Increasing the number of Myo4 motors bound to RNA improves bud localization. (A) Model system for modifying Myo4 motor copy number on ASH1 RNA. She3 is expressed with the RNA-binding domain from U1Ap fused to its C terminus, and ASH1 is expressed with 0, 1, 2, 4, 6, or 8 U1Ap-binding sites positioned between the stop codon and 3′ UTR. Both constructs are expressed in ash1Δ she2Δ cells to ensure that Myo4 binds to ASH1 mRNA only through the interaction between She3-U1Ap and the U1Ap-binding sites tagged to ASH1 RNA. (B) Increasing the number of Myo4 bound to ASH1 RNA enhances bud localization of Ash1. She3-U1Ap and ASH1-HA tagged with the indicated number of U1A aptamers were expressed in ash1Δshe2Δ yeast cells where ADE was under the control of HO promoter. She2 was also coexpressed with She3-U1Ap and ASH1-HA as a control (bottom). Cells were spotted onto selective media plates with or without adenine. (C) U1A tags do not affect Ash1 expression. Protein extracts were prepared from cells expressing ASH1-HA tagged with the indicated number of U1A aptamers. Equal amounts of protein extracts were analyzed by Western blotting with anti-HA antibody. (D) Bud transport of ASH1 mRNA is improved by increasing Myo4 binding sites on ASH1 RNA. She3-U1Ap was expressed with U1A-tagged ASH1-HA in ash1Δshe2Δ cells. wt indicates cells coexpressing She2 with She3-U1Ap and ASH1 as a control. The cells were synchronized and fixed after release from synchronization. ASH1 mRNA was detected by FISH, and the percentage of cells showing bud-localized ASH1 mRNA among ∼100 cells was determined in each cell sample (n = 3). Bud localization was further subdivided to localization confined to the distal tip of buds and that dispersed throughout the bud.