Sequence-encoding full-length Myo4p (1,472 amino acids) was cloned into pVL1392 with a C-terminal FLAG tag to facilitate purification by affinity chromatography. A second full-length Myo4p construct contained a biotin tag at the N terminus for attachment to streptavidin Qdots (Invitrogen). The biotin tag is an 88–amino acid sequence segment from the Escherichia coli
biotin carboxyl carrier protein, which, when expressed in Sf9 cells, is biotinated at a single Lys residue (Cronan, 1990
; Li and Cronan, 1992
). A truncated Myo4p construct lacking the motor domain and neck (K924 to the end of MYO4
) with C-terminal FLAG tag was also cloned into pAcSG2 for baculovirus expression. Yeast CaM and the yeast light chain Mlc1p were separately cloned into the pAcUW51 plasmid under the polyhedrin promoter. A C-terminally HIS-tagged Mlc1p was cloned into pAcSG2 (BD) and a C-terminal Myc-tagged yeast CaM in pacUW51. These constructs were used to facilitate identification of the bound light chains by Western blotting. For bacterial expression, yeast CaM and Mlc1p were each cloned into pNew (a kanamycin-resistant plasmid with T7 promoter). SHE3
with a C-terminal His tag was cloned into the baculovirus vector pAcSG2 as previously described (Hodges et al., 2008
The SHE2 gene was amplified by PCR from S. cerevisiae genomic DNA and verified as the coding sequences found at the Saccharomyces genome database. For cellular studies, SHE2 was cloned into a pRS314 derivative with ADE2 selection behind the inducible GAL1 promoter. For bacterial expression, the SHE2 gene was cloned into pGEX-2T. A mutant She2p called WT* had four Cys residues mutated to Ser (C14S, C68S, C106S, and C180S). All additional variations of She2p were made on the WT* backbone for both the cellular and in vitro experiments. This includes two point mutants (S120Y and L130Y), a Δhelix construct (lacking residues 174–179; VQFAIK), and a construct containing a C-terminal YFP (She2p-GSG-YFP-FLAG). All changes were verified by DNA sequencing. She2p constructs cloned with fluorescent proteins at either the N or C terminus were shown to be completely functional in Δshe2 yeast cells.
actin was obtained from genomic DNA and cloned into the pAcSG2 baculovirus vector following a FLAG and recombinant Tobacco Etch Virus (rTEV) cleavage site (DYKDDDDK-ENLYFQ-MDSEV …). This construct allowed the actin to be purified on a FLAG column followed by cleavage of the tag with rTEV protease (between Q and M), removing all nonnative residues before the start codon of the yeast gene. Met was included in the actin sequence because actin retains its initiator Met in yeast (Cook et al., 1991
Budding yeast tropomyosin (Tpm1p) was a PCR product made using S. cerevisiae
genomic DNA. To mimic acetylation, an Ala-Ser was added at the N terminus before the start codon (MASMDSEV …) (Maytum et al., 2001
). The construct was cloned into the pET3 vector.
Protein expression and purification
Myosin constructs were expressed in Sf9 cells using the baculovirus system and purified on a FLAG affinity column essentially as previously described (Fig. S1; Krementsova et al., 2006
). In brief, Sf9 cells were infected with recombinant viruses for the Myo4p heavy chain, She3p, yeast CaM, and MLC1p and incubated with shaking for ~72 h at 27°C. Cells were pelleted and resuspended in 10 mM imidazole, pH 7.4, 0.3 M NaCl, 5 mM MgCl2
, 7% sucrose, 1 mM EGTA, 2 mM DTT, 25 µg/ml yeast CaM, 25 µg/ml MLC1p, and protease inhibitors (0.5 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 5 µg/ml leupeptin, and 0.78 mg/ml benzamidine). The cells were lysed by sonication, and the lysate was centrifuged for 15 min at 250,000 g
in the presence of 2 mM MgATP. After incubation with FLAG affinity resin (Sigma-Aldrich) for 30 min, the resin was sedimented for 5 min at 1,000 rpm and washed with buffer (10 mM imidazole, pH 7.4, 0.3 M NaCl, and 1 mM EGTA). Bound protein was eluted using a 0.1-mg/ml solution of FLAG peptide in the same buffer. Fractions were pooled, concentrated in 50% glycerol, and stored at −20°C.
