC complex T. M. Li M. J. Kessler F. D.J. Plant Scholar). of the region in a for the transit peptide T. Li M. M. Kessler F. D.J. J. Biol. Chem... Scholar). In Tic110 the complex in the stroma in to the translocation The protein a complex with and and at of the Tic110 residues and are in the T. M. Li M. J. Kessler F. D.J. Plant Scholar). The interaction with a proposed and of the TIC was localized to the region of Tic110 in F. Kuchler M. Sveshnikov D. Oppermann U. Li Y. Soll J. J. Biol. Chem... 2004; 279: 34756-34762Google Scholar). of a of Tic110 that the two hydrophobic transmembrane helices at the N terminus in of a to transit L. A. R. Wagner R. Kuchler M. Hormann F. Sveshnikov D. Soll J. EMBO J.. 2002; 21: Scholar). Two been proposed to the topology of The on the of the two hydrophobic transmembrane helices located at the N been shown to essential on the protein to the inner envelope membrane J. Soll J. M. EMBO J.. Scholar), but on protein Whereas that resides in the the other that in the intermembrane The was developed from with a protein in form in T. Li M. M. Kessler F. D.J. J. Biol. Chem... Scholar) and limited proteolysis with and inner envelope membrane J. Biol. Chem... 1998; Scholar). The experiments by our to L. A. R. Wagner R. Kuchler M. Hormann F. Sveshnikov D. Soll J. EMBO J.. 2002; 21: Scholar, J. Soll J. M. EMBO J.. Scholar). The of Tic110 in the In of a of the topology of Tic110 is we that the features of Tic110 as a channel and, in show that Ca2+ has a dramatic effect on Tic110 channel and been to the topology of the protein at the inner envelope membrane of pea chloroplasts and to on we a in has amphipathic transmembrane This to two large regions located in the intermembrane space of chloroplasts and a large in the The the results on six in the Tic110 of are in the stromal the potential to regulation inner envelope intermembrane mass and Tic110 residues and were J. Soll J. M. EMBO J.. Scholar). The region of the region of the Tic110 protein from pea residues was by J. Soll J. M. EMBO J.. Scholar) and into The were into was in as The protein was by in The protein was by in and a In the of the cell was to a was with The protein was with and to a to was in to to the of the of in inner envelope of were from chloroplasts from pea as Soll J. Biol... Scholar). The Tic110 was and protein was from in the of into and were as A. J. S. Scholar) in and proteins were by a The and the protein were to a of and with of a and The were at for at were and with The were in and by and with were from by in and in a for was to a of to the addition of an of to of The was for at and for The were as Soll J. Wagner R. J. Biol. Chem... 2006; Scholar). a of a of the of the and was from were and on a in a in of were in of and with and for at The was by and The were and in in the of experiments were also with and In the proteolysis experiments with were in of and with and at After the was by the addition of In a were in of and with and of at After was The were in in the of The were in with and to and by of Tic110 were from and to The protein from the was by a combination of and in the at the by the were also by The localization of for of the of as the Scholar) or was to the mass and in the Tic110 from a region of Tic110 was to and the molecular of the was with the molecular mass in the the structure and that the proteolysis in residues that not to structure A. B. P. M. 2004; Scholar), a is of the the or was a were with in a for and at in the The was by in the of were a The protein was by structure of was by The protein was and the protein was to The was at a with a cell a from to The the of at a of and was by the at in the from the on the The of the structure was with the Scholar), Scholar), J. Scholar), and B. Biol. 1994; Scholar) of Tic110 by J. D.J. Scholar) was as to known to the Tic110 The of the Tic110 family was by J. J. Biol... Scholar) and and were from the by the Li 2006; Scholar), P. J. R. M. J. 2001; Scholar), and and S. Scholar) respectively. The B. J. Biol... 2001; Scholar), J. Biol... Scholar), G. A. 21: Scholar), and J. P. Scholar) were to the The J. J. Biol... Scholar), J. J. Biol... 2001; Scholar), R. L. on Scholar), and R. A. M. L. 2008; Scholar) were in an to a to of in was with or at After the protein was and the was with and in a were by and the of the redox a of or into was with to the and with of or from in the of or The of stromal and to Tic110 was in the of Redox of Tic110 in redox of Tic110 was in chloroplasts in the were or with or at After of and was The chloroplasts were to and Tic110 was by of from from pea the two transmembrane helices at the N was into The of with a in of molecular mass of and with that of the proteins the not was to the of The to the residues of the the and the the and to of the Tic110 A at the the of