Figure 5. Aggregation analysis and Ade⁺ phenotype of Mca1₄₅₄-Sup35C.

(A) Full-length Mca1₄₅₄ (N-terminal extension of MGKMSLEVYLNYHQRRPTRFTI), the N-terminal extension and Q/N-rich region of Mca1₄₅₄ (Mca1₄₅₄N) and Mca1C (caspase domain of Mca1) were fused to Sup35C and expressed in a sup35Δ strain. Soluble and aggregated proteins were separated by centrifugation analysis and detected using polyclonal anti-Sup35C antibodies and immunoblotting. Mca1₄₅₄-Sup35C fusions revealed aggregation dependent on the Q/N-rich region of Mca1₄₅₄. Strains used in this assay (from top to bottom): Y133 (endogenous Sup35p; [PSI⁺]), Y312 (p2HG-SUP35C), Y320 (p2HG-MCA1₄₅₄-SUP35C), Y316 (p2HG-MCA1₄₅₄N-SUP35C), Y322 (p2HG-MCA1C-SUP35C). (B) Full-length Mca1₄₅₄ (N-terminal extension of MGKMSLEVYLNYHQRRPTRFTI), the N-terminal extension and Q/N-rich region of Mca1₄₅₄ (Mca1₄₅₄N) and Mca1C (caspase domain of Mca1) were fused to Sup35C and constitutively expressed in a sup35Δ strain harboring the chromosomal ade1–14 mutation. Growth of two independent clones was analyzed on media lacking adenine after 12 days of incubation. Strains used in this assay: Y133 (endogenous Sup35p; [PSI⁺]), Y81 ([psi⁻][pin⁻]), Y320 (p2HG-MCA1₄₅₄-SUP35C), Y316 (p2HG-MCA1₄₅₄N-SUP35C), Y322 (p2HG-MCA1C-SUP35C), Y312 (p2HG-SUP35C).