All She2p variants were expressed as GST fusion proteins in E. coli BL21(DE3). A 5-ml overnight culture grown in Lennox-L broth was diluted 50 times into enriched media and grown for 3.5 h at 37°C. After induction with 50 µM IPTG, the cells were grown overnight at 25°C. Cells were lysed by sonication in PBS with protease inhibitors and loaded onto a 20-ml glutathione Sepharose 4B column (GE Healthcare). The column was washed with 5 vol PBS and drained. 90 U thrombin (Haematologic Technologies, Inc.) in 5 ml PBS was added to the column, and the slurry was incubated for 1 h at room temperature with occasional mixing. The column was drained and washed with ~25 ml PBS. 1 mM DTT was added followed by 2 µg/ml leupeptin to inactivate the thrombin. Thrombin was removed by passing the mixture over a benzamidine Sepharose 6B column (GE Healthcare). The protein was concentrated in a 50-ml Centricon (Millipore) to ~5 ml and stored in 50% glycerol, 10 mM imidazole, pH 7.4, 0.2 M NaCl, and 1 mM DTT.
Chicken skeletal actin was prepared from acetone powder (Pardee and Spudich, 1982
). Yeast actin was expressed using the baculovirus/insect cell expression system. Sf9 cells were infected with recombinant baculovirus encoding yeast actin and harvested 72 h later. Cells were lysed using 1 M Tris-HCl, pH 7.5, 0.6 M KCl, 0.5 mM MgCl2
, 0.5 mM NaATP, 1 mM DTT, 4% Triton X-100, 1 mg/ml Tween 20, 0.5 mM AEBSF, 0.5 mM tosyllysine chloromethyl ketone, and 5 µg/ml leupeptin and stirred for 1.5 h at 4°C. The lysate was clarified for 1 h at 177,700 g
. The supernatant was dialyzed overnight against a buffer containing 300 mM NaCl, 10 mM imidazole, pH 7.5, 0.2 mM CaCl2
, 0.5 mM NaATP, 0.2 mM DTT, and 1 µg/ml leupeptin. After clarifying at 25,000 g
, the supernatant was incubated with 3 ml FLAG resin for 1 h. The resin was washed with the same buffer as above but without NaATP and DTT. Actin was eluted using 100 µg/ml FLAG peptide in 300 mM NaCl, 10 mM imidazole, pH 7.5, 0.2 mM CaCl2
, 0.5 mM NaATP, and 1 µg/ml leupeptin. Actin was then dialyzed against G buffer overnight (5 mM Tris, pH 8.3, 0.2 mM CaCl2
, 0.2 mM DTT, 0.2 mM NaATP, and 1 µg/ml leupeptin), clarified for 30 min at 400,000 g
, and concentrated. The actin concentration was determined by the Bradford assay, and rTEV protease (Miller and Trybus, 2008
) was added at a 1:8 molar ratio (rTEV protease/actin) and incubated overnight. G-actin was polymerized by adding 50 mM KCl, 4 mM MgCl2
, and 1 mM MgATP.
Tpm1p was expressed in E. coli
BLR(DE3) cells grown in Lennox-L broth. They were induced with 0.4 mM IPTG and grown overnight at 27°C before being harvested and frozen. Tpm1p was purified using a protocol similar to that in Maytum et al. (2000)
. In brief, cells were lysed by sonication. The clarified supernatant was boiled for 10 min and clarified for 10 min at 10,000 rpm. The soluble Tpm1p was precipitated by lowering the pH to 4.5. The Tpm1p was further purified using a 1-ml MonoQ column with 0–1 M NaCl gradient with the protein eluting at ~250 mM NaCl. Only fractions that showed a single band by SDS-PAGE and that had a 260:280-nm absorbance ratio below 0.8 were kept. Tpm1p was concentrated and stored in 50% glycerol with 0.2 M NaCl, 10 mM imidazole, pH 7.4, 1 mM EGTA, 1 µg/ml leupeptin, and 1 mM DTT at −20°C.
