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Fig. 7 Q/N-rich regions from Ccr4p, Pop2p and Dhh1p contribute to efficient accumulation of these proteins in P-bodies. (B) Localization of GFP-tagged full-length Ccr4p (pMR212), Pop2p (pMR214), Dhh1p (pMR210) or truncated versions of these proteins (Ccr4
N(148-837) from pMR218, Pop2N(147-433) from pMR215, Dhh1C(1-427) from pMR211) before and after osmotic shock. All GFP-fusions were expressed in BY4741 cells and localization was examined in cells during normal growth (normal) or 20-40 minutes after osmotic shock (stress). (B) Localization of GFP-tagged Ccr4N(148-837) and Ccr4N2 (aa 250-837; pMR213) in fixed cells with DAPI-stained nuclear DNA (C) Localization of C-terminally GFP-tagged full-length Pop2p (pMR216) or Pop2Np (pMR217); DAPI-stained nuclear DNA in blue. (D) Localization of GFP-Ccr4, GFP-Ccr4N and Dcp2-RFP in ccr4 cells (Y10387) 30 minutes after hypo-osmotic shock (E) Anti-GFP western blot analysis of full-length and truncated Lsm4, Ccr4, Pop2 and Dhh1 proteins. Curiously, GFP-Ccr4 (122 kDa) migrates faster than GFP-Ccr4N (106 kDa), but slower than GFP-Ccr4N2 (95 kDa; Fig. 7E), and all three GFP-Ccr4 proteins migrate faster than their predicted molecular weights. The presence or absence of the highly polar N-terminal region of Ccr4p causes a change in the effective charge of the entire protein (predicted charges at pH 7 are –5.8, –4.0 and –5.5 respectively) and might affect protein conformation resulting in unusual migration during SDS-PAGE. Nop1p was used as a loading control.
