Schemes of EJC complex structure, in situ distribution, and applied experimental approaches. (A) The minimal EJC core consists of a tetrameric complex, comprising eIF4A3, MLN51, Magoh, and Y14 (Tange et al. 2005). Two additional layers of EJC factors cover the EJC core: RNPS1, Acinus, Pinin, and REF/Aly comprise the outer shell and UAP56 and NXF1/p15 are transiently interacting factors. The investigated proteins in this study are highlighted in white. The figure is modified from Tange et al. (2005). Please note that the model does not reflect the asymmetry of the EJC. For a detailed structural view on the EJC please see Chang et al. (2007) and Le Hir and Andersen (2008). (B) The in situ distribution of EJC proteins is exemplified with a GFP–Magoh-expressing living cell. The dotted line indicates the outline of the cell, the arrows point at speckles or the nucleoplasm. spe: speckles; nuc: nucleoplasm; cyto: cytoplasm; and scale bar = 5 μm. (C) Scheme of FRAP, CP, and FCS to illustrate how the different parameters (half-times of recovery, diffusion correlation times, free, transiently bound/slowly mobile and immobilized fractions) were obtained. For FRAP, fluorescent molecules within a region of interest were bleached with a high intensity laser pulse. The exchange of bleached against unbleached molecules was observed over time and half-times of recovery and immobilized fractions were determined. For CP, a laser beam was placed at a point of interest, which was illuminated continuously to bleach the pool of fluorescent molecules within the spot. The exchange of fluorescent against bleached molecules was measured within the spot over time and the fractions of fully free and transiently bound or immobilized molecules were determined. Immobilization refers to the absence of exchange within the observation period; it does not imply that the proteins will never be replaced. FCS and two color FCCS were measured for 60 sec and (co)diffusion correlation times were obtained from correlated fluctuation curves. For details see the text and Materials and Methods.
