1. Volume 1, Issue 5, Pages 483-494 (May 2012)
Scp160-Dependent mRNA Trafficking Mediates Pheromone Gradient Sensing and Chemotropism in Yeast 
Rita Gelin-Licht, Saurabh Paliwal, Patrick Conlon, Andre Levchenko, Jeffrey E. Gerst 
Cell Reports 
Volume 1, Issue 5, Pages (May 2012)
DOI: /j.celrep
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2. Cell Reports 2012 1, 483-494DOI: (10.1016/j.celrep.2012.03.004)
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3. Figure 1
SRO7 mRNA and Protein Localize to the Shmoo Tip in Mating Factor-Treated Yeast
(A) Wild-type yeast expressing the RFP-SEC4 or RFP-SRO7 genes bearing MS2-CP binding sites upstream to their 3′ UTRs and MS2-CP-GFP from plasmids were grown to early log phase on selective medium and either treated with α factor (5 μM; +) or maintained in the absence of mating factor (−) for 1.5 hr, and then examined by fluorescence microscopy. mRNA is indicated by GFP fluorescence, Protein by RFP fluorescence, and Merge combines the fluorescence and transmitted light windows. White arrowheads indicate colocalization of the mRNA granule and RFP protein at the shmoo (in treated cells) or bud (in untreated cells) tip.
(B) Wild-type yeast expressing RFP-SRO7 bearing MS2-CP binding sites upstream to its 3′ UTR and MS2-CP-GFP from plasmids were treated with α factor (5 μM) and examined by time-lapse fluorescence microscopy. Time is given in minutes. White arrowheads indicate the localization of mRNA granules.
See corresponding Movie S1.
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4. Figure 2
SRO7 mRNA Localization Is SHE2 and SHE3 Independent in Mating Factor-Treated Yeast
she2Δ, she3Δ, and myo4Δ cells expressing RFP-SRO7 bearing MS2-CP binding sites upstream to the 3′ UTR and MS2-CP-GFP from plasmids were either treated with α factor (5 μM; +) or left untreated (−), and examined using a DeltaVision imaging system. The white arrowheads indicate colocalization of the mRNA granule and RFP-Sro7 protein at the shmoo tip. Merge combines the fluorescence and transmitted light windows.
See corresponding Movie S2 for SRO7 mRNA and protein in she3Δ cells.
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5. Figure 3
SRO7 mRNA Localization Correlates with ER Localization in a MYO4- and SEC3-Dependent Manner
Wild-type (WT), sec3Δ, and myo4Δ yeast all expressing SRO7 bearing MS2-CP binding sites (upstream of the 3′ UTR), SEC63-RFP, and MS2-CP-GFP from plasmids were either treated with α factor (5 μM; +) or left untreated (−) and examined by fluorescence microscopy using a DeltaVision imaging system. White arrowheads indicate localization of the mRNA granule to either the shmoo or bud tips. Merge combines the fluorescence and transmitted light windows.
See corresponding movie for SRO7 mRNA and ER localization in untreated WT cells (Movie S3), untreated sec3Δ cells (Movie S4), α factor-treated WT cells (Movie S5), and α factor-treated sec3Δ cells (Movie S6).
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6. Figure 4 Scp160 Is Required for SRO7 mRNA Localization
(A) SRO7 mRNA and protein are mislocalized in cells lacking SCP160. scp160Δ and scp160Δ cells overexpressing SCP160 (+SCP160) from a multi-copy plasmid were transformed with single-copy plasmids expressing SRO7 bearing MS2-CP binding sites upstream of the 3′ UTR, and MS2-CP-GFP. Cells were grown to mid-log phase and either treated with α factor (5 μM; +) or left untreated (−) and then examined by fluorescence microscopy. Merge combines the fluorescence and transmitted light windows.
(B) Scp160-GFP also localizes to cER present at the bud and shmoo tips. Cells expressing SCP160-GFP from the SCP160 locus were grown and treated either with (+) or without 5 μM α factor (−), and examined using fluorescence microscopy after 1.5 hr.
