CD79b Y195 Controls BCR Internalization.

<p><u>Panel A</u>, Amino acid sequences of the cytoplasmic domains of CD79a and CD79b. YxxØ putative AP2 binding motifs underlined. <u>Panel B</u>, Confocal laser scanning microscopic analysis of the endocytosis of indicated MHC class II-CD79 chimeric proteins. Plasma membrane is yellow (anti-MHC class II-Alexa 488+ post-endocytic labeling of external MHC class II-CD79 with an Alexa 594-labeled antibody). Internalized MHC class II-CD79 is green (anti-MHC class II-Alexa 488 only). <u>Panel C</u>, Quantification of MHC class II-CD79 endocytosis. 100+ cells from across 3 independent experiments were scored for internalization. Reported is the percent of cells showing internalized MHC class II-CD79 for each construct. Statistical comparisons were made between the construct with both CD79 cytoplasmic domains and cells expressing other constructs.</p

Receptor Endocytosis of Isolated CD79a but not CD79b.

<p>Endocytosis of the indicated MHC class II-CD79 fusion proteins was analyzed (<u>Panel A</u>) and quantitated (<u>Panel B</u>) as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054938#pone-0054938-g001" target="_blank">Figure 1</a>. Statistical comparisons were made between the reporter proteins with both CD79 cytoplasmic domains and other reporter proteins.</p

AP2-mediated CD79-driven Endocytosis is Regulated in Cis via ITAM-embedded DCSM and QTAT motifs.

<p><u>Panel A</u>, Binding of AP2-btn to bead-captured GST bearing the indicated CD79a or CD79b cytoplasmic domains with the indicated mutations was determined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054938#pone-0054938-g003" target="_blank">Figure 3</a>. Binding is expressed as a percentage of wild type CD79a binding and represents the mean of 3 independent experiments ± SEM. Statistical comparisons were measured between the non-mutated CD79a or CD79b_DCSM cytoplasmic domains and other samples. <u>Panels B and C</u>, Endocytosis of the indicated MHC class II-CD79 fusion proteins was analyzed and quantitated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054938#pone-0054938-g001" target="_blank">Figure 1</a>. Statistical comparisons were made between the reporter protein expressing the wild type CD79a or CD79b cytoplasmic domains and other reporter proteins. <u>Panel D</u>, Diagrammatic representation of <i>cis</i> and <i>trans</i> regulation of BCR AP2 binding and endocytosis. The open arrows indicate <i>cis</i> regulation by the DCSM and QTAT regulatory motifs. The closed arrows represent <i>trans</i> regulation of the endocytic activity of each cytoplasmic domain by the presence of the partner cytoplasmic domain.</p

Ultrastructural Colocalization of Ligand-BCR Complexes and AP2 in Clathrin-Coated Pits.

<p>A20µWT B cells or primary murine splenocytes were pulsed with anti-IgM-btn followed by anti-biotin-15 nm gold (arrow heads), incubated 2 minutes at 37° and then plasma membrane rips were prepared as previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054938#pone.0054938-Caballero1" target="_blank">[10]</a>. The exposed cytoplasmic face of the plasma membrane was stained with anti-AP2 and Protein A-5 nm gold (arrows). The percent of BCR-containing CCP that also stained for AP2 is indicated below each image. Inset: AP2 and BCR co-localization within electron dense membrane regions lacking discernable CCP architecture. Shown are representative images from 1 of 3 experiments, with 1,000+ BCR-bound gold particles or 100+ CCP photographed cumulatively.</p

AP2µ Directly Binds the YxxØ Motif Centered on the Membrane-Proximal Tyrosine of Isolated CD79a.

<p><u>Panel A</u>, The binding of AP2µ-btn to bead-captured GST bearing 21 amino acid peptides centered on the five CD79 YxxØ motifs was determined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054938#pone-0054938-g003" target="_blank">Figure 3</a>. Binding is expressed as a percentage of CD79a–AP2 interactions and represents the mean of 3 independent experiments ± SEM. Statistical comparisons were measured between CD79a and other samples. <u>Panel B</u>, For both CD79a and CD79b, a positive or negative regulatory motif lies within +/−10 amino acids of the tyrosine residue of the membrane-proximal YxxØ AP2 binding motif. In this example, the motif is arbitrarily depicted as being downstream of the YxxØ motif.</p

C-terminal Deletions Localize the Motif that Drives AP2 Binding to the Membrane Proximal YxxØ Motif of CD79a.

