Figure 6

Endoplasmic reticulum luminal binding protein is localized to the novel protein bodies, but does not specifically interact with elastin-like polypeptide. (A) The tobacco endoplasmic reticulum- (ER-)resident chaperone binding protein (BiP) fused with cyan fluorescent protein (CFP) (pBCK) is appropriately localized to the ER. (B) An ER-targeted yellow fluorescent protein-elastin-like polypeptide (YFP-ELP) fusion (pPYEK) accumulated in the induced protein bodies (PBs) located in the cytoplasm. (C-E) When co-expressed, pBCK co-localized with the PBs induced by pPYEK expression, suggesting that the novel PBs originate from the ER. (F) Western blot analysis comparing BiP accumulation in leaves transiently expressing ER-targeted GFP in the absence (pPGK) or presence (pPGEK) of an ELP tag, with or without co-agro-infiltration of the p19 suppressor of gene silencing. Total protein extracts (30 μg/lane) were resolved by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by immunoblotting using an anti-BiP antibody. (G) Leaf extracts expressing pPGK or pPGEK were immunoprecipitated with anti-ELP antiserum. The supernatants (Sup) and immunoprecipitates (IP) were separated by SDS-PAGE and probed with anti-BiP antibody. Bar, 10 μm (A, B); 5 μm (C-E).