Protein Complex within Escherichia coli Inner Membrane

Abstract

The TolA, TolB, TolQ, and TolR proteins are involved in maintaining the integrity of the Escherichia coli outer membrane and in the import of group A colicins and filamentous phage DNA. TolA, TolQ, and TolR are localized in the inner membrane while TolB is periplasmic, although a small amount of membrane-associated TolB is always found. In vivo cross-linking experiments with formaldehyde were performed in order to determine the proteins interacting with TolA. In wild-type strains, two specific complexes of 65 and 71 kDa, comprising TolA, were identified. These complexes were absent in a tolQ strain, while only the 65-kDa complex was absent in a tolR strain. When the tol strains were transformed with plasmids encoding TolR or TolQ, the specific complexes were restored. Moreover, immunoprecipitation experiments with the antiserum directed against and TolR were with These with TolR and proteins of the or the of the were to in vivo two specific complexes with TolR and TolQ, the with The TolA, TolB, TolQ, and TolR proteins are involved in maintaining the integrity of the Escherichia coli outer membrane and in the import of group A colicins and filamentous phage DNA. TolA, TolQ, and TolR are localized in the inner membrane while TolB is periplasmic, although a small amount of membrane-associated TolB is always found. In vivo cross-linking experiments with formaldehyde were performed in order to determine the proteins interacting with TolA. In wild-type strains, two specific complexes of 65 and 71 kDa, comprising TolA, were identified. These complexes were absent in a tolQ strain, while only the 65-kDa complex was absent in a tolR strain. When the tol strains were transformed with plasmids encoding TolR or TolQ, the specific complexes were restored. Moreover, immunoprecipitation experiments with the antiserum directed against and TolR were with These with TolR and proteins of the or the of the were to in vivo two specific complexes with TolR and TolQ, the with The TolA, and proteins are group A colicins to in the outer membrane to in the inner membrane or the The of the proteins is although proteins to a in maintaining the integrity of the outer The tol a in are to and and proteins the The proteins to the group A colicins and and the of and to the outer The of the TolA, TolQ, and TolR inner membrane proteins is a with membrane to a and a involved in import The to in with colicins A and TolR inner membrane to a and the of the the to to and to and a to the TolB is periplasmic, although a of is membrane-associated of the and is involved in the import of group colicins and and of phage and The of the is to the of and outer membrane the inner to the outer the of to the and are and are TolR and in the membrane of and proteins are Moreover, the of and to to of TolA, TolQ, and TolR and TolQ, and or the proteins are the inner and outer and A TolA, and a complex with a The of a complex the experiments in the was with of the the was to with with the with the membrane in the of TolR a in a of and TolR vivo cross-linking experiments with and with the outer membrane In the in vivo cross-linking experiments were performed in order to the and proteins were with with and TolR and two specific complexes cross-linking with In the of cross-linking performed two the or of the with and TolR and the strains and plasmids in the are in In the tol the tolR and the of were were The specific were the immunoprecipitation were with of and with strains and and and and and tol and in a of plasmids in were the of the the of the was in the and and was the of the the the of the tol to to to the tol was the of the of the of the and the and the of the tolQ the and TolB proteins and small of TolR the of the were the with transformed strains and A and was to the and while the tolR was was the the of the of the TolQ, and was a of the of tolR was of was to and the was of two encoding to of A and are in the to the of the of to to the of and the of TolA, the was the was in the of The of was the of was In the was and was two was the the with a the a and are in the the in to of in the of a was transformed with were to a and to formaldehyde cross-linking experiments were performed and were in to of and The was with and in the to of was to a of and the were to The were and with The were in and to the formaldehyde or to the and was were in to of and the was to and the were The and were were against to a of in with a of the proteins were to and and were performed in and was and and or with formaldehyde were in were in with and in order to The was with to The were with and with In the the were with and or in order to the or to the and to the of 65 and 71 kDa, TolA, in vivo cross-linking with formaldehyde was performed in order to the proteins interacting with TolA. The was with was with the with the or with formaldehyde and were and in cross-linking in of the wild-type These complexes were a to formaldehyde and were the to were absent with These and complexes are formaldehyde were a in were in The were the two complexes were with in the membrane and with in the of TolA, TolQ, and TolR were and to and kDa, and in to and the specific complexes of 65 and 71 to with TolR or with In order to complexes were absent strains tolR and the the two specific complexes were while only the complex was in tolR A the 65-kDa with a wild-type strain. the was transformed with plasmids or TolQ, and proteins or and The 65-kDa complex was TolR was in was in in the the of TolR is to the 65-kDa of in and strains and strains in vivo with were and the was with the were and with The is the in vivo cross-linking was performed the strain. In strain, the two specific complexes were experiments were with transformed with the and complexes were in is the of the tolQ TolR the tolQ of TolR a and the complex to the of with the 65-kDa complex to the of TolR with TolA. were and the of cross-linking experiments were the