<p>Binding of AP2µ-btn to bead-captured GST bearing the indicated C-terminal deletions of CD79a was determined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054938#pone-0054938-g003" target="_blank">Figure 3</a>. Data is the mean of 3 independent experiments ± S.E.M. and data were normalized to the binding of AP2 to full length CD79a.</p

Hierarchical Binding of AP2µ to BCR-derived Minimal YxxØ Motifs.

<p><u>Panel A</u>, Amino acid sequences of CD79a and CD79b cytoplasmic domains. YxxØ putative AP2 binding motifs underlined. <u>Panel B</u>, Diagram of the synthetic peptide–AP2µ binding assay. <u>Panel C</u>, 18 amino acid long peptides of the form [xxYxxØ]₃ and corresponding to each of the five putative AP2µ binding motifs of CD79 were synthesized and used at a final concentration of 250 µM to block the binding of AP2µ-btn to beads coated with GST-TGN38 (GST–[SDYQRL]₃). Peptides were also tested across a decreasing range of concentration (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054938#pone.0054938.s002" target="_blank">Figure S2</a>). Data is presented as the level of BCR-derived inhibitor peptide binding to AP2µ-btn (percent inhibition of binding to bead-associated target) and is the mean of 3 independent experiments ± S.E.M. and data were normalized to the background binding of AP2 to the non-AP2 binding target GST–[SD<u>G</u>QRL]₃.</p

AP2µ Binds to Isolated CD79a but not CD79b.

<p><u>Panel A</u>, Amino acid sequences of CD79a and CD79b cytoplasmic domains. YxxØ putative AP2 binding motifs underlined. <u>Panel B</u>, AP2µ expressed as a Gal4 activation domain fusion protein was assayed for specific interaction with the cytoplasmic domain of either CD79a or CD79b fused to the Gal4 DNA binding domain. Growth on histidine deficient (His-) plates indicates an AP2–CD79 interaction. The cytoplasmic domain of TGN38 contains a known AP2 binding YxxØ motif and served as a positive control, while the cytoplasmic domain of OCA2 contains a dileucine motif (which does not bind AP2µ) and served as a negative control. Data are representative of 2 experiments. <u>Panel C</u>, Diagram of the GST-CD79 cytoplasmic domain–AP2µ direct binding assay. <u>Panel D</u>, The cytoplasmic domains of CD79a and CD79b were expressed as GST fusion proteins in BL21 <i>E. coli</i> cells. GST-fusion proteins were captured from cell lysates on glutathione beads and the resulting matrix was tested for binding to <i>in vitro</i> translated, biotin-labeled AP2µ. The AP2 binding motif from TGN38, (SDYQRL)₃, and a non-AP2-binding derivation containing a tyrosine to glycine substitution, (SDGQRL)₃, fused to GST served as positive and negative controls, respectively. Binding is expressed as a percentage of (SDYQRL)₃–AP2 interactions. Data is the mean of 3 independent experiments ± S.E.M. Statistical comparisons were measured between SDYQRL and other samples.</p

Additional file 2: Figure S2. of The pesticidal Cry6Aa toxin from Bacillus thuringiensis is structurally similar to HlyE-family alpha pore-forming toxins

Ab initio models of Cry6Aa. The five models constructed with reference to database information (db) and five models constructed entirely de novo (dn) are shown. The predicted N-terminal domains are shown in light gray and marked “N,” while the C-terminal domains are shown in black and marked “C.” (PPTX 774 kb

Additional file 4: Figure S4. of The pesticidal Cry6Aa toxin from Bacillus thuringiensis is structurally similar to HlyE-family alpha pore-forming toxins

Western blot of wild-type and L259D mutant Cry6Aa. Expression of the wild-type Cry6Aa (WT) and the L259D mutant confirmed by western blotting. (PPTX 1138 kb