Strains and plasmids
All strains used in this study were derived from W303 and are shown in .
MYO4-13xMYC,
MYO4-TAP,
MYO2-1/2TAP (IgG-binding domain and TEV site), and
MYO4-1/2TAP were made by PCR-mediated gene modification by the method of
Longtine et al. (1998). p
HO-ADE-HO 3′ UTR is described in
Irie et al. (2002).
STE2-3xHA-U3 was also made using PCR-based modification by inserting U3 zipcode into the AscI site in pFA6a-3HA-His3MX6 (
Longtine et al., 1998). Plasmid U1A-GFP-TAP-NLS was generated as in
Takizawa and Vale (2000), with a TAP sequence replacing GST. This construct was subcloned into vector pRS304 (PT87), digested with HindIII for integration into
trp1 locus, and used to transform
ash1Δ (YPT86). Plasmid p
GAL-4×U1A-U3-t
ADH1 (PT185) was made by inserting four repeats of a U1A binding sequence from U1A pre-mRNA 3′ UTR (
Allain et al., 1996) into the EcoRI site of pRS423 (2μ) and sequentially inserting a
GAL1 promoter into XhoI–EcoRI sites, U3 into BamHI–SacI sites, and
ADH1 terminator into a SacI site. To construct plasmid p
GAL-4×U1A-
ADH2 (77NT)-t
ADH1 (PT229), U3 in PT185 was replaced by a 77-nucleotide fragment from
ADH2 starting seven nucleotides upstream of the stop codon by inserting
ADH2 (77NT)-
SpeI-t
ADH1-
SacI in place of U3-
SacI-t
ADH1-
SacI. Plasmid p
GAL-1×U1A-U3-t
ADH1 (PT204) was derived from PT185 in which 4×U1A was replaced by a single U1A binding sequence with additional restriction sites so that a 36-nucleotide spacer was located in between 1xU1A and U3. Plasmid p
GAL-U3-t
ADH1 (PT244) used for RNA overexpression was derived from PT229 in which EcoRI–4×U1A-
ADH2 (77NT)–SpeI was replaced with MfeI–U3–SpeI. The construction of plasmid
MYO4-HA has been described previously (
Dunn et al., 2007). Cross-linking experiments to examine the effect of RNA binding on She2 oligomerization, and the effect with various She2 mutants, were performed with She2 expressed on CEN/ARS vectors. All other analyses were performed with She2 expressed from high-copy 2μ plasmids. Plasmid p
GPD-
SHE2-
1/2TAP (PT228) was made by inserting XhoI–SHE2–SacI into pRS424 (2μ) and ligating KpnI–pGPD–XhoI upstream of
SHE2 start codon (PT94), and inserting XbaI–
1/2TAP-SpeI into an NheI site engineered immediately upstream of
SHE2 stop codon. To make plasmid
SHE2-1/2TAP (PT240), a PCR clone of
SHE2 including 1 kb each of the 5′ UTR and 3′ UTR sequences was subcloned into pRS314 (CEN/ARS, PT52), and XbaI–
1/2TAP–XbaI was inserted into an NheI site engineered immediately upstream of a
SHE2 stop codon. Mutations of
SHE2 were made by PCR-mediated mutagenesis. The plasmids
SHE2 (N36S)-
1/2TAP (AAC mutated to AGC, PT243),
SHE2 (T47Y)-
1/2TAP (ACG mutated to TAT, PT245),
SHE2 (R63K)-
1/2TAP (AGA mutated to AAA, PT246), and
SHE2 (L130Y)-
1/2TAP (TTG mutated to TAC, PT239) were then made by ligating the mutated
SHE2, which included 1 kb of 5′ UTR, with
1/2TAP, and inserting XhoI–5′ UTR-
SHE2-1/2TAP–XbaI in place of wild-type XhoI–5′ UTR-
SHE2–NheI in PT52. For high-level expression of
SHE2 L130Y, plasmid p
GPD-
SHE2 (L130Y)-
1/2TAP (PT249) was made from PT94 in which wild-type
SHE2 was replaced with
SHE2 (L130Y)-
1/2TAP. To make plasmid
SHE3-U1A (PT248), a PCR clone of
SHE3 including 1 kb each of 5′ UTR and 3′ UTR was subcloned into pRS314 (CEN/ARS), and a U1Ap sequence was inserted into an NheI site engineered just upstream of the stop codon. To express
ASH1-3×HA with 0 (PT290), 1 (PT291), 2 (PT292), 4 (PT293), 6 (PT294), and 8 (PT295) repeats of the 22-nucleotide U1Ap binding sequence (
Nagai, 1996), a PCR clone of
ASH1 including 937 bp of 5′ UTR and 563 bp of 3′ UTR was subcloned into pRS313, and an AscI–
EcoRI–U1A-GCGC–MfeI–AscI aptamer was generated by PCR. The U1A repeats were made by digesting the aptamer with either EcoRI or MfeI, ligating the two digested products, and performing PCR with 5′–AscI–EcoRI–U1A and 3′–AscI–MfeI–GCGC-U1A primers. The repeats of interest were then digested with AscI and subcloned into pFA6a-3HA-His3MX6 (
Longtine et al., 1998) so that the U1A repeats were immediately 3′ of the stop codon. These subclones were used as templates for PCR to obtain BglII–3×HA-(U1A-GCGC)
n–MfeI–AscI–BglII and inserted into a BamHI site engineered immediately upstream of the stop codon of
ASH1.
