Deletion analysis of ER-lumen-oriented loops. (A) Predicted Shr3 structure (AlphaFold) and the positions of the internal deletions in loops L1 and L3. The helical segment predicted in L1 is colored with yellow for non-polar, gray for polar, blue for negatively-, and red for positively charged residues. Molecular graphics and analyses were performed with UCSF ChimeraX. (B) Helical wheel projection of the L1 α-helix with non-polar (yellow), polar (gray), negatively (blue), and positively charged (red) residues indicated. (C) Serial dilutions of cell suspensions from strain JKY2 (shr3Δ) carrying pRS316 (VC), pPL210 (SHR3), pAR41 (shr3Δ90), pAR42 (shr3Δ91), pAR43 (shr3Δ92), pAR44 (shr3Δ93), or pAR45 (shr3Δ94) spotted on SAD containing D-histidine (D-his), SD + L-canavanine (L-Can), SD + AzC, and YPD + MM plates. Plates were incubated at 30°C for 2 d and photographed. Bottom: Immunoblot analysis of Shr3 proteins in extracts prepared from the strains; the levels of Dpm1 were used as loading controls. The blots were developed using α-Shr3 and α-Dpm1 antibodies. The signal intensities of the immunoreactive forms of Shr3 and Dpm1 were quantified and the Shr3 signals were normalized with respect to Dpm1; the mean values are plotted and the error bars show standard deviation (n = 3; biological replicates). Source data are available for this figure: SourceData F2.