Figure 1.
Myo2 binds with its CBD to Pea2.(A) Two-channel microscopy of yeast cells expressing GFP-Pea2 together with Spa2-mCherry (left), Cdc42GTP-sensor PBDGic2 (middle), or Myo2-mCherry (right). Shown are images of cells during bud site formation (left column), bud growth (middle column), or mitosis (right column). Scale bar: 5 µm. (B) Cutouts of a split-Ub array of diploid yeast cells expressing genomic Myo2CRU (CRU; C-terminal half of Ubiquitin (Cub)-R-Ura3; left panel) or a CRU fusion to a plasmid-based cargo binding domain of Myo2 (CBD; right panel) together with the indicated Nub fusions (Nub; N-terminal half of Ubiquitin). Four independent matings were arrayed as a quadruplet on medium containing 5-FOA. Colony growth indicates interaction between the fusion proteins. Cells coexpressing interacting Nub fusions are displayed in the left row (stars indicate novel binding partners). Cells coexpressing noninteracting Nub fusions are displayed in the right row. Due to the presence of the native Myo2, the interaction signals between CBDCRU and the respective Nub fusions always appear weaker. Complete analysis is shown in Fig. S1. (C) Split-Ub assay of cells coexpressing CBDCRU or CBDCRU containing selected point mutations together with the indicated Nub fusions. Cells were grown to OD600 = 1 and 4 µl or 4 µl of a 10-fold serial dilution spotted on medium containing 5-FOA. (D) Extracts of yeast cells expressing Pea2-9myc (lanes 1–3) were incubated with Glutathione-coupled Sepharose beads exposing bacterially expressed GST (lane 1), GST-CBD (lane 2), or GST-CBDY1415R (lane 3). Glutathione eluates were separated by SDS-PAGE and probed with anti-myc antibodies (lanes 1–3). The asterisk indicates a degradation product. Ponceau staining of the supernatants after elution and Western blot of the yeast extract are shown in Fig. S2 A. (E) Split-Ub assay as in B but with cells coexpressing Myo2CRU and the indicated Nub fusions in the presence (upper panel, PEA2) or absence (lower panel, pea2Δ) of PEA2. (F) Bacterially expressed GST-CBD (lanes 1–3) or GST (lanes 4–6) coupled to Glutathione Sepharose beads were incubated with bacterially expressed 6xHis-Pea2221–420 (lanes 1 and 4), 6xHis-Pea2221–350 (lanes 2 and 5), or 6xHis-Pea2351–420 (lanes 3 and 6). Glutathione eluates were separated by SDS-PAGE and stained with anti-His antibody after transfer onto nitrocellulose. Inputs of 6xHis-Pea2 fragments, GST-CBD, and GST are shown in Fig. S2 B. Black line indicates that intervening lanes have been spliced out. (G) As in C but with haploid cells expressing Nub-Pea2, Nub-Pea2Δ286–330, or Nub as negative control together with CRU fusions to CBD (first panel), Pea2 (second panel), Spa21–535 (third panel), or Bud6 (fourth panel).

Myo2 binds with its CBD to Pea2. (A) Two-channel microscopy of yeast cells expressing GFP-Pea2 together with Spa2-mCherry (left), Cdc42GTP-sensor PBDGic2 (middle), or Myo2-mCherry (right). Shown are images of cells during bud site formation (left column), bud growth (middle column), or mitosis (right column). Scale bar: 5 µm. (B) Cutouts of a split-Ub array of diploid yeast cells expressing genomic Myo2CRU (CRU; C-terminal half of Ubiquitin (Cub)-R-Ura3; left panel) or a CRU fusion to a plasmid-based cargo binding domain of Myo2 (CBD; right panel) together with the indicated Nub fusions (Nub; N-terminal half of Ubiquitin). Four independent matings were arrayed as a quadruplet on medium containing 5-FOA. Colony growth indicates interaction between the fusion proteins. Cells coexpressing interacting Nub fusions are displayed in the left row (stars indicate novel binding partners). Cells coexpressing noninteracting Nub fusions are displayed in the right row. Due to the presence of the native Myo2, the interaction signals between CBDCRU and the respective Nub fusions always appear weaker. Complete analysis is shown in Fig. S1. (C) Split-Ub assay of cells coexpressing CBDCRU or CBDCRU containing selected point mutations together with the indicated Nub fusions. Cells were grown to OD600 = 1 and 4 µl or 4 µl of a 10-fold serial dilution spotted on medium containing 5-FOA. (D) Extracts of yeast cells expressing Pea2-9myc (lanes 1–3) were incubated with Glutathione-coupled Sepharose beads exposing bacterially expressed GST (lane 1), GST-CBD (lane 2), or GST-CBDY1415R (lane 3). Glutathione eluates were separated by SDS-PAGE and probed with anti-myc antibodies (lanes 1–3). The asterisk indicates a degradation product. Ponceau staining of the supernatants after elution and Western blot of the yeast extract are shown in Fig. S2 A. (E) Split-Ub assay as in B but with cells coexpressing Myo2CRU and the indicated Nub fusions in the presence (upper panel, PEA2) or absence (lower panel, pea2Δ) of PEA2. (F) Bacterially expressed GST-CBD (lanes 1–3) or GST (lanes 4–6) coupled to Glutathione Sepharose beads were incubated with bacterially expressed 6xHis-Pea2221–420 (lanes 1 and 4), 6xHis-Pea2221–350 (lanes 2 and 5), or 6xHis-Pea2351–420 (lanes 3 and 6). Glutathione eluates were separated by SDS-PAGE and stained with anti-His antibody after transfer onto nitrocellulose. Inputs of 6xHis-Pea2 fragments, GST-CBD, and GST are shown in Fig. S2 B. Black line indicates that intervening lanes have been spliced out. (G) As in C but with haploid cells expressing Nub-Pea2, Nub-Pea2Δ286–330, or Nub as negative control together with CRU fusions to CBD (first panel), Pea2 (second panel), Spa21–535 (third panel), or Bud6 (fourth panel).

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