with was in with to of the tolQ or to of in the of of and were with the amount of in order to the of the of in strains and and strains in vivo with were and was the and were and with The is the in the plasmids and the tol of the and the and transformed with plasmids and tolR and tolQ were In the of the TolQ, and proteins in the with in and in the and specific complexes was in the with the These and TolR the and and the the were the TolB in the of a complex of in and in vivo with were was with and the are the in and TolR with with TolR and with TolA, was and are and was to the proteins with immunoprecipitation experiments were with in vivo cross-linking with formaldehyde and were with the and were with The were in and to The were in two in order to the proteins or to the the is to the In with formaldehyde two and The was to to of the to a of in while a TolB was with the of with formaldehyde two proteins and TolR the cross-linking was of specific of and or and was in order to the of and In experiments with was the to and and TolR were with the in vivo cross-linking with of and or with formaldehyde and in The of was in and to the in vivo specific to the of the TolA, TolB TolB, TolQ, and or were and in and of with and the were in the and in order to the of interacting with and The plasmids and and with and in the of the to were experiments TolA, and and were the the wild-type the a membrane The of transformed with and plasmids was the two the and of were group A was in with a to of the of the transformed and in a and were with the in vivo cross-linking specific with to of complexes the and or TolR were The of the and complexes were of The of the complexes were in with The and complexes to the and the and to the the and TolR were with the was the of with and of in and in vivo with were and was with The of the and complexes comprising TolA, and are and of the complexes and in with TolA, and or in a in vivo cross-linking experiments with two proteins interacting with were The were with a of cross-linking to with and with the outer membrane in wild-type strains, two specific complexes with of 65 and 71 with the tol strains with formaldehyde were in order to the proteins interacting with TolA. TolR was involved in a 65-kDa complex comprising was absent a tolR the and complexes were absent a tolQ the of the tolQ TolR in was involved in the complex TolA. the tolQ or tolR were to The 65-kDa and complexes to the of and TolR or and complex is a TolQ, and to of in vivo cross-linking with and TolR were the with TolA. In and TolR with order to of the proteins were involved in the two the or of the were and of two formaldehyde complexes with to of and with or TolR were was the of with and The and the in is the in with of TolR and of the of the and a was the and against the of with the of the the are specific to or These and with and TolQ, Moreover, the of of the inner membrane of the complex was with a with A was is a of the in the of These and In the of the the with and In of the the and the of with and TolR the inner In in vivo cross-linking with and a complex of in a amount or the cross-linking of to the of TolR with the of The of a TolQ, and complex was the of the proteins in the of The to the complex the the inner and outer of Escherichia coli Moreover, the TolB was with the outer membrane the the complex localized the and were to a the are involved and or and A the complex the of TolA, TolQ, and TolR to the of with TolR and with of and with and TolR of the only to with TolA. the of coli and is with TolA, a is a of in the of in of and of with two is is to with the or cross-linking experiments with tolQ of interacting with two the of TolR and and are and were with or was in with the of the tol and the These two are in the is to to while a membrane of the were to outer membrane and with the of the the membrane integrity was and to colicins A and a and are the is the of and the complex are in order to proteins to maintaining outer membrane integrity and to group A colicins and filamentous phage the coli The TolA, and proteins are group A colicins to in the outer membrane to in the inner membrane or the The of the proteins is although proteins to a in maintaining the integrity of the outer The tol a in are to and and proteins the The proteins to the group A colicins and and the of and to the outer The of the TolA, TolQ, and TolR inner membrane proteins is a with membrane to a and a involved in import The to in with colicins A and TolR inner membrane to a and the of the the to to and to and a to the TolB is periplasmic, although a of is membrane-associated of the and is involved in the import of group colicins and and of phage and The of the is to the of and outer membrane the inner to the outer the of to the and are and are TolR and in the membrane of and proteins are Moreover, the of and to to of TolA, TolQ, and TolR and TolQ, and or the proteins are the inner and outer and A TolA, and a complex with a The of a complex the experiments in the was with of the the was to with with the with the membrane in the of TolR a in a of and TolR In vivo cross-linking experiments with and with the outer membrane In the in vivo cross-linking experiments were performed in order to the and proteins were with with and TolR and two specific complexes cross-linking with In the of cross-linking performed two the or of the with and TolR and the strains and plasmids in the are in In the tol the tolR and the of were were The specific were the immunoprecipitation were with of and with strains and and and and and tol and in a of plasmids in were the of the the of the was in the and and was the of the the the of the tol to to to the tol was the of the of the of the and the and the of the tolQ the and TolB proteins and small of TolR the of the were the with transformed strains and A and was to the and while the tolR was was the the of the of the TolQ, and was a of the of tolR was of was to and the was of two encoding to of A and are