Reagents and antibodies
Anti-HA (HA.11) and anti-myc (9E10) antibodies were obtained from Covance. Anti-GFP (JL8) was purchased from Takara Bio Inc. Anti-digoxigenin antibody was obtained from Jackson ImmunoResearch Laboratories, Inc. Anti–mouse Alexa Fluor 488 and a ProLong Antifade kit were obtained from Invitrogen. The preparation of antibodies against Myo4, She3, and She2 has been described in
Dunn et al. (2007). IgG Sepharose 6 Fast Flow, protein G Sepharose 4 Fast Flow, Superdex 200 10/300 GL, and high- and low-molecular weight gel filtration calibration kits were obtained from GE Healthcare. Detection reagents for Western blots, EDC, and
N-hydroxysuccinimide (NHS) were obtained from Thermo Fisher Scientific. Calmodulin affinity resin was obtained from Agilent Technologies. Anti-sense digoxigenin (DIG)-labeled probes were made using MAXIscript kit from Applied Biosystems.
RNP complex purification
Cells containing pGAL-1x/4×U1A-U3 or pGAL-4×U1A-ADH2 (77 NT) along with U1Ap-GFP-TAP, or those containing pGAL-U3 and MYO4-TAP (or -1/2TAP) were grown overnight in synthetic media containing 2% raffinose. Cultures were adjusted to ~OD600 0.2 in rich media containing 2% raffinose and incubated at 30°C. At ~OD600 1.0, galactose was added to 1% and further incubated 1.5 h at 30°C. Cells expressing U1Ap-GFP-TAP only or MYO4-1/2TAP or MYO2-1/2TAP were incubated in rich media containing 2% dextrose at 30°C until OD600 reached ~1.2–1.5. The cells were harvested by centrifugation, washed with HCB wash buffer (25 mM Hepes-KOH, pH 7.5, 0.15 M KCl, and 2 mM MgCl2), resuspended in 1/2 pellet volume of HCB extract buffer (25 mM Hepes-KOH, pH 7.5, 0.15 M KCl, 2 mM MgCl2, 0.1% NP-40, 1 mM DTT, 5 mM ATP, 0.2 mg/ml heparin, 20 mM vanadyl ribonucleoside complexes, 0.4 mM 4-[2-aminoethyl] benzenesulfonyl fluoride hydrochloride [AEBSF], and 2 µg/ml of aprotinin, leupeptin, and pepstatin), and frozen in liquid nitrogen. The frozen cell extracts were lysed using a mortar and pestle with liquid nitrogen and thawed with added HCB extract buffer (1 ml per culture).
The thawed lysates were centrifuged for 5 min at 4,000 relative centrifugal force (rcf), and supernatants were centrifuged again for 20 min at 16,100 rcf. Supernatants were then protein quantified and incubated with IgG sepharose for 2 h at 4°C. The beads were washed with HCB (25 mM Hepes-KOH, pH 7.5, 0.15 M KCl, 2 mM MgCl2, 0.1% NP-40, and 1 mM DTT) followed by TCB (25 mM Hepes-KOH, pH 7.5, 0.15 M KCl, 0.5 mM EDTA, 0.1% NP-40, and 1 mM DTT), then incubated with AcTEV protease in TCB at 16°C for 2 h. After collecting the TEV-eluted fraction, IgG beads were rinsed with HCB, and this solution was combined with the TEV eluate. The TEV eluate was used for further experiments or adjusted so that the final composition was identical to CCB (25 mM Hepes-KOH, pH 7.5, 0.15 M KCl, 2 mM MgCl2, 1 mM imidazole, 2 mM CaCl2, 0.1% NP-40, and 1 mM DTT) and incubated with calmodulin resins for 1.5 h at 4°C. The beads were then washed with CCB and the purified complex was either eluted with CEB (25 mM Hepes, pH 7.5, 0.15 M KCl, 2 mM MgCl2, 1 mM imidazole, 10 mM EGTA, 0.1% NP-40, and 1 mM DTT), elution buffer (50 mM Tris-Cl, pH 8.0, 0.15 M NaCl, 12.5 mM EDTA, and 0.1% SDS), or 1× SDS-PAGE sample buffer.