Yeast CaM and Mlc1p were expressed in E.coli BL21(DE3) grown in enriched media overnight. Cells were lysed by sonication in 50 mM Tris-Cl, pH 7.5, 2 mM EDTA, 0.2 mM AEBSF, and 1 µg/ml leupeptin and clarified for 30 min at 27,000 g. The supernatant was boiled for 5 min, cooled, and clarified for 15 min at 27,000 g. For yeast CaM, 5 mM CaCl2 and 1 mM DTT were added, and the protein bound to a Phenyl-Sepharose CL-4B column (GE Healthcare) was preequilibrated with 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 5 mM CaCl2, and 1 mM DTT. The column was washed with the same buffer containing 0.1 mM CaCl2 and then the same low-calcium buffer with 0.5 M NaCl. Bound protein was eluted with 50 mM Tris-Cl, pH 7.5, 1 mM DTT, and 1 mM EGTA. The protein was stored in 50% glycerol, 10 mM imidazole, pH 7.4, 0.2 M NaCl, 1 mM EGTA, and 1 mM DTT at −20°C. For Mlc1p, the clarified supernatant after boiling was precipitated with ammonium sulfate to 80% saturation, pelleted for 15 min at 27,000 g, and dialyzed against 20 mM imidazole, pH 7.4, 200 mM NaCl, and 1 mM EGTA. Mlc1p was stored in 50% glycerol, 10 mM imidazole, pH 7.4, 0.2 M NaCl, 1 mM EGTA, and 1 mM DTT at −20°C.
ASH1 mRNA reporter system
A two-plasmid system was used to follow ASH1
mRNA localization. The two plasmids pG14-MS2-GFP with LEU2
selection and YEplac195 lacZ
selection were gifts from R. Long (Medical College of Wisconsin, Milwaukee, WI; Bertrand et al., 1998
). pG14-MS2-GFP expresses an MS2 coat protein–GFP under a constitutive promoter. The MS2-GFP chimera contains an NLS to confine unbound MS2-GFP to the nucleus. YEplac195 lacZ
expresses the ASH1
mRNA along with six stem loop–binding motifs for the MS2 coat protein under control of a galactose-inducible promoter. The URA3
marker in YEplac195 lacZ
was replaced with HIS3
Visualization of transformed yeast
The mutant yeast strain K5477 (MATα ade2 his3 leu2 trp1 ura3 can1-100 she2::URA [W303a background]) was used. The yeast strain she2Δ was transformed with three plasmids. Two were required for the ASH1 reporter system. The third plasmid contained the various forms of She2p, including a WT used as a positive control and a plasmid without insert as the negative control. Transformants were grown on the appropriate complete synthetic medium lacking the nutrients needed to maintain the plasmids. To induce expression of the various versions of She2p along with the reporter mRNA, a single colony was grown overnight in the same synthetic medium containing 2% galactose as the sole carbon source. 4 h before visualization, the yeast cells were streaked in a fresh patch to ensure exponential growth when viewed and scored. The live cells were mounted on a slide in the appropriate liquid medium containing galactose and viewed with an inverted microscope (TE2000-E2; Nikon) using a Plan Apo 60× oil objective lens and differential interference contrast and FITC filters. A fluorescence illuminator (X-Cite 120; EXFO) and a 14-bit camera (CoolSNAP HQ2; Photometrics) were used. Images were processed using NIS Elements (Nikon) and ImageJ (National Institutes of Health) software. Actively budding yeast were scored according to the location and number of fluorescent particles seen. Actively budding yeast cells that contained one fluorescent mRNA particle were scored according to whether the particle was located in the bud tip (correct localization) or the mother (incorrect localization).
