![A subset of Ergs accumulates at ERSESs.(A and B) Merged FMs of cells expressing Erg6-mCherry and Sec61-GFP (A) or GFP-D4H (B). Individual channels and merged magnified areas are shown. Average ± SEM frequencies of colocalization of cortical Erg6-mCherry with Sec61-GFP (A) or GFP-D4H (B) are indicated. n > 75 rims or patches. (C) Confocal FMs of cells expressing Sec61-YFP, Abp1-CFP, and Erg6-mCherry. Individual channels and merged magnified time lapses are shown. Arrowheads point to contacts. Average ± SEM frequencies of colocalization of Erg6-mCherry with Abp1-CFP are indicated. n > 100 from three experiments. (D) Immunoblots of anti-GFP agarose precipitates (IP [GFP]) from yeast expressing protein A (ProtA), GFP, and/or HA-tagged proteins (+) or nontagged (−), probed for the indicated epitopes. 10 µg of total protein was loaded as input. (E and G) Diagram of interactions and two-hybrid assays of Ergs versus Myo5 (E) or Oshes (G). Blue indicates interaction. BICOID was used as negative control. TH1, tail homology 1; SH3, Src homology 3; CA, central and acidic; ANK, ankyrin repeats; PH, Pleckstrin homology; N, N-terminal domain. (F) Protein–protein interaction network. The purple lines are reported interactions (Encinar del Dedo et al., 2017; Mo and Bard, 2005). Red lines are described here. (H) Individual channels and merged FMs of cells expressing Erg6-mCherry and Osh2-GFP. Individual channels and merged magnified insets are shown. Average ± SEM frequencies of colocalization of cortical Erg6-mCherry with Osh2-GFP indicated. n > 125 from three experiments. (I) Average ± SEM and t test P values of cortical GFP-D4H patch RFI normalized to WT (pOSH2) in osh2Δ cells expressing the empty plasmid or plasmids encoding OSH2 alleles. Significant differences are red (n > 210). Merged FMs of the corresponding cells and merged and individual channels of magnified areas shown. (J) Serial dilutions of erg6Δ cells bearing an empty plasmid (p) or plasmids encoding the WT ERG6 or a catalytically inactive mutant (erg6-D152L) grown at the indicated temperatures. (K) GFP-D4H cortical patch RFI normalized to the WT (pERG6) for erg6Δ cells bearing an empty plasmid (p) or plasmids encoding ERG6 or erg6-D152L.](https://cdn.rupress.org/rup/content_public/journal/jcb/220/10/10.1083_jcb.202010016/6/jcb_202010016_fig3.png?Expires=1773828795&Signature=l~HpWHZAXlMT~-CN0rtRre-BL-~yiceHyK3Ls4pbq0nkDjNt1boYhj0SyCPZvzXWqlNLKeN~04y0smK5-dzddWIO~tOMlmeuRKcDiN6wdFRDw2nl0uLlNuIkWq5AH2dciUkCGmV3NJA0sKL4b0Sz~8-xCWhQH9qyk3FMRt8RxONyDcAhZ-akJbQW1ca-tBJS4SgocYZcDOq3XOHR-fmcSrH0gJMAb9R2kcb~Gy8f03orzJejytY6XatFoRc88BYgMo-tycH6SBcLOyLcy2eDrHO71hmo62KzM9JERH~GUzjXPMBwIabzEZ4gA5HA5QQS1JP3SyKhH4bhkB2BfvUSBQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
A subset of Ergs accumulates at ERSESs. (A and B) Merged FMs of cells expressing Erg6-mCherry and Sec61-GFP (A) or GFP-D4H (B). Individual channels and merged magnified areas are shown. Average ± SEM frequencies of colocalization of cortical Erg6-mCherry with Sec61-GFP (A) or GFP-D4H (B) are indicated. n > 75 rims or patches. (C) Confocal FMs of cells expressing Sec61-YFP, Abp1-CFP, and Erg6-mCherry. Individual channels and merged magnified time lapses are shown. Arrowheads point to contacts. Average ± SEM frequencies of colocalization of Erg6-mCherry with Abp1-CFP are indicated. n > 100 from three experiments. (D) Immunoblots of anti-GFP agarose precipitates (IP [GFP]) from yeast expressing protein A (ProtA), GFP, and/or HA-tagged proteins (+) or nontagged (−), probed for the indicated epitopes. 10 µg of total protein was loaded as input. (E and G) Diagram of interactions and two-hybrid assays of Ergs versus Myo5 (E) or Oshes (G). Blue indicates interaction. BICOID was used as negative control. TH1, tail homology 1; SH3, Src homology 3; CA, central and acidic; ANK, ankyrin repeats; PH, Pleckstrin homology; N, N-terminal domain. (F) Protein–protein interaction network. The purple lines are reported interactions (Encinar del Dedo et al., 2017; Mo and Bard, 2005). Red lines are described here. (H) Individual channels and merged FMs of cells expressing Erg6-mCherry and Osh2-GFP. Individual channels and merged magnified insets are shown. Average ± SEM frequencies of colocalization of cortical Erg6-mCherry with Osh2-GFP indicated. n > 125 from three experiments. (I) Average ± SEM and t test P values of cortical GFP-D4H patch RFI normalized to WT (pOSH2) in osh2Δ cells expressing the empty plasmid or plasmids encoding OSH2 alleles. Significant differences are red (n > 210). Merged FMs of the corresponding cells and merged and individual channels of magnified areas shown. (J) Serial dilutions of erg6Δ cells bearing an empty plasmid (p) or plasmids encoding the WT ERG6 or a catalytically inactive mutant (erg6-D152L) grown at the indicated temperatures. (K) GFP-D4H cortical patch RFI normalized to the WT (pERG6) for erg6Δ cells bearing an empty plasmid (p) or plasmids encoding ERG6 or erg6-D152L.