For protein identification, protein bands were cut out from a Coomassie blue–stained SDS-polyacrylamide gel and subjected to LC-MS/MS at the W.M. Keck Foundation Biotechnology Laboratory at Yale University.
Sucrose gradients
RNP complexes eluted from calmodulin resins with CEB or from IgG resins with TEV were added to the top of 10–50% sucrose gradients made with the buffer compositions in the RNP complexes. In some cases, TEV-eluted RNP complexes were incubated with RNaseA (to 0.3 mg/ml) for 1 h before ultracentrifugation to detach RNA cargo from the complex. Purified wild-type She2 and L130Y were loaded on 5–20% sucrose gradients made with 20 mM Hepes-KOH, pH 7.5, 0.1 M NaCl, and protease inhibitors. Protein standards were also loaded on parallel gradients to estimate the S values. The gradients were spun for ~16 h at 40,000 rpm in a SW50.1 rotor (Beckman Coulter). Fractions were then collected with a peristaltic pump, and TCA precipitated or was directly used for further experiments.
Immunoprecipitation
Yeast lysates were prepared as previously described. Equal amounts of total protein in cell extracts were used in parallel immunoprecipitation experiments. RNaseA (to 0.3 mg/ml) was added to select extracts before further incubation. Cell extracts were precleared by incubating with protein G beads for 1 h at 4°C with gentle rotation. Supernatants were incubated with mouse anti-myc antibody for 1.5 h at 4°C and further incubated with added protein G beads for 1 h at 4°C. The beads were then washed extensively with HCB and incubated with elution buffer.
She2 purification
For analytical ultracentrifugation and concentration-dependent cross-linking analysis of purified wild-type She2, yeast cells containing wild-type SHE2-1/2TAP on a high-copy plasmid (2µ) were grown overnight in synthetic media containing 2% dextrose. The overnight cultures were diluted in rich media containing 2% dextrose to ~OD600 0.2 and harvested by centrifugation at ~OD600 2.0. The cell pellet was washed with HB (25 mM Hepes-KOH, pH 7.5, 0.15 M potassium acetate, 2 mM magnesium acetate, 1 mM EGTA, and 1 mM DTT) without DTT, resuspended in 1/2 pellet volume of HB with protease inhibitors, and frozen with liquid nitrogen. The cell extract was lysed using a mortar and pestle with liquid nitrogen, thawed, and centrifuged for 5 min at 4,000 rcf, 10 min at 48,384 g, and 1 h at 50,000 rpm in a Ti 60 rotor (Beckman Coulter). The clear lysate was then passed over IgG Sepharose columns and washed with 60× column volume of HB and 25× column volume of TB (25 mM Hepes-KOH, pH 7.5, 0.15 M potassium acetate, 1 mM EGTA, 0.5 mM EDTA, and 1 mM DTT) at 4°C. The beads were then mixed with TB containing AcTEV protease and incubated overnight at 4°C with gentle shaking. After the TEV eluate was collected, the beads were rinsed with TB and the eluate was either used for cross-linking or concentrated with a centrifugal filter (Amicon Ultra; Millipore) for analytical ultracentrifugation.
For cross-linking analyses of the She2 mutants, SHE2-1/2TAP was expressed on low-copy plasmids (CEN/ARS), and cell lysates were prepared as previously described but without high speed centrifugation. The mutated She2 were then 1/2TAP purified and the TEV eluates were used for cross-linking experiments.
For gel filtration and sucrose gradient analysis, wild-type SHE2-1/2TAP and SHE2 (L130Y)-1/2TAP were expressed on high-copy plasmids and cultured as previously described. The cell pellets were then resuspended with HNB (20 mM Hepes-KOH, pH 7.5, 0.1 M NaCl, 0.5 mM EDTA, 0.1% NP-40, and 2 mM DTT) with protease inhibitors, and cell lysates were prepared. The clear lysates were incubated with IgG sepharose beads at 4°C for 2 h, washed with HNB, and incubated with HNB containing AcTEV protease at 16°C for 2 h. The TEV eluates were quantified and the protein purity was confirmed by silver staining and Coomassie staining.