in the to the of the of to to the of and the of TolA, the was the was in the of The of was the of was In the was and was two was the the with a the a and are in the the in to of in the of a was transformed with were to a and to formaldehyde cross-linking experiments were performed and were in to of and The was with and in the to of was to a of and the were to The were and with The were in and to the formaldehyde or to the and was were in to of and the was to and the were The and were were against to a of in with a of the proteins were to and and were performed in and was and and or with formaldehyde were in were in with and in order to The was with to The were with and with In the the were with and or in order to the or to the and to the and the strains and plasmids in the are in In the tol the tolR and the of were were The specific were the immunoprecipitation were with of and with of plasmids in were the of the the of the was in the and and was the of the the the of the tol to to to the tol was the of the of the of the and the and the of the tolQ the and TolB proteins and small of TolR the of the were the with transformed strains and A and was to the and while the tolR was was the the of the of the TolQ, and was a of the of tolR was The of was to and the was of two encoding to of A and are in the to the of the of to to the of and the of TolA, the was the was in the of The of was the of was In the was and was two was the the with a the a and are in the the in to of in the of a was transformed with were to a and to formaldehyde cross-linking experiments were performed and were in to of and The was with and in the to of was to a of and the were to The were and with The were in and to the formaldehyde or to the and was were in to of and the was to and the were The and were were against to a of in with a of the proteins were to and and were performed in and was and and or with formaldehyde were in were in with and in order to The was with to The were with and with In the the were with and or in order to the or to the and to the of 65 and 71 kDa, TolA, in vivo cross-linking with formaldehyde was performed in order to the proteins interacting with TolA. The was with was with the with the or with formaldehyde and were and in cross-linking in of the wild-type These complexes were a to formaldehyde and were the to were absent with These and complexes are formaldehyde were a in were in The were the two complexes were with in the membrane and with in the of TolA, TolQ, and TolR were and to and kDa, and in to and the specific complexes of 65 and 71 to with TolR or with In order to complexes were absent strains tolR and the the two specific complexes were while only the complex was in tolR A the 65-kDa with a wild-type strain. the was transformed with plasmids or TolQ, and proteins or and The 65-kDa complex was TolR was in was in in the the of TolR is to the 65-kDa vivo cross-linking was performed the strain. In strain, the two specific complexes were experiments were with transformed with the and complexes were in is the of the tolQ TolR the tolQ of TolR a and the complex to the of with the 65-kDa complex to the of TolR with TolA. were and the of cross-linking experiments were the with was in with to of the tolQ or to of in the of of and were with the amount of in order to the of the of in strains and and strains in vivo with were and was the and were and with The is the in the plasmids and the tol of the and the and transformed with plasmids and tolR and tolQ were In the of the TolQ, and proteins in the with in and in the and specific complexes was in the with the These and TolR the and and the the were the TolB in the of a complex of in and in vivo with were was with and the are the in and TolR with with TolR and with TolA, was and are and was to the proteins with immunoprecipitation experiments were with in vivo cross-linking with formaldehyde and were with the and were with The were in and to The were in two in order to the proteins or to the the is to the In with formaldehyde two and The was to to of the to a of in while a TolB was with the of with formaldehyde two proteins and TolR the cross-linking was of specific of and or and was in order to the of and In experiments with was the to and and TolR were with the in vivo cross-linking with of and or with formaldehyde and in The of was in and to the in vivo specific to the of the TolA, TolB TolB, TolQ, and or were and in and of with and the were in the and in order to the of interacting with and The plasmids and and with and in the of the to were experiments TolA, and and were the the wild-type the a membrane The of transformed with and plasmids was the two the and of were group A was in with a to of the of the transformed and in a and were with the in vivo cross-linking specific with to of complexes the and or TolR were The of the and complexes were of The of the complexes were in with The and complexes to the and the and to the the and TolR were with the was the of with and of in and in vivo with were and was with The of the and complexes comprising TolA, and are and of the complexes and in with TolA, and or in a of 65 and 71 kDa, TolA, in vivo cross-linking with formaldehyde was performed in order to the proteins interacting with TolA. The was with was with the with the or with formaldehyde and were and in cross-linking in of the wild-type These complexes were a to formaldehyde and were the to were absent with These and complexes are formaldehyde were a in were in The were the two complexes were with in the membrane TolR and with in the of TolA, TolQ, and TolR were and to and kDa, and in to and the specific complexes of 65 and 71 to with TolR or with In order to complexes were absent strains tolR and In the the two specific complexes were while only the complex was in tolR A the 65-kDa with a wild-type strain. the was transformed with plasmids or TolQ, and proteins or and The 65-kDa complex was TolR was in was in in the the of TolR is to the 