Except where noted, strains were constructed using conventional genetic crosses, the PCR method (Baudin et al., 1993; Longtine et al., 1998) for deletion and tagging of chromosomal genes, and/or plasmid transformations (Table 2). The cdc15-2 marker was introduced as described by Nishihama et al. (2009). In addition to the PCR tagging plasmids described by Longtine et al. (1998), similar plasmids contained CFP sequences (unpublished data), TAP tag sequences (provided by P. Walter, University of California, San Francisco, San Francisco, CA), URA3-kanMX6 (unpublished data), VN173 (Sung and Huh, 2007), or VN155^I153L (Table 2).

Constructed by transforming EcoRV-digested YIp128-P_GAL1RHO1^Q68L into YEF473B.

Constructed by transforming NcoI-digested pRS306-3HA-RHO1 into a YEF473-background rho1Δ::His3MX6/RHO1 heterozygote and selecting Ura ⁺ His⁺ segregants.

Constructed by digesting pRS315-cyk3^TGcΔ with SphI (at −384 relative to the CYK3 start codon) and SacII (at 2,935, 464 bp downstream of the stop codon), transforming into strain RNY2150, and selecting Ura⁻ colonies on an SC + 5-FOA plate. Correct integration was confirmed by showing kanamycin sensitivity and PCR amplifying genomic DNA using appropriate primers.

Constructed by crossing YOC2439 (MATa ade2 his3 lys2 trp1 ura3 Δfks1::HIS3 Δfks2::LYS2 ade3::FKS1-GFP::LEU2; a gift from Y. Ohya, University of Tokyo, Tokyo, Japan) and RNY2046.