(C) Scp160 associates with Myo4. Cells expressing FLAG-SCP160 from its chromosomal locus were grown to mid-log phase, either treated with 5 μM α factor for 1.5 hr or left untreated, lysed, and incubated with anti-Flag M2 affinity gel to immunoprecipitate proteins. Untreated wild-type control cells were grown and processed in parallel. Samples of the total cell lysate (TCL) and each immunoprecipitate (IP) were separated on a 7% SDS-PAGE gel, blotted, and probed with anti-Flag and anti-Myo4 antibodies.
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7. Figure 5
mRNA Binding by Scp160 Is Modulated by the Mating Pheromone Response
(A) Precipitation of Flag-tagged Scp160. Untreated and α factor-treated wild-type cells expressing FLAG epitope-tagged Scp160, as well as untreated cells expressing both FLAG-Scp160 and Gpa1Q323L, and a wild-type control were grown to mid-log phase, lysed, and incubated with anti-Flag M2 affinity gel to immunoprecipitate proteins. Samples of the total cell lysate (TCL) and each immunoprecipitate (IP) were separated on a 9% SDS-PAGE gel, blotted, and probed with anti-Flag antibody. The filled arrow indicates Scp160; the open arrow indicates an ∼100 kDa anti-Flag cross-reacting protein (Lang and Fridovich-Keil, 2000) that served as an internal control for loading.
(B and C) SRO7 and SEC3 mRNA binding to Scp160 is increased upon pheromone treatment or Gα activation. RNA was extracted from the TCL samples and immunoprecipitates, and used as a template for reverse transcriptase in either semiquantitative (B) or real-time PCR (C). Specific primer pairs were used to detect the SRO7, SEC3, and HOM2 mRNAs (as labeled) in (B), and the SRO7 (blue) and SEC3 (red) mRNAs in (C).
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8. Figure 6
RNA Binding by Scp160 Modulates Mating Efficiency and Is Required for Chemotropism
(A) Mating efficiency is affected by the loss of Scp160, its RNA-binding function, or removal of the 3′ UTRs belonging to mating pathway components. Mating between scp160Δ (MATa) and wild-type BY4742 (MATα) cells, scp160Δ (MATa) and scp160Δ (MATα) cells, FLAG-SCP160KH14Δ (MATa) and wild-type BY4742 (MATα) cells, myo4Δ (MATa) and myo4Δ (MATα) cells, SCP160-GFP (MATa) and SCP160-GFP (MATα) cells, FUS3-GFP (MATa) and FUS3-GFP (MATα) cells, KAR3-GFP (MATa) and KAR3-GFP (MATα) cells, SCP160-GFP (MATa) and KAR3-GFP (MATα) cells, and SCP160-GFP (MATa) and FUS3-GFP (MATα) cells was compared in a quantitative fashion with mating between control BY4741 (MATa) and BY4742 (MATα) wild-type cells. Mating efficiency of the wild-type control cells was designated as 100%. The average of three independent experiments is shown for each cross and includes error bars indicating the SD.
(B) Cells lacking Scp160 or its RNA-binding function are deficient in chemotropism. Wild-type, scp160Δ, and SCP160KH14Δ-expressing yeast were coincubated in the same experiment, and exposed to a gradient created between a high (750 nM) and low pheromone source (100 nM) in a microfluidic chamber for 6 hr. Shmoo orientation relative to the direction of the gradient was determined. Representative photos of cells in the microfluidic chamber for each cell type are shown. Red arrows indicate the direction of shmoo orientation.
(C) Cells lacking Scp160 show an increased number of projections and reduced viability. Wild-type and scp160Δ cells were compared in terms of the number of projections and cell death. Cells that formed a single projection and sensed gradient are shown in blue, cells that formed multiple projections are shown in black, and cells that underwent cell death are in red. The numbers on top reflect the total number of cells scored for each category. The numbers on bottom indicate pheromone concentration (in nanomolars).
