Table 2.
Plasmids used in this studya
| Plasmid | Mutation introduced | Genotype | Reference or source |
|---|---|---|---|
| pMSG370 | WT | CEN URA3 pRS316-ENV7 | 43 |
| pMSG470 | WT | 2 μm URA3 pRS426-ENV7 | 43 |
| pSMG44 | WT | CEN URA3 pRS316-YCK3 | This study |
| pSMG45 | WT | 2 μm URA3 PPGK-YCK3-3×HA | This study |
| pSMG46 | WT | 2 μm URA3 PPGK-3×HA-YCK3 | This study |
| pSMG47 | WT | pET24d(+)-YCK3-6×His | This study |
| pSMG34 | WT | pET24d(+)-ENV7-6×His | 44 |
| pSMG17 | WT | 2 μm URA3 PPGK-ENV7-3×HA | 44 |
| pSMG19 | WT | CEN URA3 PGAL1- ENV7-GFP | 44 |
| pSMG30 | Env7-C13-15S-HA | 2 μm URA3 PPGK-ENV7-C13-15S-3×HA | 44 |
| pSMG48 | Env7-(1-304)-HA (truncated) | 2 μm URA3 PPGK-ENV7-(1-304)-3×HA | This study |
| pSMG49 | Env7-S331A-HA | 2 μm URA3 PPGK-ENV7- S331A-3×HA | This study |
| pSMG50 | Env7-S323A-S328A-S331A-HA | 2 μm URA3 PPGK-ENV7-S323A-S328A-S331A-3×HA | This study |
| pSMG51 | Env7-T351A-T352A-S353A-HA | 2 μm URA3 PPGK-ENV7-T351A-T352A-S353A-3×HA | This study |
| pSMG52 | Env7-6×-HA (sextuple mutant, overexpressed) | 2 μm URA3 PPGK-ENV7-S323A-S328A-S331A-T351A-T352A-S353A-3×HA | This study |
| pSMG53 | Env7-5×-A323S-HA (add-back) | 2 μm URA3 PPGK-ENV7-A323S-S328-AS331A-T351A-T352A-S353A-3×HA | This study |
| pSMG54 | Env7-5×-A328S-HA (add-back) | 2 μm URA3 PPGK-ENV7-S323A-A328S-S331A-T351A-T352A-S353A-3×HA | This study |
| pSMG55 | Env7-5×-A331S-HA (add-back) | 2 μm URA3 PPGK-ENV7-S323A-S328A-A331S-T351A-T353A-353A-3×HA | This study |
| pSMG56 | Env7-5×-A353S-HA (add-back) | 2 μm URA3 PPGK-ENV7-S323A-S328A-A331S-T351A-T352A-353S-3×HA | This study |
| PSMG57 | Env7-6× (sextuple mutant, native) | CEN URA3 pRS316-ENV7-A323S-S328A-S331A-T351A-T352A-S353A | This study |
| pSMG58 | Env7-5×-A323S (add-back) | CEN URA3 pRS316-ENV7-A323S-S328A-S331A-T351A-T352A-S353A | This study |
| pSMG59 | Env7-5×-A328S (add-back) | CEN URA3 pRS316-ENV7-S323A-A328S-S331A-T351A-T352A-S353A | This study |
| pSMG60 | Env7-5×-A331S (add-back) | CEN URA3 pRS316-ENV7-S323A-S328A-A331S-T351A-T352A-S353A | This study |
| pSMG61 | Env7-S331d-HA (overexpressed) | 2 μm URA3 PPGK-ENV7-S331d-HA | This study |
| pSMG62 | Env7-5×-A331D-HA (overexpressed) | 2 μm URA3 PPGK-ENV7-S323A-S328A-A331D-T351A-T352A-S353A-3×HA | This study |
| pSMG63 | Env7-S331D (native) | CEN URA3 pRS316-ENV7-S331D | This study |
| pSMG64 | Env7-5×-A331D (native) | CEN URA3 pRS316-ENV7-A323S-S328A-A331D-T351A-T352A-S353A | This study |
| pSMG65 | Env7-GFP (WT, native) | CEN URA3 pRS316-ENV7-GFP | This study |
| pSMG66 | Env7-S331D-GFP (native) | CEN URA3 pRS316-ENV7-S331D-GFP | This study |
| pSMG67 | Env7-5×-A331D-GFP (sextuple mutant, native) | CEN URA3 pRS316-ENV7-S323A-A328S-A331D-T351A-T352A-S353A-GFP | This study |
| pSMG68 | Env7-S323A-S331A-HA | 2 μm URA3 PPGK-ENV7-S323A-S331A-3×HA | This study |
aEnv7 amino acid change(s) introduced is shown in boldface. Overexpressed HA-tagged Env7 and Yck3 were expressed from yeast 2 μm URA3 PPGK plasmids. Native levels of Env7, Yck3, and GFP-tagged Env7 were expressed from their native promoters in yeast CEN URA3 pRS316 plasmids.