two and a and into a Tic110 translation in two are and respectively. The proteins, be by we of the and of Tic110 from The protein was from by and by The protein was by with and into of in the or of The to the of and The was in the and of A in the of and molecular in the of that and of and in the not the that the is in The the of mass of the of and in the of In the of proteins show a experiments of and and in form or in was with and its was by in a and protein is at the of the the protein with at However, is not into the and at the the with of After were with an in and and the were to a In at that the of the protein is not to on the of the The of is at the of the as a in and not show that is a the of in to a protein of The is in with of are to the that Tic110 is an with a to that of a of the the gave a at the that the is at between and were not to of such as or that is in in combination with that disulfide are not in the the in the of and were not in in the or a and and of to and was by and the for and in the of The in of and the of the Tic110 that and, a that the of not the of the proteins and not the for and suggest an of the Tic110 proteins with the In the protein was with in an to of as L. A. R. Wagner R. Kuchler M. Hormann F. Sveshnikov D. Soll J. EMBO J.. 2002; 21: Scholar). The of the protein into the was by in a In the of at the of the in the of the protein to the of the This shows that its by to The was also with the of from pea was to to at the of the in the or of has no to to that the interaction of with the not on the the were with or for at the were to The results that is not but is in the with and on and but not on suggest that Tic110 regions to and between residues and an in and in the of structure of was by in in the and of The the of the protein with of at and and at The and for the results with the the for for and for are in the of that the of the structure are In and of Tic110 in of or with channel activity be and The channel of a was large and with an on the of of After of be by the The of were from channel between to The of the of the channel that the be to a channel as for other protein translocation P. B. P. Wagner R. 2002; Scholar, A. M. M. J. R. Wagner R. Scholar). Moreover, the of a large with and was by of and large were in experiments and were to two with the membrane to proteins in the This since a potential with was by were with and A in Tic110 channel activity was a event with a of in and other with respect to the of and a the channel was at or is the be to the the that to be of experiments were for the were into two on the of were and a However, from the of the we that in experiments only or two were into the The of processes as well as the of of the The of the Tic110 channel be to be on the of In the channel of the and were to other A and and were also with the of other protein translocation and P. B. P. Wagner R. 2002; Scholar, A. M. M. J. R. Wagner R. Scholar). of Tic110 was and/or In and the of as from the potential was on the of the of and were the B. of Scholar), to a of and respectively. as an of in the of Ca2+ was Ca2+ Tic110 the addition of in a dramatic in the potential from to to an of This effect be to in composition but to in the on the of the channel Moreover, the of Ca2+ in the a effect on channel of a with the the channel the activity of However, of the to and of a was in of the This of the channel be back to a well by the addition of see The effect to be to since or a of the no effect not Tic110 in to the of Tic110 in inner envelope was with that were not to the inner envelope membrane of chloroplasts and whose activity is with an of as and and be with is proposed to to the inner envelope membrane at the stromal not to the A. Benz P. Balsera M. Soll J. Bolter B. J. Biol. Chem... 2008; 283: 6656-6667Google Scholar, M. A. Soll J. Bolter B. Biol... 2007; Scholar), and as for the proteolysis experiments the of the was with the to the shows of Tic110 and in with and is that Tic110 is to the In is that be with an of and and no activity is of the The with shows the of the inner envelope experiments results that parts of Tic110 are in the intermembrane space J. Soll J. M. EMBO J.. Scholar). of Tic110 an Tic110 that gave rise to two of and Whereas the was the was the This was by to to residues and and with or not of and were also by were and of that in regions in of Tic110 by of of of of proposed in a new The by of Tic110 that are in the membrane the proteolysis were mapped the Tic110 as are proposed on a combination of molecular mass and structure to residues that are located in regions and are not in structure a and has been as the pea the the of be results in a of the by The be residues of The residues be and the basis of the molecular mass by the is was to the of the other The results are summarized