Fig 2. CLD1 is an Allele-specific Suppressor of Hypomorphic COQ7.

(A) Schematic of genomic library screening protocol: coq7-19(Δcoq7) yeast carrying the hypomorphic coq7-22(P_ADH1-COQ7-HA) allele on a low copy number (CEN) plasmid were transformed with a library of genomic fragments cloned into high-copy plasmid pRS424 (2μ origin of replication). Suppressors of the ‘hypomorphic growth on 3% ethanol’ phenotype of the parent strain were then isolated. (B) One library suppressor clone (sup+) isolated conferred allele-specific suppression to coq7-9 (P_COQ7-COQ7-Myc (CEN)), coq7-10 (P_COQ7-COQ7-HA (CEN)), coq7-11_i(W120R), but not coq7-5_i(H183L); indicating suppression did not depend upon the HA epitope tag, the ADH1 promoter, or simply alter plasmid copy number of hypomorphic P_ADH1-COQ7-HA(CEN). Both the coq7-5 and coq7-11 alleles were re-integrated (subscript ‘i’) into the wild type COQ7 locus. Growth enhancement was more pronounced at 35°C for coq7-10 and coq7-11. [vector: pRS424 (2μ)] (C) Chromosomal map showing genetic landscape of library suppressor clone (sup+) and the fragment boundaries used for various plasmid constructs. 1D, 2C, 2G, 4a and 4b indicate independent PCR amplicons. (D) Growth suppression requires residual COQ7 activity to mediate enhanced growth on 3% ethanol (30°C). Strain genotype (top) and transformed plasmid identity (see panel C) are indicated. (Prom.—promoter). (E) The CLD1 locus is sufficient to confer growth enhancement on 3% ethanol to coq7-11 (30°C, left panel) and coq7-10 (35°C, right panel). Strain genotype (top) and transformed plasmid identity (refer to panel C) are indicated. (F) Quantification of coq7-11_i(W120R) growth inhibition in 3% ethanol (30°C) and its suppression by overexpression of CLD1. Neither the CLD1 promoter nor the cld1 loss-of-function 2C amplicon are able to suppress the slow growth phenotype of coq7-11_i(W120R). (wt, wild type). Part of the promoter of CLD1 was removed when constructing the 1D amplicon to avoid incorporation of the Ty1 LTR into the clone and we presume this is the reason we did not observe the same degree of suppression as the original library clone (sup+). Disruption of just CLD1 within the sup+ clone background did not confer any suppression (see later, main text). (G) The ability of CLD1 to suppress hypomorphic coq7-11i (W120R) is independent of strain background. TA405 [COQ7/coq7-11_i(W120R)] heterozygous diploids were sporulated and four independent haploid coq7-11_i(W120R) isolates retained (left). Plasmid DNA (both CEN and 2μ) containing various CLD1 derivatives were transformed into TA405[coq7-11_i(W120R)] or control SEY6210[coq7-11i (W120R)] yeast (right) and then growth enhancement on 3% ethanol quantified at 30°C after 8 days.