Figure 7

L535Q KASH5 does not insert spontaneously into protein-free liposomes. A protein-free liposome system was used to check for the spontaneous membrane insertion of L535Q KASH5. Liposomes were added either during the in vitro transcription/translation reaction [co-translational (A)] or after the proteins were translated [post-translational (D)]. Both samples were then subjected to proteinase K digestion in the presence or absence of TritonX100 detergent (PKT and PK, respectively). Co- or post-translational translocation assays were dissolved in loading buffer and subjected to immunoblot analysis with an anti-opsin or an anti-V5 antibody to detect the protected fragment (PF). For all samples, protein expression levels were determined by running an aliquot of the in vitro transcription/translation reaction (Input IVT). An opsin-tagged Cytochrome B5 was used as a positive control for spontaneous insertion into liposomes, which showed insertion in both the co-translational (B) and post-translational setup (E) as evidenced by the ~ 9 kDa protected fragment (cB5 PF). For wild-type and L535Q KASH5 proteins, the expected size of the protected fragment is also ~ 9 kDa. Neither wild-type nor L535Q KASH5 showed any spontaneous insertion activity, either co-translationally (C), or post-translationally (F). The experiment was repeated five times.