Table 1.
Plasmids and strains used in this study
| Plasmid or strain | Genotype or relevant characteristics | Source or reference |
|---|---|---|
| Plasmid | ||
| pRS415 | Single-copy E. coli/S. cerevisiae shuttle vector with LEU2 | (139) |
| Derivative | ||
| pGH315 | PAH1 with native promoter in pRS415 | (59) |
| pGH315-D398E | pGH315 with the D398E mutation in PAH1 | (98) |
| pGH315-D400E | pGH315 with the D400E mutation in PAH1 | (98) |
| pGH315-T443L | pGH315 with the T443L mutation in PAH1 | This study |
| pGH315-R445H | pGH315 with the R445H mutation in PAH1 | This study |
| pGH315-K496A | pGH315 with the K496A mutation in PAH1 | This study |
| pGH315-G529A | pGH315 with the G529A mutation in PAH1 | This study |
| pGH315-N530A | pGH315with the N530A mutation in PAH1 | This study |
| pGH315-D534A | pGH315 with the D534A mutation in PAH1 | This study |
| YCplac33 | Single-copy E. coli/S. cerevisiae shuttle vector with URA3 | (140) |
| Derivative | ||
| YCplac33-SEC63-GFP | YCplac33 with SEC63-GFP fusion | (33) |
| pET-15b | E. coli IPTG-inducible expression vector with N-terminal His6-tag fusion | Novagen |
| Derivative | ||
| pGH313 | pET-15b with PAH1 coding sequence | (1) |
| pGH313-D398E | pGH313 with the D398E mutation in PAH1 | (70) |
| pGH313-T443L | pGH313 with the T443L mutation in PAH1 | This study |
| pGH313-K496A | pGH313 with the K496A mutation in PAH1 | This study |
| pGH313-N530A | pGH313 with the N530A mutation in PAH1 | This study |
| pGH313-ΔAH | pGH313 with the ΔAH (Δ2-18) mutation in PAH1 | This study |
| Strain | ||
| S. cerevisiae | ||
| RS453 | MATaade2-1 his3-11,15 leu2-3112 trp1-1 ura3-52 | (141) |
| Derivative | ||
| SS1026 | pah1Δ::TRP1 | (29) |
| W303-1A | MATaade2-1 can1-100 his3-11,15 leu2-3112 trp1-1 ura3-1 | (142) |
| Derivative | ||
| GHY66 | pah1Δ::URA3 app1Δ::natMX4 dpp1Δ::TRP1/Kanrlpp1Δ::HIS3/Kanr | (8) |
| E. coli | ||
| DH5⍺ | F- Φ80 lacZΔM15Δ (lacZYA-argF)U169 deoR recA1 endA1 hsdR17 (rk- mk+) phoA supE44 λ−thi-1 gyrA96 relA1 | (124) |
| NiCo21 (DE3)pLysSRARE2 | can::CBD fhuA2 [Ion] ompT gal (λ DE3) [dcm] amA::CBD sly::CBD glmS6Ala ΔhsdS λ DE3 = λ sBamHIo ΔEcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 Δnin5 pLysSRARE2 | New England Biolabs |