Figure 1.
Figure 1. Epo1p interacts with members of the polarisome and Scs2p. (A) Split-Ub interaction assay of 48 yeast strains each coexpressing Epo1CRU with a different Nub fusion. Shown are quadruplets of each strain after 3 d of growth on medium containing 5-FOA. White boxes indicate the fusions that induce the growth of the strain reflecting the interaction between Nub and Cub fusion. The identities of all Nub fusions are revealed in Table S2. (B) Split-Ub interaction assay between Epo1CRU and selected Nub fusion proteins in WT, Δpea2, Δspa2, Δkel1, and Δbem3 cells. Cells were grown to OD600 of 1 and 4 µl of this, and 10-fold serial dilutions were spotted on 5-FOA plates. Nub without a C-terminally attached ORF (Nub−) serves as a control for the specificity of the Split-Ub assays. (C) As in B, but selected interactions of Pea2CRU were compared between WT and Δepo1 cells. (D) Domain structure of Epo1p. Shown as blue rectangles are the three predicted coiled-coil (CC) regions. Numbers indicate amino acid positions of the putative start and end points of each domain. (E) As in A, but with fragments of Epo1p as CRU fusions and 16 independently generated diploids for each experiment shown after 4 d of growth. (F) Protein extracts of bacterial cells expressing his6-Pea2 (lanes 1–4) or yeast cells expressing MYC-tagged Kel1p (lanes 5–8) were incubated with glutathione-coupled Sepharose beads exposing bacterially expressed GST (lanes 1 and 5), GST-Epo1761–943 (lanes 2 and 6), GST-Epo1761–867 (lanes 3 and 7), or GST-Epo1852–943 (lanes 4 and 8). Glutathione eluates were separated by SDS-PAGE and probed with anti-His (lanes 1–4) or anti-MYC (lanes 5–8) antibodies after Western blotting. (G) As in F, except bacterially expressed MBP-Epo1 (lanes 1 and 2) or MBP-Epo11–760 (lanes 3 and 4) were precipitated with bacterially expressed and Sepharose bead immobilized GST (lanes 2 and 4) or GST-Pea2p (lanes 1 and 3). The inputs for the experiments in F and G are shown in Fig. S1. (H) Pea2p mediates the interaction between Epo1p and Spa2p. As in F, except bacterially expressed his6-Spa21–535-SNAP (lanes 1, 2, 7, and 8) or his6-Spa21–488-SNAP (lanes 4, 5, 9, and 10) were first incubated with his6-Pea2 (lanes 1 and 4) or left untreated (lanes 7 and 9) before being incubated with bacterially expressed and immobilized GST-Epo1761–943. The glutathione eluates are shown in lanes 1, 4, 7, and 9. The inputs for the experiment in lane 1 are shown in lanes 2 and 3. The inputs for the experiment in lane 4 are shown in lanes 5 and 6. The inputs for the experiment in lanes 7 and 9 are shown in lanes 8 and 10, respectively. The asterisk indicates a degradation product. Lanes 1–6 show cutouts of the same gel with the vertical line indicating the removal of an empty lane. (I) Architecture of the ER–cell tip tethering complex. Edges connecting nodes indicate direct (black) or potentially indirect (green) interactions. Blue rectangles indicate coiled-coil regions shown in D.

Epo1p interacts with members of the polarisome and Scs2p. (A) Split-Ub interaction assay of 48 yeast strains each coexpressing Epo1CRU with a different Nub fusion. Shown are quadruplets of each strain after 3 d of growth on medium containing 5-FOA. White boxes indicate the fusions that induce the growth of the strain reflecting the interaction between Nub and Cub fusion. The identities of all Nub fusions are revealed in Table S2. (B) Split-Ub interaction assay between Epo1CRU and selected Nub fusion proteins in WT, Δpea2, Δspa2, Δkel1, and Δbem3 cells. Cells were grown to OD600 of 1 and 4 µl of this, and 10-fold serial dilutions were spotted on 5-FOA plates. Nub without a C-terminally attached ORF (Nub−) serves as a control for the specificity of the Split-Ub assays. (C) As in B, but selected interactions of Pea2CRU were compared between WT and Δepo1 cells. (D) Domain structure of Epo1p. Shown as blue rectangles are the three predicted coiled-coil (CC) regions. Numbers indicate amino acid positions of the putative start and end points of each domain. (E) As in A, but with fragments of Epo1p as CRU fusions and 16 independently generated diploids for each experiment shown after 4 d of growth. (F) Protein extracts of bacterial cells expressing his6-Pea2 (lanes 1–4) or yeast cells expressing MYC-tagged Kel1p (lanes 5–8) were incubated with glutathione-coupled Sepharose beads exposing bacterially expressed GST (lanes 1 and 5), GST-Epo1761–943 (lanes 2 and 6), GST-Epo1761–867 (lanes 3 and 7), or GST-Epo1852–943 (lanes 4 and 8). Glutathione eluates were separated by SDS-PAGE and probed with anti-His (lanes 1–4) or anti-MYC (lanes 5–8) antibodies after Western blotting. (G) As in F, except bacterially expressed MBP-Epo1 (lanes 1 and 2) or MBP-Epo11–760 (lanes 3 and 4) were precipitated with bacterially expressed and Sepharose bead immobilized GST (lanes 2 and 4) or GST-Pea2p (lanes 1 and 3). The inputs for the experiments in F and G are shown in Fig. S1. (H) Pea2p mediates the interaction between Epo1p and Spa2p. As in F, except bacterially expressed his6-Spa21–535-SNAP (lanes 1, 2, 7, and 8) or his6-Spa21–488-SNAP (lanes 4, 5, 9, and 10) were first incubated with his6-Pea2 (lanes 1 and 4) or left untreated (lanes 7 and 9) before being incubated with bacterially expressed and immobilized GST-Epo1761–943. The glutathione eluates are shown in lanes 1, 4, 7, and 9. The inputs for the experiment in lane 1 are shown in lanes 2 and 3. The inputs for the experiment in lane 4 are shown in lanes 5 and 6. The inputs for the experiment in lanes 7 and 9 are shown in lanes 8 and 10, respectively. The asterisk indicates a degradation product. Lanes 1–6 show cutouts of the same gel with the vertical line indicating the removal of an empty lane. (I) Architecture of the ER–cell tip tethering complex. Edges connecting nodes indicate direct (black) or potentially indirect (green) interactions. Blue rectangles indicate coiled-coil regions shown in D.

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