Figure 9. DEK5 topology. (A–D) Confocal... | Rockefeller University Press

Figure 9.

$Figure 9. DEK5 topology. (A–D) Confocal micrographs after transient expression of GFP fusion proteins in N. benthamiana leaves. (A) Empty vector is pB7FWG2. (B) DEK5N-GFP has the N-terminal region shown in Fig. 7 E. (C) DEK5NΔTMD-GFP has an in-frame deletion of the predicted TMD. (D) DEK5TP+TMD-GFP includes on the predicted chloroplast targeting sequence and TMD. Scale bars are 5 µm in all panels. (E) Immunoblots of total leaf protein with antibodies detecting DEK5, Cyt f, and H3 proteins. (F) Subfractionation of isolated whole chloroplasts (CP) into thylakoid (Thy), soluble (Sup), IEM, and OEM fractions. HSP70 is a soluble, stromal protein. (G) Washes of isolated chloroplasts using chaotropic agents or freeze–thaw lysis (TE). Immunoblots have membrane pellets (P) and soluble (S) fractions. OEC23 is a peripheral thylakoid membrane protein. (H and I) Protease protection assays with purified chloroplasts treated with thermolysin (H) or trypsin (I) proteases (+) or the protease plus protease inhibitors (+In).$

DEK5 topology. (A–D) Confocal micrographs after transient expression of GFP fusion proteins in N. benthamiana leaves. (A) Empty vector is pB7FWG2. (B) DEK5N-GFP has the N-terminal region shown in Fig. 7 E. (C) DEK5NΔTMD-GFP has an in-frame deletion of the predicted TMD. (D) DEK5TP+TMD-GFP includes on the predicted chloroplast targeting sequence and TMD. Scale bars are 5 µm in all panels. (E) Immunoblots of total leaf protein with antibodies detecting DEK5, Cyt f, and H3 proteins. (F) Subfractionation of isolated whole chloroplasts (CP) into thylakoid (Thy), soluble (Sup), IEM, and OEM fractions. HSP70 is a soluble, stromal protein. (G) Washes of isolated chloroplasts using chaotropic agents or freeze–thaw lysis (TE). Immunoblots have membrane pellets (P) and soluble (S) fractions. OEC23 is a peripheral thylakoid membrane protein. (H and I) Protease protection assays with purified chloroplasts treated with thermolysin (H) or trypsin (I) proteases (+) or the protease plus protease inhibitors (+In).