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![[Figure 6]](ji5021fig6mag.jpg)
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Figure 6
In vivo cleavage of a model substrate by wild-type AaRseP and PA-inserted mutants. (a) Design of the model substrate. The wild-type RseA from E. coli is a type II membrane protein composed of 216 amino-acid residues. The full-length RseA binds σE and RseB in the cytoplasmic and periplasmic domains, respectively. DegS cleaves RseA at the C-terminal side of Val148 and liberates RseB. The model substrate, HA-MBP-RseA(LY1)148, contains the first transmembrane region of lactose permease (LacY TM1; LY1) with a recombinant cytoplasmic domain composed of HA-tagged MBP. The C-terminal periplasmic domain was truncated at Val148 to mimic the DegS-cleaved product. (b) Immunoblotting with anti-HA antibody detected the model substrate. The full-length model substrate appeared at the `Uncleaved' position. `Cleaved' indicates the model substrate that was cleaved by RseP within the membrane, as illustrated in (a). Immunoblotting with anti-His antibody detected His-tagged EcRseP, AaRseP and their derivatives. Immunoblotting of the cytoplasmic protein SecB serves as a loading control. Molecular-size marker positions are shown in kDa on the left. |
![[Figure 6]](ji5021fig6.jpg)
 | STRUCTURAL BIOLOGY |
ISSN: 2059-7983
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