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As a potential transcription factor, STM was expected to direct GFP fluorescence into the plant cell nucleus. However, both fusion proteins, GFP-STM and STM-GFP remained in the cytoplasm (Figure 1B and C).
0663
The cytoplasmic retention of the chimeric GFP-STM protein was further substantiated by confocal laser microscopy scanning and staining of nuclei with propidium iodide. The comparison of a median Z-stack projection through the nucleus either analysed for GFP-STM fluorescence (Figure 1F), the corresponding propidium iodine stains (Figure 1G) and their overlay in Figure 1H confirm exclusion of GFP fluorescence from the nuclear compartment.
0114
Crystallization trials were conducted for various forms of CgE, gE, and gE-gI (including CgE [residues 213–390], gE [residues 21–419], gE2 [residues 21–390], gE-gI [gE plus gI residues 21–266], gE2-gI2A [gE2 plus gI residues 21–208], and gE2-gI2B [gE2 plus gI residues 21–201]) both alone and complexed with wtFc or heterodimeric Fc. The only isolated protein to crystallize was CgE (described above), and the only complex of the six possible gE-gI/Fc complexes that crystallized was one that contained gE residues 21–419 and gI residues 21–266 and wtFc (residues 223–447). The complex crystals grew from drops containing a 2:1 molar ratio of gE-gI and wtFc mixed with an equal volume of well solution (0.1 M MES [pH 6.0] or 0.1 M HEPES [pH 7.0] and 0.9–1.1 M sodium malonate), resulting in a final pH of approximately 7.5. Microseeding increased the reproducibility of crystal growth.
0114
The structural information available on protein–protein interactions involving ZF domains is also limited [25]. The X-ray structure of the Munc13–1/RIM2α heterodimer described here provides the first high-resolution view of a complex directly involving a member from the family of ZF domains that includes α-RIMs and other Rab effectors. The structure shows that two surfaces at opposite sides of the RIM2α ZF domain interact with two different structural motifs of Munc13–13–150. Thus, the N-terminal loop region of the RIM2α ZF domain binds to the tip of the C2A-domain β-sandwich, whereas the crevice formed by the C-terminal β-hairpin and helix a2 binds the C-terminal helix of Munc13–13–150. Interestingly, in the complex between the α subunit of Hif-1 (Hif-1α) and the TAZ1 domain of CREB-binding protein (a ZF domain unrelated to the RIM2α ZF domain), Hif-1α also wraps around the TAZ1 domain, interacting with surfaces at opposite sides of the domain [31]. An emerging theme from these observations is that, because of their small size, ZF domains may often use multiple surfaces to increase the affinity and specificity of interactions with target proteins, although further research will be necessary to assess the generality of this notion.
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(E) Superposition of the structure of the RIM2α ZF domain observed in the heterodimer (blue) and its solution structure determined in isolation by NMR spectroscopy (red) [20].
0416
The effect of depleting RCK/p54 on localization of Ago2 was next examined by immunofluorescence analysis of HeLa cells expressing Myc-Ago2 and siRNAs against RCK/p54. As shown in Figure 4B, 4C, 4F, and 4G, depleting RCK/p54 disrupted the cellular P-body structures. In these P-body-deficient cells, Ago2 proteins were diffused throughout the cytoplasm and no longer accumulated at specific foci (Figure 4A and 4E). This cytoplasmic redistribution of Ago2 suggests that its localization to P-bodies is driven in mammalian cells by factors such as RCK/p54.
0416
We next visualized Lsm1 and Ago2 by immunofluorescence using antibodies against Lsm1 and Myc tag and found that Lsm1 and Ago2 co-localized in P-bodies (Figure 5B). To determine the status of P-body structures and RISC localization in cells after Lsm1 knockdown, Lsm1-depleted cells were analyzed by immunofluorescence. We found that P-body structures were drastically disrupted, Ago2 was diffused throughout the cytoplasm, and Lsm1 and Ago2 were minimally co-localized (Figure 5B). These results show that P-body structures were drastically disrupted and Ago2 was diffuse throughout the cytoplasm when Lsm1 was depleted by RNAi.