in experiments that regions of Tic110 residues and are located in the of by that regions of Tic110 are in the we experiments in as a is a that with of for After the of mass in to the of that with is an to residues in regions of Tic110 the were with and the was by and of molecular mass was with A was also and to the in the of with stromal the Tic110 a of or of molecular In an to the experiments with the results with limited of was The was to at in specific regions of Tic110 to the to be by The experiments that to Tic110 in with that the in Tic110 are The not its mass that the is located between residues and Two are in region in pea Tic110, and is that of is located in the region of and by no or structure is for Tic110, we the of a of the protein by with Tic110 is in Curr. Biol... 2004; Scholar). A of the Tic110 family is as structure that Tic110 is an protein and This is in with from for a in land but not pea no large were in the protein were between and and Two the not at the of the hydrophobic transmembrane of the N terminus and in a region to the and the two hydrophobic helices to be transmembrane and a but region in pea to be by the P. J. R. M. J. 2001; Scholar). in and in the pea are potential for or in a disulfide Sequence of Tic110 by the Li 2006; Scholar), for in of proteins, six to a of structure are also in the at a not of the pea Tic110 by the and S. Scholar), on the of that the for the region of residues an with the region and are two other regions with but In an in of helices by shows that amphipathic features to the pea A and A and are by the and for into the membrane. proteins to Tic110 were not in we by were to proteins of with were as by the of the and that for The of the and as well as and are proteins that of is known that in and or are the sequence. in of a of hydrophobic residues P. Scholar). The of the well with the in Moreover, suggest that Tic110 six for that to be and to by of the and be parts of the region composed of residues interact with regions that residues and are located in the parts of the region composed of residues and are located in the stroma to interact with the complex T. M. Li M. J. Kessler F. D.J. Plant parts of the region composed of residues are located in the to be to the TOC complex T. M. Li M. J. Kessler F. D.J. Plant of the region composed of residues is located in the stroma to interact with F. Kuchler M. Sveshnikov D. Oppermann U. Li Y. Soll J. J. Biol. Chem... 2004; 279: 34756-34762Google and in an to the The two transmembrane helices were as essential for Tic110 to the inner not is to to a of Tic110 is to be located between residues and the amphipathic helices are The combination of with structure and of amphipathic helices an for the limited proteolysis experiments with inner envelope that the regions of residues and are the there are six transmembrane helices in our for Tic110 with at two large regions in the and a in the stroma Redox in of Tic110 family shows six residues in This to the in vitro redox of we with the protein or The of an disulfide was by a in of the form with the in to the molecular mass results show that was in form with other proteins T. G. R. Scholar), Tic110 as a that was with the the of in with that Tic110 was in redox The and of the and of Tic110 are of the between the and a of Tic110 was by and, was with and The of the form with is for the of at redox-active disulfide the disulfide to a Tic110 was with with an of for by The results show that was by in form or into of a disulfide Tic110 was also by by and The of Tic110 was in vitro and two of the family in with the not of Tic110 is that Tic110 is a potential of in the stroma of Tic110 a redox of Tic110 was in chloroplasts from pea in the the and of Tic110, were with and The of a disulfide is in the in of Tic110 chloroplasts with or to with and with The results that Tic110 was in a in chloroplasts in the In an to the to the form and the of the chloroplasts were with and with to with in the of Tic110 was with the of a disulfide in the The results a of the inner envelope membrane of chloroplasts the basis for a of The of the region of Tic110 in the and at the results on the This is on the Tic110 is to that the inner envelope of chloroplasts the two transmembrane to and show channel activity in and the Tic110 helices with amphipathic The is the in the channel of Tic110 protein been in The large of Tic110 activity of in that the Tic110 is but the for the transport of such as with respect to and In the of and potential for Tic110 L. A. R. Wagner R. Kuchler M. Hormann F. Sveshnikov D. Soll J. EMBO J.. 2002; 21: Scholar) are in with our However, the and of and in the channel not been previously. the complex of the or and of the inner show for P. B. P. Wagner R. 2002; Scholar, A. M. M. J. R. Wagner R. Scholar, M. Wagner R. P. B. A. P. Science.. 