(A) Domain organization of dynein and Lis1 constructs used in this study. Dynein's AAA+ domains are labeled AAA1–6. MTBD: microtubule binding domain; CC: coiled coil; LisH: Lis-homology …
(A) SDS-PAGE of dynein and Lis1, affinity purified from S. cerevisiae. (B) Comparison between re-projections of the dynein and dynein–Lis1 reconstructions and the best-matching reference-free class …
(A) Zoomed out view of dynein–Lis1; only the portion of the crystal structure corresponding to AAA4 is displayed, in yellow (PDB ID: 4AKG [Schmidt et al., 2012]). (B) Close-up of the N-terminal …
(A) The Lis1 β-propeller engages dynein primarily at a surface helix connecting AAA3 and AAA4 (yellow arrowhead, see Video 3). Inset: a zoomed out view. (B) (Left) View along the axis highlighted in …
(A) Sequence identity (100%, purple; 0%, white) mapped onto the Lis1 homology model. The alignment was carried out with the following species: M. musculus, H. sapiens, S. cerevisiae, A. nidulans, D. …
Histogram showing the velocity distribution of single TMR-labeled GST-dynein331kDa molecules in the absence of Lis1 (black) and with 200 nM wild-type Lis1 (light gray), Lis1R378A (medium gray) and …
(A) Cryo-NS maps of S. cerevisiae dynein in 100 μM ADP displaying the linker next to either AAA5 (left) or AAA4 (right). The S. cerevisiae linker domain (lacking nucleotide at AAA1, PDB ID: 4AKG [Sch…
(A) Diagram of a microtubule-gliding assay. Monomeric GFP-dynein molecules are immobilized on the coverslip via anti-GFP antibodies (Y shape). Dynein-driven gliding of fluorescently labeled (purple …
Microtubule-stimulated ATPase activity of dynein monomers carrying (A) wild-type AAA+ modules, (B) a hydrolysis deficient E1849Q mutation in AAA1 (Kon et al., 2004), (C) a hydrolysis deficient …
(A–D) SDS-PAGE of fractions eluted from size-exclusion chromatography runs of Lis1 mixed with each of the dynein constructs used in the ATPase assays. Lis1 co-elutes with all of the constructs.
(A) A short linker construct was designed by docking the crystal structure of the D. discoideum linker (purple ribbon) (PDB ID: 3VKG [Kon et al., 2012]) into our EM map of dynein alone and …
(A) Single-molecule motility assays. Kymographs of GST-dimerized full-length and short linker dyneins. Horizontal scale bar = 2 μm, vertical = 30 s. (B) Velocity and run length for short linker and …
(A–G) Current view of dynein's mechanochemical cycle. (A) ATP binding to AAA1 induces the low-affinity conformation in dynein's microtubule-binding domain and (B) release from the microtubule. (C) …
The movie shows the 3D reconstruction of dynein in complex with Lis1 with 360° rotation about the Y-axis. After this rotation, the EM density is made transparent to display the docked dynein crystal …
The movie shows the 3D reconstruction of dynein alone with 360° rotation about the Y-axis. After this rotation, the EM density is made transparent to display the docked dynein crystal structure …
The movie shows the 3D reconstruction of dynein–Lis1, with the crystal structure of the dynein motor domain and the Lis1 homology model docked in. After a few frames, the EM density disappears to …
Yeast strains
Strain | Genotype | Figure(s) |
---|---|---|
Reference | ||
RPY753 | MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PGAL1-ZZ-Tev-GFP-3xHA-GST-DYN1331kDa-gs-DHA, pac1Ä::URA3, ndl1Ä::cgLEU2 | Figure 2, Figure 2—figure supplement 1,2, Figure 5—figure supplement 1 |
Huang et al., 2012 | ||
RPY816 | MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PGAL1-ZZ-Tev-PAC1, dyn1Ä::cgLEU2, ndl1Ä::HygroR | Figures 1–5, Figure 2—figure supplement 1,2, Figure 1—figure supplement 1, Figure 4—figure supplement 1, Figure 5—figure supplement 1 |
