Cellular characterization of Aß₄₂ mutants selected for their toxicity.

<div><p><b>A</b>: Peptide sequences of Aß alleles. </p> <p>Amino acid substitutions in mutated alleles are shown at the top. The number of asterisks (*) is proportional to their toxicity in yeast. Non-toxic mutations are boxed in red.</p> <p><b>B</b>: Comparison of the toxicities of Aß alleles in yeast. </p> <p>BY4742 cells carrying various plasmids were serially diluted and spotted on galactose (inducing) and dextrose (non-inducing) media. Cell growth was observed after 48h.</p> <p><b>C</b>: Expression of mutated Aß. </p> <p>BY4742 WT cells expressing the highly toxic Aß alleles after 6 hours of induction were collected to extract total protein. Equal quantities of protein were separated by SDS-PAGE (12%), transferred to nitrocellulose membranes, and probed with an anti-Aß antibody (Santa-Cruz Biotechnology). The bands correspond to the precursor (P), glycosylated (G) and mature (M) forms.</p> <p><b>D</b>: Aggregation patterns of GFP-tagged Aß alleles in WT yeast. </p> <p>WT cells were grown for 6 h in galactose medium to induce the expression of chimeric proteins and were examined by epifluorescence microscopy.</p> <p><b>E</b>: Ile31 and Leu34 are essential for Aß toxicity. </p> <p>Site-directed mutagenesis of Ile31 and Leu34 was performed with degenerate primers. The resulting harmless mutants were sequenced. The nucleotide substitutions and the corresponding changes in amino acids are shown.</p> <p><b>F</b>: Harmless mutations do not prevent toxicity in <i>trans</i>. </p> <p>BY4742 WT cells co-transformed by the indicated plasmids were serially diluted and spotted on inducing and non-inducing media. Growth was observed after 72 h.</p> <p><b>G:</b> Harmless mutations prevent toxicity in <i>cis</i>.</p> <p> BY4742 WT cells transformed by the Aß double mutant were serially diluted and spotted on inducing and non-inducing media. Growth was observed after 48 h. H: Aß mutants are toxic without GFP. BY4742 deleted for a gene involved in Aß-GFP toxicity and carrying different alleles of Aß without GFP were serially diluted and spotted on galactose (inducing) and dextrose (non-inducing) media. Cell growth was observed after 48 h.</p>