S according to two sources: proteins identified by mass spectrometry to
S depending on two sources: proteins identified by mass spectrometry to become insoluble and detergent resistant in [Cin+] cells, and Q/N-rich proteins identified bioinformatically [38]. These candidate proteins were then experimentally tested for prion-like features using a array of assays: 1) overexpression of fluorescently tagged proteins to test for distinct cellular foci; 2) induction of lasting phenotypes upon transient protein overexpression with 40 unique environmental and pressure conditions getting tested; and 3)Cell-free extract source Sc [psi-] Sc [PSI+] Sp wild-type Sp pREP41-ScSup35-GFP Sp pREP41-ScSup35-GFP, GdnHCl treated Sc: S. cerevisiae; Sp: S. pombeTotal Ura+ colonies 75 84 88 72[PSI+] colonies 0 7 0 6OPEN ACCESS | www.microbialcellMicrobial Cell | January 2017 | Vol. four No.T. Sideri et al. (2016)Prion propagation in fission yeastinheritance of induced phenotypes in a non-Mendelian VEGF165 Protein medchemexpress manner. Sadly, none from the 80 candidate proteins showed constructive results in all 3 of those assays and no protein seemed consequently sufficiently promising to further pursue. Soon after these initial attempts leading to damaging results, we applied the PLAAC algorithm that accurately predicts PrDs depending on the comprehensive sequence and functional data from S. cerevisiae prion-forming proteins [49]. A PLAAC screen of your entire fission yeast proteome identified 295 proteins that contained putative PrDs (Supplemental Table 1). Two of these proteins, Fib1 and Myo1, had been included among the 80 candidate proteins utilized within the initial screen. We looked for enriched functions among these proteins employing the AnGeLi tool [50]. The 295 proteins have been strongly enriched for Ser, Pro, Asp and Thr residues (p 9.9 x 10-12 to 0.002) and under-enriched for Lys, Leu, Ile and Glu residues (p 7.5 x 10-10 to 0.001). Furthermore, these proteins had been enriched for characteristics diagnostic of plasma membrane and cell surface proteins, including the Pfam MASP1 Protein site domain `Ser-Thr-rich glycosyl-phosphatidyl-inositol-anchored membrane family’ (p 0.0009), GPI anchor surface proteins (p 0.0007), plus the GO cellular element `anchored element of external side of plasma membrane’ and related categories (p sirtuininhibitor0.004). We performed some initial in vivo tests on 30 proteins with high PLAAC scores to recognize by far the most promising prion candidates. Following overexpression on the respective proteins, the cells were subject to a number of analyses, including assaying an array of growth phenotypes and have been also screened for the presence of detergent-resistant forms from the protein employing semi-denaturing detergent agarose gel electrophoresis (SDD-AGE). Ctr4 contains predicted prion-forming domain in disordered region According to these preliminary analyses, we focused on the Ctr4 copper transporter protein which contains one strongly predicted 55 amino-acid PrD (residues 55-109), consisting of ten Asn but no Gln residues (Figure 2A). Notably, thisFIGURE 2: Sequence capabilities of Ctr4 (A) The 289 amino acid Ctr4 protein consists of a 55 amino acid prion-forming domain (PrD, red) as predicted by the PLAAC algorithm [49]. (B) The predicted PrD of Ctr4 (red bar) coincides using the highest predicted unfolded region (disordered, blue curve) in accordance with the DISOPRED3 algorithm [51]. The yellow trace will be the location of predicted protein binding web sites inside disordered regions. (C) DISOPRED3 predictions of intrinsically disordered regions in two prion-forming proteins of S. cerevisiae, Rnq1 (left) and Sup35 (rig.