An analytical ultracentrifuge (Optima XL-I; Beckman Coulter) was used to determine the sedimentation coefficients of the expressed constructs. Sedimentation velocity runs were performed in the An-60 Ti rotor at 40,000 rpm and 20°C in 10 mM Hepes, pH 7.0, 0.3 M NaCl, 1 mM DTT, 1 mM EGTA, and 1 mM NaN3
. Sedimentation values were corrected for density and viscosity of the solvent. Sedimentation coefficients were determined by curve fitting to one or more species using the dc/dt program (Philo, 2000
). Sedimentation equilibrium data were collected at 15,000 rpm and 4°C, 10 mM Hepes, pH 7.0, 0.3 M NaCl, 1 mM DTT, 1 mM EGTA, and 1 mM NaN3
. The data were analyzed with Origin software provided with the Optima XL-I ultracentrifuge. For molecular mass calculations, the fit to the mean of three independent scans at equilibrium was determined for each protein sample, and the mean and SD of those values were calculated for each construct.
0.3 µM Myo4p–She3p was mixed with various She2p constructs (~1.2 µM monomer) and incubated for 10 min in 10 mM imidazole, pH 7.5, 1 mM EGTA, 1 mM DTT, and 0.2 M NaCl. Both proteins were preclarified before mixing together. 2.4 µM actin was then added. The mixtures were pelleted for 20 min at 400,000 g. The supernatant was removed, and the pellet was washed twice with buffer. Protein content of the supernatant and pellet was assessed by SDS-PAGE.
FLAG resin pull-down assay
A truncated Myo4p construct lacking the motor domain and neck was coexpressed with She3p and assessed for binding to various She2p constructs. Both proteins were clarified at 400,000 g for 15 min before mixing. Myo4p–She3p and She2p (each at 1 µM) were mixed together in 250 µl and loaded onto 0.3-ml FLAG columns (Sigma-Aldrich). The columns were washed with 10 mM imidazole, pH 7.5, 1 mM EGTA, and 0.2 M NaCl, and bound proteins were eluted with 0.1 mg/ml FLAG peptide in wash buffer. Fractions were analyzed on SDS gels.
Myo4p–She3p and She2p-WT* were dialyzed into 10 mM MOPS, pH 7, 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, and 1 mM DTT. Myo4p–She3p and She2p were mixed at a molar ratio of 6:1, incubated for 30 min in ice, and then diluted in the same buffer containing 50% glycerol to a final concentration of 60–120 µg/ml. 5–10 µl of each sample was sprayed on a freshly split mica surface, dried for 2 h under vacuum, rotary shadowed with platinum at an angle of 7°, and replicated with carbon in a Balzers 410 freeze-fracture machine. Replicas were photographed in an electron microscope (410; Philips) operating at 60 kV at a magnification of 75,300. The negatives were digitally scanned at 1,000 pixels per inch. Images were obtained from areas at the edge of each droplet that showed distinct molecules and a clear background.
For experiments in which run lengths were measured, motion was followed from the YFP signal on She2p. She2p-YFP was clarified at 400,000 g for 20 min to remove aggregates. 0.2 µM Myo4p–She3p was mixed with 0.05 µM She2p-YFP (moles monomer) and was allowed to incubate on ice for at least 1 h. Flow cells were prepared by introducing the following solutions into the flow cell: 0.1 mg/ml N-ethylmaleimide–modified skeletal muscle myosin (5-min incubation), 5× rinse of 1 mg/ml BSA (2 min), Alexa Fluor 594 phalloidin yeast actin–Tpm1p filaments or chicken skeletal muscle actin filaments (2–5 min), 5× rinse with motility buffer, and, finally, Myo4p–She3p diluted in motility buffer with either 10 µM, 1 mM, or 2 mM MgATP. Motility buffer consists of 50 mM KCl, 25 mM imidazole, pH 7.4, 4 mM MgCl2, 1 mM EGTA, 50 mM DTT, 1 mg/ml BSA, 0.2 mg/ml yeast CaM, 0.2 mg/ml Mlc1p, an ATP-regenerating system (0.5 mM phosphoenolpyruvate and 100 U/ml pyruvate kinase), and an oxygen-scavenging system (3 mg/ml glucose, 0.1 mg/ml glucose oxidase, and 0.18 mg/ml catalase). The final concentrations were 20 nM Myo4p–She3p and 5 nM She2p-YFP. N-ethylmaleimide–modified skeletal muscle myosin forms a strong and ATP-insensitive bond with actin and was used to attach the actin filaments to the coverslip.