Julie Huang, Harvard Medical School | ||
RPY842 | MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PGAL1-ZZ-Tev-PAC1-g-1xFLAG-ga-SNAP-KanR, dyn1Ä::cgLEU2, ndl1Ä::HygroR | Figures 3,5, Figure 3—figure supplement 1, Figure 5—figure supplement 1 |
Huang et al., 2012 | ||
RPY844 | MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PAC11-13xMYC-TRP1, PGAL1-ZZ-Tev-GFP-3xHA-DYN1331kDa, pac1Ä::HygroR | Figures 1,4, Figure 1—figure supplement 1, Figure 3—figure supplement 1 |
Huang et al., 2012 | ||
RPY1198 | MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PAC11-13xMYC-TRP1, PGAL1-ZZ-Tev-GFP-3xHA-DYN1331kDa-gs-DHA-KanR, pac1Ä::HygroR | Figure 5, Figure 5—figure supplement 1 |
Huang et al., 2012 | ||
RPY1245 | MATa, ura3-52, lys2-801, leu2-Ä1, his3-Ä200, trp1-Ä63, SPC110-GFP::TRP1, HXT1-tdTomato::HIS3 | Figure 2 |
Jeff Moore, University of Colorado | ||
RPY1248 | MATa, ura3-52, lys2-801, leu2-Ä1, his3-Ä200, trp1-Ä63, SPC110-GFP::TRP1, HXT1-tdTomato::HIS3, dyn1Ä::URA3 | Figure 2 |
This work | ||
RPY1302 | MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PAC11-13xMYC-TRP1, PGAL1-ZZ-Tev-DYN1331kDa, pac1Ä::HygroR | Figures 1,3 |
This work | ||
RPY1400 | MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PAC11-13xMYC-TRP1, PGAL1-ZZ-Tev-GFP-3xHA-DYN1331kDa-L2441ybbR, pac1Ä::HygroR | Figure 3, Figure 3—figure supplement 1 |
This work | ||
RPY1422 | MATa, his3-11,15, ura3-52, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PGAL1-ZZ-Tev-GFP-3xHA-DYN1314kDa-gs-DHA, pac1Ä::HygroR | Figures 4,5, Figure 4—figure supplement 1, Figure 5—figure supplement 1 |
This work | ||
RPY1436 | MATa, his3-11,15, ura3-52, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PAC11-13xMYC-TRP1, PGAL1-ZZ-Tev- DYN1314kDa, pac1Ä::HygroR | Figure 5 |
This work | ||
RPY1439 | MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PGAL1-ZZ-Tev-GFP-3xHA-GST-DYN1314 kDa-gs-DHA-KanR, pac1Ä:URA3, ndl1Ä::cgLEU2 | Figure 5—figure supplement 1 |
This work | ||
RPY1509 | MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PAC11-13xMYC-TRP1, PGAL1-ZZ-Tev-DYN1331kDa-gs-DHA-KanR, pac1Ä::HygroR | Figure 5—figure supplement 1 |
This work | ||
RPY1510 | MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PAC11-13xMYC-TRP1, PGAL1-ZZ-Tev-DYN1314kDa-gs-DHA-KanR, pac1Ä::HygroR | Figure 5—figure supplement 1 |
This work | ||
RPY1523 | MATa, ura3-52, lys2-801, leu2-Ä1, his3-Ä200, trp1-Ä3, SPC110-GFP::TRP1, HXT1-tdTomato::HIS3, pac1Ä::URA3 | Figure 2 |
This work | ||
RPY1524 | MATa, ura3-52, lys2-801, leu2-Ä1, his3-Ä200, trp1-Ä63, SPC110-GFP::TRP1, HXT1-tdTomato::HIS3, PAC1R378A | Figure 2 |
This work | ||
RPY1525 | MATa, ura3-52, lys2-801, leu2-Ä1, his3-Ä200, trp1-Ä63, SPC110-GFP::TRP1, HXT1-tdTomato::HIS3, PAC1R275A,R301A,R378A,W419A,K437A | Figure 2 |
This work | ||
RPY1543 | MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PGAL1-ZZ-Tev-PAC1R275A, dyn1Ä::cgLEU2, ndl1Ä::HygroR | Figure 2—figure supplement 1 |
This work | ||
RPY1544 | MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PGAL1-ZZ-Tev-PAC1R378A, dyn1Ä::cgLEU2, ndl1Ä::HygroR | Figure 2, Figure 2—figure supplement 1,2 |
This work | ||
RPY1545 | MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PGAL1-ZZ-Tev-PAC1W419A, dyn1Ä::cgLEU2, ndl1Ä::HygroR | Figure 2—figure supplement 1 |
This work | ||
RPY1546 | MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PGAL1-ZZ-Tev-PAC1K437A, dyn1Ä::cgLEU2, ndl1Ä::HygroR | Figure 2—figure supplement 1 |
This work | ||