0004
To dissect and understand the relationship between RNAi function and P-bodies, we affinity-purified RISC using Myc-Ago2 and expression vectors of the YFP-tagged P-body proteins, Lsm1, RCK/p54, Dcp2, and eIF4E. Ago2 interacted with these various P-body components in ways that were RNA-dependent or RNA-independent (Figure 1A).
0006
In a converse experiment, ephrin-B1 was co-immunoprecipitated with an anti-Cx43 antibody (Figure 6Cb).
0019
It remains to be determined to what extent PDE4D9, which is activated after both β1AR and β2AR stimulation (Figure 5) and which also showed interaction with β1AR in co-IPs of exogenous proteins (Figure 3A and B), can substitute for interaction with the βARs in vivo.
0077
The model of AIRE–PHD1 complexed with H3K4me0 was in perfect agreement with the experimental chemical shift perturbation data, as the peptide-binding region coincided with the binding surface identified by NMR spectroscopy (Fig 3A). In fact, the H3K4me0 peptide induced chemical shift changes in AIRE–PHD1 residues that map only on one side of the protein surface, involving residues in the N terminus of the PHD finger, the first β-strand, and the loop connecting the first and the second β-strands (D297, G305, G306, L308, C310, D312 and G313; Fig 2; supplementary Fig S5 online). A similar pattern of chemical shift changes indicated the same binding site for H3K4me1.
0276
The samples were fractionated in SDS-PAGE and stained with Coomassie Blue.
0030
To determine whether the Bcl-XL/Bax heterodimer also prevented the subsequent oligomerization of Bax, we examined oligomerization by cross-linking. In these experiments, the cross-linker was added to reactions containing an equal amount of membrane-bound Bax in the absence or presence of Bcl-XL (Figure S3). In these reactions, membrane-bound Bcl-XL inhibited Bax oligomerization, as detected by cross-linking concomitant with inhibition of dye release from liposomes. Taken together, these results suggest that, when bound to Bcl-XL, Bax function is neutralized, both in recruitment of other Bax molecules through autoactivation and in oligomerization to permeabilize membranes (Figure 6C, step 6).
0019
(B) Bax was incubated in the presence of liposomes, tBid, and increasing concentrations of Bcl-XL. Immunoprecipitations and immunoblotting were performed as in (A) without the addition of 2% CHAPS.
0019
(C) Bax was incubated for 2 h with liposomes (without tBid) at increasing concentrations of Bcl-XL or Bcl-XL Y101K. Immunoprecipitations and immunoblotting were performed as in (B).
0030
(D and E) Bcl-XL inhibits liposome-induced cross-linking of Bax. (D) Bax (100 nM) was incubated with liposomes for 2 h either alone (left panel), with 20 nM tBid (middle panel), or with 20 nM tBid and 100 nM Bcl-XL (right panel). Cross-linking with DSS was performed for 30 min at room temperature with or without 2% CHAPS to solubilize the liposomes prior to cross-linking, as indicated. Results were analyzed by immunoblotting. (E) Bax (100 nM) was incubated with or without liposomes for 2 h. Cross-linking and immunoblotting were performed as in (D).
0030
To investigate further the effects of the membrane surface on Bax and the inhibition of these effects by Bcl-XL, cross-linking experiments using disuccinimidyl suberate (DSS) were performed. Cross-linking of Bax into higher-order structures after Bax binds to membranes has been observed previously [18].
0077
Data were recorded on home-built or Bruker spectrometers with 11.7, 14.1, 17.6, and 22.3 T field strengths and processed with NMRPipe (Delaglio et al., 1995). Backbone chemical shift assignments were obtained using standard triple resonance experiments. Peptide titration experiments were performed by mixing two stock solutions (in 50 mM sodium phosphate, 100 mM NaCl, 2 mM DTT, 5% D2O, and 30 μM DSS [pH 6.9]) containing 235 μM 15N-enriched α-parvin-CHC and either no or a maximum concentration of LD peptide at the required protein/ligand ratios (Figure S2). Phase-sensitive gradient-enhanced 1H-15N HSQC spectra (Kay et al., 1992) were recorded at 25°C.