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9. Figure 7
A Model for the Role of Scp160 in Pheromone-Mediated Chemotropic Growth
Left panel illustrates untreated budding yeast cells that have the inactive α factor pheromone receptor (Ste2) present at the cell surface in a complex with the inactive G protein α,β,γ subunits (e.g., Gpa1-GDP, Ste18, Ste4, respectively). Scp160 binds to mRNAs, interacts either directly or indirectly with a myosin motor protein (i.e., Myo4), and resides on cER membranes. The existence of a putative adaptor required for Scp160 to bind to ER membranes and/or the myosin motor is illustrated. Center panel shows that upon pheromone (α factor) binding to the receptor, GDP-GTP catalyzed exchange on Gpa1 results in the release of Ste18/Ste4 to activate MAP kinase cascade involved in actin polarization, cell wall integrity, and cell-cycle control (not shown). In addition, Gpa1-GTP binds to Scp160 and increases the amount of associated mRNAs (i.e., SRO7, SEC3, FUS3, STE7, etc.). Right panel illustrates Scp160 and cER that are targeted to the shmoo tip along actin filaments that are oriented along the polarization axis via Ste18/Ste4 signaling. This targeting is likely to involve a myosin motor, like Myo4. mRNA and cER delivery allows for the localized translation and enrichment of polarity/secretion and mating pathway components at the site of polarization (to facilitate shmoo growth).
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10. Figure S1
Scp160 Is Required for SRO7 mRNA Localization in MATα Cells, Related to Figure 4
MATα wild-type or MATα scp160Δ cells were transformed with plasmids expressing MS2 aptamer-tagged RFP-SRO7 mRNA and MS2-CP-GFP. Cells were grown to mid-log phase and both mRNA and protein localization examined with a DeltaVision imaging system (fluorescence microscopy). As seen with MATa cells, Scp160 is required for both RFP-SRO7 mRNA localization to the bud tip and RFP-Sro7 protein to the bud in MATα cells.
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11. Figure S2
FUS3 mRNA and Protein Localize to the Shmoo Tip, Related to Figure 4
(A) Wild-type yeast expressing a mCherry-FUS3 gene fusion bearing MS2-CP binding sites upstream to the FUS3 3′UTR and MS2-CP-GFP from a single-copy plasmid were grown to mid-log phase at 26°C on synthetic selective medium. Cells were then switched to medium lacking methionine and either treated with α-factor (5 μM; +) or maintained in the absence of mating factor (-) for 1.5hrs. Both mRNA and protein localization were examined using a DeltaVision imaging system (fluorescence microscopy). Merge indicates merger between the GFP (mRNA), RFP (protein), and transmitted light windows. Note the localization of FUS3 mRNA and protein to the shmoo tip in α-factor-treated cells.
(B) Fus3 protein levels are unchanged in the absence of Scp160 or its RNA-binding function. BY4741 wild-type, scp160Δ (BY4741 background), W303 wild-type, and scp160KH14Δ (W303 background) cells were grown to mid-log phase and treated with 10μM α-factor for 1hr to induce FUS3 expression. Cells were harvested and lysed for whole cell protein extraction. The lysate was separated on a 4%–20% gradient SDS-PAGE gel and analyzed by immunoblotting with anti-Fus3 (1:500) and, as a loading control, anti-GAPDH (1:5000) antibodies. Note that steady-state levels of Fus3 protein are unchanged in the mutants versus their respective wild-type controls.
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12. Figure S3
Scp160, but Not She2, Binds the mRNAs of Mating-Induced Genes in Response to Mating Factor Signaling, Related to Figure 5
Untreated and α-factor-treated wild-type cells expressing FLAG epitope-tagged Scp160, untreated cells expressing both FLAG-Scp160 and Gpa1Q323L, and untreated wild-type control cells (control) were grown to mid-log phase, lysed, and incubated with anti-Flag M2 affinity gel to immunoprecipitate FLAG-Scp160 and bound RNA, as described in Figure 5A. In parallel, untreated and α-factor-treated wild-type cells expressing myc epitope-tagged She2, untreated cells expressing both myc-She2 and Gpa1Q323L, and untreated wild-type control cells were were grown to mid-log phase, lysed, and incubated with anti-myc antibodies and Protein G-agarose. Real-time PCR was performed on the extracted and reverse-transcribed RNA and is presented as a histogram of the relative fold-increase in binding between the samples. The data illustrate binding of the FUS3 (blue), STE7 (red), and KAR3 (yellow) mRNAs to Scp160 in cells expressing FLAG-Scp160 (A) or to She2 in cells expressing myc-She2 (B). Either α-factor-treatment or the presence of activated Gpa1 induced mRNA binding to Scp160, but not to She2.
