Extended Data Table 1 ∣. Engineering and testing of dynein mutants.
a, MT stimulated ATPase activity and velocity analysis of Dyn and DynRK+7hep under different salt conditions. An increase in salt concentration reduces the MT affinity (KM-MT) of Dyn and DynRK+7hep, but it does not alter their directionality. KM-MT of Dyn without added salt is similar to that of DynRK+7hep under 50 mM KAc (Two-sided t-test, p = 0.055). ATPase data was collected from three independent experiments (mean ± s.d.). n = 67, 55, 52, 58 from left to right. b, The list of yeast strains used in this study. These strains were produced by homologous recombination using the template strain (MATa his3-11,5 ura3-1 leu2-3, 112 ade2-1 trp-1 pep4::HIS3 prb1Δ) expressing the tail-truncated yeast cytoplasmic dynein (331Dyn1) under the galactose promoter (pGal) or full-length dynein (471DYN1) under the native promoter. A ZZ-Tev tag was inserted at the N-termini of these constructs for affinity purification. The DHA tag was used to label the motor with fluorescent dyes functionalized with alkyl chloride (F: Figure, EDF: Extended Data Figure, EDT: Extended Data Table).
| a | ||||
|---|---|---|---|---|
| Dyn - 0 mM KAc | Dyn - 50 mM KAc | DynRK+7hep-50 mM KAC | DynRK+7hep-150 mM KAC | |
| kcat (s−1 per head) | 7.3 ± 0.1 | 7.9 ± 0.1 | 6.8 ± 0.2 | 8.5 ± 0.2 |
| kbasai(s−1 per head) | 1.1 ± 0.1 | 2.4 ± 0.1 | 4.6 ± 0.6 | 6.6 ± 0.3 |
| KM (nM) | 19 ± 3.4 | 630 ± 65 | 19 ± 5.1 | 75 ± 4.3 |
| + Runs (nm s−1) | N.A. | N.A. | 18.2 ± 10.9 | 23.9 ± 13.8 |
| − Runs (nm s−1) | 88.2 ± 30.2 | 95.5 ± 51.5 | 10.6 ± 3.6 | 33.3 ± 11.1 |
| + Runs (%) | 0 | 0 | 81 | 83 |
| − Runs (%) | 100 | 100 | 8 | 6 |
| Diffusive (%) | 0 | 0 | 10 | 11 |
| b | ||||
| Strain ID | Construct Name | Description | Source | Dataset |
| VY208 | GFP-GST-Dyn-DHA | GFP-3xHA-GST-331DYN1-gs-DHA | ref. 4 | F1c,d, F4c,d, EDF3d |
| VY268 | DHA-GST-Dyn | DHA-GST-331DYN1 | ref. 4 | F3c F4c,e, EDT1a |
| VY209 | FRB-Dyn-DHA | 3xHA-FRB-331DYN1-gs-DHA | ref. 4 | F2e,f, EDF6c, EDF7 |
| Y144 | GFP-Dyn-FKBP-DHA | GFP-3xHA-331DYN1-gsgsgsgs-FKBP12 URA3 | ref. 16 | F3a,b,e, EDF9 |
| Y124 | GFP-Dyn−3hep-DHA | GFP-3xHA-331DYN1-(−3hep3036/3292)-331DYN1-gs-DHA | This study | F1c,d, EDF3d |
| Y200 | GFP-GST-Dyn+3hep-DHA | GFP-3xHA-GST-331DYN1-(+3hep3085/3159)-331DYN1-gs-DHA | This study | F1c,d, EDF3d |
| Y123 | GFP-Dyn+7hep-DHA | GFP-3xHA-331DYN1-(+7hep3085/3159)-331DYN1-gs-DHA | ref. 12 | F4b, EDF3d |
| Y197 | GFP-GST-DynRK-DHA | GFP-3XHA-GST-331DYN1I3101P/P3103A.P3229A/R3231P | This study | F4b |
| Y187 | GFP-GST-DynRK+7+hep-DHA | GFP-3xHA-GST-331DYN1I3101P/P3103A-(+7hep3085/3159)-331DYN1P3229A/R3231P-gs-DHA | This study | F3c, F4a,b,c,e |
| Y198 | GFP-DynRK+7hep-DHA | GFP-HA-331DYN1I3101P/P3103A-(+7hep3085/3159)-331DYN1P3229A/R3231P-gs-DHA | This study | F3a,b,e, EDF8, EDF9 |
| Y201 | FRB-DynRK+7hep-DHA | 3xHA-FRB-331DYN1I3101P/P3103A-(+7hep3085/3159)-331DYN1P3229A/R3231P-gs-DHA | This study | F2e,f, EDF6c, EDF7 |
| Y202 | GFP-471DynRK+7hep-DHA | GFP-3xHA-471DYN1I3101P/P3103A-(+7hep3085/3159)-331DYN1P3229A/R3231P-gs-DHA | This study | F4d, EDT1a |