Construction of Gap1 Mutant Alleles

The plasmids used in this study are listed in Table S2. All derive from the centromere-based pRS416 [34] (link) or pFL38 [35] (link) vectors carrying the URA3 gene. The 64 mutant gap1 alleles were constructed by recombination in yeast between two partially overlapping PCR fragments corresponding to the 5′ and 3′ regions of the GAL-GAP1-GFP gene (Fig. S1). The overlapping sequence was 40 bp long and contained the sequences so as to introduce 3 or 4 consecutive alanine substitutions. The pCJ130 recipient plasmid, a pRS416 vector containing the GAL-YCH1-GFP gene, was linearized with BamHI and treated with alkaline phosphatase. Each mutant gap1 gene was purified by cloning into E. coli and verified by sequencing. The sequences of the 128 oligonucleotides used to construct the 64 mutant genes are available upon request.