2006; Scholar). The Tic110 to the of the for the a of with is a of In an has also been for the complex, of in the channel the The channel activity of an to the on of the protein the that the of are to of Tic110, the in the residues for channel activity are located of to the terminus of the pea The that Ca2+ as an of and is since its in the import is has been shown that Ca2+ the import of at the inner envelope membrane of level F. Soll J. Vothknecht U.C. Plant J.. Scholar). a of the TIC complex, is as a protein whose with the TIC machinery is by and (5Chigri F. Hormann F. Stamp A. Stammers D.K. Bolter B. Soll J. Vothknecht U.C. Proc. Natl. Acad. Sci. U. S. A... 2006; 103: 16051-16056Google Scholar). by and Tic110, Ca2+ a effect on the import such as the of other the of and the interaction with proteins, a on channel in The of transmembrane helices in of chloroplasts is also by limited proteolysis experiments in in inner envelope of pea Its to that the envelope and the in with and that of are in the intermembrane The results presently are to of J. Soll J. M. EMBO J.. Scholar) but of J. Biol. Chem... 1998; Scholar). In the the the of the chloroplasts by the in the is that the of Tic110 to was to an of the and that of the we show that the be well by a of an of and After the addition of to envelope that the no were envelope Moreover, the not the as shown by the to of a TIC complex proposed to to the stromal of the inner envelope of chloroplasts the membrane. we are of the between our results and of J. Biol. Chem... 1998; Scholar), we no for Two features the the of residues in the membrane and the of that of the A large of the are in the two to function as that and as on other molecular P. Scholar). The region in Tic110 be for protein since the of residues is in of the of the The between and be to a composition of the TIC complex, that not for the or of the region of Tic110 be in form in was shown by T. Li M. M. Kessler F. D.J. J. Biol. Chem... Scholar). is that Tic110 has the to hydrophobic regions in an that and in The of regions in a membrane is not F. J. Curr. 2002; Scholar, L. 2004; Scholar), since or other residues for in the a in also in the of the the topology of the channel of the inner envelope is the of of the since the that transport proteins protein import are by B. Y. 2004; Scholar), F. Wagner R. 1998; Scholar), and R. Soll J. Biol. Chem... The and of amphipathic helices in membrane proteins been M. S. J. Biol... 2007; Scholar). respect to the of Tic110 into the membrane and the of its amphipathic A the is well by J. Soll J. M. EMBO J.. Scholar). that a Tic110 residues in the protein was located in the membrane of pea chloroplasts and, other residues residues was to results suggest the of an essential region between residues and in Tic110 that the to the and to The localization of of Tic110 was also in a with chloroplasts T. M. Li M. J. Kessler F. D.J. Plant Scholar). In that were and are to the and The results from the suggest that residues from to the in Tic110 also for membrane The regions into the membrane that the results are the amphipathic helices and that in we between or into the be in a In the of the pea be to the and to with in inner envelope a that with the results A form of a transmembrane the other be located in the the experiments show a molecular with that in the region is from the The proposed of Tic110 of the and the two the region is located in the stroma or intermembrane In our the are since a large of the protein is located in the stroma and for interaction with and T. M. Li M. J. Kessler F. D.J. Plant Scholar) and other of the TIC the other the of regions of Tic110 in the intermembrane space be essential to and form supercomplexes with the TOC machinery T. M. Li M. J. Kessler F. D.J. Plant Scholar). The interaction of Tic110 with the transit peptide in the intermembrane space with 2007; and the stroma the import The experiments a redox-active disulfide in Tic110 stromal are such a disulfide is in accord with the redox regulation of the import proposed to at the inner envelope M. Biol... 2008; Scholar). the residues in Tic110 are to plants, the redox machinery for import was proteins that redox regulation by are in the and in the light. The that Tic110 is in the in chloroplasts in the a role in with as a is that to Tic110 an is to Tic110 channel activity or the a of the TIC has been as a S. J. F. J. D. S. Proc. Natl. Acad. Sci. U. S. A... 2008; Scholar). is that proteins been to be in the T. A. A. A. Biol... Scholar) and F. J. Plant 2002; F. Y. T. J. Biol. Chem... Scholar), was in the has been linked to A. J. S. Proc. Natl. Acad. Sci. U. S. A... 2008; Scholar). is that the activity of the TIC complex is to and are to L. and P. for the experiments and the P. Benz and A. for and of with