RPY1547 | MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PGAL1-ZZ-Tev-PAC1 R275A,R301A,R378A,W419A,K437A, dyn1Ä::cgLEU2, ndl1Ä::HygroR | Figure 2, Figure 2—figure supplement 1,2 |
This work | ||
RPY1548 | MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PGAL1-ZZ-Tev-PAC1R301A, dyn1Ä::cgLEU2, ndl1Ä::HygroR | Figure 2—figure supplement 1 |
This work | ||
RPY1553 | MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PAC11-13xMYC-TRP1, PGAL1-ZZ-Tev-GFP-3xHA-DYN1331kDaE1849Q, pac1Ä::HygroR | Figure 4, Figure 4—figure supplement 1 |
This work | ||
RPY1554 | MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PAC11-13xMYC-TRP1, PGAL1-ZZ-Tev-GFP-3xHA-DYN1331kDaE2819Q, pac1Ä::HygroR | Figure 4, Figure 4—figure supplement 1 |
This work | ||
RPY1555 | MATa, his3-11,15, ura3-52, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PGAL1-ZZ-Tev-GFP-3xHA-DYN1314kDaK3438E,R3445E,F3446D-gs-DHA, pac1Ä::HygroR | Figure 4—figure supplement 1, Figure 5—figure supplement 1 |
This work | ||
RPY1557 | MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PAC11-13xMYC-TRP1, PGAL1-ZZ-Tev-GFP-3xHA-DYN1331kDaK3438E,R3445E,F3446D-gs-DHA-KanR, pac1Ä::HygroR | Figure 4, Figure 4—figure supplement 1 |
This work | ||
RPY1623 | MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PGAL1-ZZ-Tev-GFP-3xHA-GST- DYN1331kDaR2857A,N2858A,K2859A,R2861A,S2862A-gs-DHA, pac1Ä::URA3, ndl1Ä::cgLEU2 | Figure 1—figure supplement 2 |
This work |
DYN1, PAC11, PAC1, and NDL1 encode the dynein heavy chain, dynein intermediate chain, Lis1 and Nudel orthologs, respectively. DHA, SNAP, and ybbR refer to the HaloTag (Promega), SNAP-tag (NEB), and ybbR tag (Yin et al., 2005), respectively. TEV indicates a Tev protease cleavage site. PGAL1 denotes the galactose promoter, which was used for inducing strong expression of Lis1 and dynein motor domain constructs. Genes encoding proteases Pep4 and Prb1 were deleted as noted. Amino acid spacers are indicated by g (glycine), ga (glycine-alanine), and gs (glycine-serine).
Dynein:Lis1 ratios in complexes purified by size-exclusion chromatography
GST-dynein331kDa | Lis1 | Lis1 (normalized to WT ratio) | |
---|---|---|---|
WT Lis1 | 0.82 ± 0.01 | 0.18 ± 0.01 | 1.00 |
Lis1R275A | 0.85 ± 0.01 | 0.15 ± 0.01 | 0.80 |
Lis1R301A | 0.88 ± 0.01 | 0.12 ± 0.01 | 0.62 |
Lis1R378A | 1.00 ± 0.00 | 0.00 ± 0.00 | 0.00 |
Lis1W419A | 1.00 ± 0.00 | 0.00 ± 0.00 | 0.00 |
Lis1K437A | 0.85 ± 0.01 | 0.15 ± 0.01 | 0.80 |
Lis15A | 1.00 ± 0.00 | 0.00 ± 0.00 | 0.00 |
In relation to Figure 2 and Figure 2—figure supplement 1. Fractions were run on SDS-PAGE, stained with SYPRO red, and the bands corresponding to GST-dynein331kDa and wild-type/mutant Lis1 were quantified using ImageJ. The quantification was done using three adjacent lanes corresponding to the peak from size-exclusion. Values are averages of the three lanes ± SD. The ratio for each mutant normalized against that of wild-type Lis1 is also shown.
ATPase assay rate measurements
Sample | Km(MT)(ìM) | kbasal(Motor domain−1.s−1) | kcat(Motor domain−1.s−1) |
---|---|---|---|
Full-length linker | 1.06 ± 0.16 | 3.51 ± 0.31 | 16.75 ± 0.49 |
+Lis1 | 1.09 ± 0.20 | 4.36 ± 0.30 | 15.06 ± 0.49 |
Short linker | 0.92 ± 0.10 | 4.45 ± 0.22 | 16.98 ± 0.32 |
+Lis1 | 2.05 ± 0.44 | 7.14 ± 0.21 | 16.12 ± 0.61 |
Full-length linker, AAA4 ATPase mutant (E2819Q) | 1.55 ± 0.14 | 4.53 ± 0.17 | 18.80 ± 0.38 |
+Lis1 | 1.10 ± 0.15 | 4.60 ± 0.19 | 13.93 ± 0.31 |
Data were fit to the following equation: kobs = (kcat − kbasal) − [MT]/(Km(MT) + [MT]) + kbasal. Km(MT) is the microtubule concentration that gives half-maximal activation. Values are the averages of triplicate readings ± SE of the fit.