Data were collected on an inverted microscope (Eclipse Ti-U; Nikon) equipped with a 100× Plan Apo objective lens (1.49 NA) and auxiliary 1.5× magnification for through-the-objective TIRF microscopy. The YFP was excited with a 473-nm laser line. Images were obtained using a camera (XR/Turbo-Z; Stanford Photonics) running Piper Control software (v2.3.39). The pixel resolution was 95.4 nm. Data were collected at 10–20 frames per second. Movement of She2p-YFP was tracked manually using ImageJ.
For experiments in which the stepping pattern of the Myo4p–She3p-She2p complex was determined, motion was followed from a Qdot attached to the N terminus of Myo4p. She2p (without YFP) was clarified at 400,000 g for 20 min before use. N-terminally biotinated Myo4p-She3p was mixed with red (655-nm emission) streptavidin Qdots (Invitrogen) at a ratio of five Qdots per Myo4p monomer. A similar solution was prepared using green (565-nm emission) Qdots, and both solutions were incubated on ice for 20 min. They were then mixed in a 1:1 ratio, and She2p was added, resulting in final concentrations of 40 nM N-terminally biotinated Myo4p-She3p and 160 nM She2p (monomer concentration). Flow cells were prepared as described above. Alexa Fluor 594 phalloidin yeast actin–Tpm1p filaments were used. The Myo4p–She3p-She2p–Qdot mixture was diluted 200-fold in motility buffer containing 1 µM MgATP and 2 µM Tpm1p and added to the flow cell.
Data were collected on an inverted microscope (TE2000; Nikon) equipped with a 100× Plan Apo objective lens (1.49 NA) and auxiliary 1.5× magnification for through-the-objective TIRF microscopy. Qdots were excited with a 488-nm argon laser line. Images were obtained using a charge-coupled device camera (XR Mega-S30; Stanford Photonics) running Piper Control software (v2.3.14). Dual-color simultaneous imaging was achieved through a beam splitter (Dual-View; Optical Insights). The pixel resolution was 58.5 nm, but data were collected using 2 × 2–pixel binning, resulting in a final resolution of 117.6 nm. Data were collected at 15 frames per second.
Movement of Qdots on actin was tracked using ImageJ and the automated motion-tracking plugin SpotTracker (Sage et al., 2005
). Data from the red and green channels were then color aligned by taking simultaneous red and green images of multicolored fluorescent microspheres at different locations within the field of view. Using a custom MATLAB program (MathWorks), a cross-correlation routine was used to determine the local displacements required to align the red image with the green one. The horizontal and vertical displacements required to correct each data point from the red channel were then calculated from nearby correlation values using 2D linear interpolation. Displacement versus time was calculated from the color-corrected data. Step size and dwell time were calculated in MATLAB using the Kerssemakers step-finding algorithm (Kerssemakers et al., 2006
Online supplemental material
Fig. S1 shows gels of expressed Myo4p–She3p and a FLAG resin pull-down assay. Fig. S2 compares the fluorescence intensity of She2p-YFP and myosin Va–YFP. Fig. S3 shows the stepping pattern of Myo4p–She3p bound to She2p. Video 1 shows a Myo4p–She3p-She2p motor complex moving processively on an actin filament. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.201106146/DC1