EDC/NHS cross-linking
EDC and NHS were dissolved in water to 1 M before usage and added to purified She2 diluted with TB (without DTT) to a final concentration of 40 mM. Cross-linking was performed at room temperature for 1 h and quenched by adding SDS-PAGE sample buffer.
Analytical ultracentrifugation
Wild-type She2 was diluted with TB to 0.15, 0.53, 1.13, and 1.90 mg/ml, and sedimentation velocity analysis was conducted at 20°C and 40,000 rpm using interference optics with an analytical ultracentrifuge (XL-I; Beckman-Coulter) at the Analytical Ultracentrifugation Facility at University of Connecticut. Double sector synthetic boundary cells equipped with sapphire windows were used to match the sample and reference menisci. The rotor was equilibrated under a vacuum at 20°C, and after a period of ~1 h at 20°C, the rotor was accelerated to 40,000 rpm. Interference scans were acquired at 60 s intervals for ~5.5 h. Data were processed using the program Sedfit, version 11.71 (National Institutes of Health).
Growth assays
Yeast cells expressing SHE3-U1A, U1A-tagged ASH1-HA, and pHO-ADE (ash1Δ she2Δ) were grown overnight at 30°C in synthetic media. The overnight cultures were pelleted, washed with water, and diluted to ~OD600 0.2 with water. Three tenfold serial dilutions were made from this culture, and 5 µl of each dilution was spotted on synthetic media plates with or without adenine and incubated at 30°C. Plates with adenine were incubated for 2 d and those without adenine for ~4 d.
Fluorescence microscopy
Exponentially growing cells were fixed by adding formaldehyde (4% final concentration) to the culture medium and incubating for 1 h at room temperature. The fixed cells were washed, spheroplasted using zymolase 100T (Seikagaku Corporation), and adsorbed onto poly-l-lysine–coated glass coverslips. For FISH, samples were sequentially incubated in hybridization mix (50% formamide, 5× SSC, 1 mg/ml yeast tRNA, 100 µg/ml heparin, 1× Denhardt’s solution, 0.1% Tween 20, 0.1% Triton X-100, and 5 mM EDTA) for 45 min at room temperature; kept in hybridization mix containing anti-sense, DIG-labeled STE2, or ASH1 RNA probes overnight at 37°C; and then were washed in 0.1× SSC at 37°C. For FISH and immunofluorescence, the prepared samples were briefly incubated in blocking buffer (50 mM Tris-Cl, pH 7.5, 0.15 M NaCl, and 5% fetal bovine serum), incubated with mouse anti-DIG antibody or anti-HA antibody in blocking buffer for 30 min at 37°C, and washed at room temperature with washing buffer (50 mM Tris-Cl, pH 7.5, 0.15 M NaCl, and 0.1% Tween 20). Samples were then incubated with goat anti–mouse Alexa Fluor 488 for 45 min at room temperature, washed again with washing buffer, and mounted on glass slides. All samples were inspected with a fluorescence microscope (TE2000; Nikon) equipped with a 100×/NA 1.4 lens (Nikon) and acquired using a charge-coupled device camera (ORCA ER; Hamamatsu Photonics) controlled by IPLab software (Scanalytics, Inc.). For imaging of cells coexpressing tagged U3 RNA and U1Ap-GFP-TAP, GFP fluorescence was observed in cells directly from galactose-induced cultures.
Online supplemental material
Fig. S1 shows that tagging the U3 localization element to
STE2 is sufficient to localize
STE2 mRNA protein to the distal tip of buds. Fig. S2 shows that RNase treatment eliminates the Myo4 detected in the heavier fractions in . Fig. S3 shows silver staining and Western analyses of U3 RNP complex purified with a single U1A aptamer tagged to U3 RNA. Fig. S4 shows that RNase treatment eliminates the small amount of Myo4-HA that co-migrates with TAP-purified Myo4 in the absence of U3 RNA overexpression. Fig. S5 shows gel filtration and sedimentation analyses of wild-type and L130Y mutant She2. Video 1 shows fluorescently labeled actin filaments moving via purified U3 RNP adsorbed to motility chambers with anti-GFP antibodies. Online supplemental material is available at
http://www.jcb.org/cgi/content/full/jcb.200912011/DC1.
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