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13. Figure S4
Scp160 Binds the 3′ UTR of Specific mRNAs via Its KH Domain, Related to Figures 5 and 6A
(A) KAR3, SCP160, and FUS3, but not FAR1, mRNA binding to Scp160 is increased upon Gα activation. Wild-type control cells (control) and wild-type cells expressing either FLAG epitope-tagged Scp160 or both FLAG-Scp160 and Gpa1Q323L were grown to mid-log phase, lysed, and incubated with anti-Flag M2 affinity gel to immunoprecipitate FLAG-Scp160 and bound RNA, as described in Figure 5A. RNA was extracted from the TCL samples and immunoprecipitates, and used as a template for RT followed by real-time PCR. Specific primer pairs were used to detect KAR3, SCP160, FUS3 and FAR1 mRNAs. All mRNAs bound to Scp160, but only the KAR3, SCP160, and FUS3 mRNAs showed increased binding in the presence of activated Gpa1.
(B) The intact KH domain of Scp160 is required for RNA binding. Wild-type cells expressing either FLAG-Scp160 or FLAG-Scp160KH14Δ together with or without Gpa1Q323L, and control wild-type cells (control) were grown to mid-log phase, lysed, and incubated with anti-Flag M2 affinity gel to immunoprecipitate FLAG-Scp160 and bound RNA, as described in Figure 5A. RNA was extracted from the TCL samples and immunoprecipitates, and used as a template for RT followed by real-time PCR. Specific primer pairs were used to detect KAR3 and SRO7 mRNAs. Both KAR3 and SRO7 mRNAs bind to Scp160, which increases significantly in the presence of activated Gpa1. However, removal of the KH14 domain abolishes the ability of Scp160 to bind these mRNAs.
(C) Scp160 binding to SRO7 mRNA requires the 3′UTR. Wild-type cells (SRO7+3′UTR) or SRO7::GFP integrated cells (which removes the SRO7 3′UTR; SRO7-3′UTR) expressing either FLAG-Scp160 or both FLAG-Scp160 and Gpa1Q323L were grown to mid-log phase, lysed, and incubated with anti-Flag M2 affinity gel to immunoprecipitate FLAG-Scp160 and bound RNA, as described in Figure 5A. RNA was extracted from the TCL samples and immunoprecipitates, and used as a template for RT followed by real-time PCR. A specific primer pair for SRO7 mRNA was used and revealed that removal of the 3′UTR (upon genome tagging) results in an mRNA that is unable to bind to Scp160.
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14. Figure S5
Quantification of Pheromone Gradient Sensing, Related to Figure 6B
(A) Polar plot of the angle of individual mating projections (formed relative to the direction of the gradient) for wild-type and SCP160KH14Δ cells. WT cells (blue; JFy4493 cells) are plotted on the outer circle (radius = 1.0), while SCP160KH14Δ-expressing cells (red) are plotted on the inner circle (radius = 0.9) for convenience. Data corresponds to cells present at the nM concentration range of mating factor after 2hrs of exposure to the gradient.
(B) Polar plot of the angle of individual mating projections formed relative to the gradient normal between wild-type and scp160Δ cells. WT cells (blue; BY4741) are plotted on the outer circle (radius = 1.0), while scp160Δ cells (red) are plotted on the inner circle (radius = 0.9) for convenience. Data corresponds to cells present at the nM concentration range of mating factor after 2hrs of exposure to the gradient.
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