65-kDa In vivo cross-linking was performed the strain. In strain, the two specific complexes were experiments were with transformed with the and complexes were in is the of the tolQ TolR the tolQ of TolR a and the complex to the of with the 65-kDa complex to the of TolR with TolA. were and the of cross-linking experiments were the with was in with to of the tolQ or to of in the of of and were with the amount of in order to the of the the plasmids and the tol of the and the and transformed with plasmids and tolR and tolQ were In the of the TolQ, and proteins in the with in and in the and specific complexes was in the with the These and TolR the and and the the were the TolB in the of a complex and TolR with with TolR and with TolA, was and are and was to the proteins with immunoprecipitation experiments were with in vivo cross-linking with formaldehyde and were with the and were with The were in and to The were in two in order to the proteins or to the the is to the In with formaldehyde two and The was to to of the to a of in while a TolB was with the of with formaldehyde two proteins and TolR the cross-linking was of specific of and or and was in order to the of and In experiments with was the to and and TolR were with the in vivo cross-linking The of with and the were in the and in order to the of interacting with and The plasmids and and with and in the of the to were experiments TolA, and and were the the wild-type the a membrane The of transformed with and plasmids was the two the and of were group A was in with a to of the and were with the in vivo cross-linking specific with to of complexes the and or TolR were The of the and complexes were of The of the complexes were in with The and complexes to the and the and to the the and TolR were with the was the of with and in vivo cross-linking experiments with two proteins interacting with were The were with a of cross-linking to with and with the outer membrane in wild-type strains, two specific complexes with of 65 and 71 with the tol strains with formaldehyde were in order to the proteins interacting with TolA. TolR was involved in a 65-kDa complex comprising was absent a tolR the and complexes were absent a tolQ the of the tolQ TolR in was involved in the complex TolA. the tolQ or tolR were to The 65-kDa and complexes to the of and TolR or and complex is a TolQ, and to of in vivo cross-linking with and TolR were the with TolA. In and TolR with order to of the proteins were involved in the two the or of the were and of two formaldehyde complexes with to of and with or TolR were was the of with and The and the in is the in with of TolR and of the of the and a was the and against the of with the of the the are specific to or These and with and TolQ, Moreover, the of of the inner membrane of the complex was with a with A was is a of the in the of These and In the of the the with and In of the the and the of with and TolR the inner In in vivo cross-linking with and a complex of in a amount or the cross-linking of to the of TolR with the of The of a TolQ, and complex was the of the proteins in the of The to the complex the the inner and outer of Escherichia coli Moreover, the TolB was with the outer membrane the the complex localized the and were to a the are involved and or and A the complex the of TolA, TolQ, and TolR to the of with TolR and with of and with and TolR of the only to with TolA. the of coli and is with TolA, a is a of in the of in of and of with two is is to with the or cross-linking experiments with tolQ was in with the of the tol and the These two are in the is to to while a membrane of the were to outer membrane and with the of the the membrane integrity was and to colicins A and a and are the is the of and the complex are in order to proteins to maintaining outer membrane integrity and to group A colicins and filamentous phage the coli in vivo cross-linking experiments with two proteins interacting with were The were with a of cross-linking to with and with the outer membrane in wild-type strains, two specific complexes with of 65 and 71 with the tol strains with formaldehyde were in order to the proteins interacting with TolA. TolR was involved in a 65-kDa complex comprising was absent a tolR the and complexes were absent a tolQ the of the tolQ TolR in was involved in the complex TolA. the tolQ or tolR were to The 65-kDa and complexes to the of and TolR or and complex is a TolQ, and to of in vivo cross-linking with and TolR were the with TolA. In and TolR with TolA. In order to of the proteins were involved in the two the or of the were and of two formaldehyde complexes with to of and with or TolR were was the of with and The and the in is the in with of TolR and of the of In the and a was the and against the of with the of the the are specific to or These and with and TolQ, Moreover, the of of the inner membrane of the complex was with a with A was is a of the in the of These and In the of the the with and In of the the and the of with and TolR the inner In in vivo cross-linking with and a complex of in a amount or the cross-linking of to the of TolR with the of The of a TolQ, and complex was the of the proteins in the of The to the complex the the inner and outer of Escherichia coli Moreover, the TolB was with the outer membrane the the complex localized the and were to a the are involved and or and A the complex the of TolA, TolQ, and TolR to the of with TolR and with of and with and TolR of the only to with TolA. the of coli and is with TolA, a is a of in the of in of and of with two is is to with the or cross-linking experiments with tolQ was in with the of the tol and the These two are in the is to to while a membrane of the were to outer membrane and with the of the the membrane integrity was and to colicins A and a and are the is the of and the complex are in order to proteins to maintaining outer membrane integrity and to group A colicins and filamentous phage the coli are to and the against and of the and and the of