Table 8.

Both the Sup35p-binding activity of Upf2p and Upf complex formation are required for efficient curing of [PSI+] prion variants in a upf2Δ strain

Prion isolate	UPF2 alleles in plasmid	Average % of Ura+ transformant clones	Phenotype of subclones from Ura− clones that lost plasmid
			Ura+	Ura−
[PSI+u1s]	Vector	96.0 ± 3.0a	0	20
	pUPF2-WT	4.6 ± 1.9b	2	52
	pUPF2-ΔAC	55.5 ± 2.5c	2	48
	pUPF2-ΔU1I	55.2 ± 1.8c	0	41
	pUPF2-ΔACU1I	89.4 ± 2.7a	0	45
[PSI+u2s]	Vector	95.4 ± 4.3a	0	20
	pUPF2-WT	5.1 ± 1.0b	0	36
	pUPF2-ΔAC	56.6 ± 1.6c	1	44
	pUPF2-ΔU1I	48.5 ± 4.5c	1	50
	pUPF2-ΔACU1I	93.9 ± 3.8a	0	41

upf2Δ strains (MS321, MS322, MS323) carrying [PSI+u1s] (cytoductants from Table 6) and upf2Δ strains carrying three [PSI+u2s] (primary isolates in upf2Δ strain MS62 from Table 5) (MS299, MS300, and MS302) were transformed with the CEN vector pRS313 or with the vector carrying the UPF2 or UPF2 mutants under its native promoter. Deletion mutants of pUPF2 were denoted as ΔAC lacking the highly acidic domain (amino acids 886–938), ΔU1I lacking the Upf1p-interacting domain (amino acids 939–1,089), and ΔACU1I for the double mutant. Transformants were selected in the presence of uracil and were replica-plated to a plate lacking uracil. More than 200 transformant clones were investigated in each case. The average percent of Ura⁺ transformants, calculated using six independent experiments, ±SD, is shown. Numbers with different letters are significantly different at a P value of <0.05 based on the Tukey test. Ura⁻ subclones that had lost each plasmid were tested for uracil auxotrophy by replica-plating to confirm that the Ura⁻ transformants had lost [PSI+].