Vps13 localization at the ER–endosome contact site. (A) Localization of Vps13–GFP. Scale bar: 1 µm. (B) Quantification of Vps13–GFP colocalizing with mCherry–Vps21 from A. (C) Localization of Vps13^ΔC–GFP (residues 1–1851). Scale bar: 1 µm. (D) Quantification of Vps13–GFP puncta localization from C. (E) The localization of Vps13^N–GFP (residues 1–39). Scale bar: 1 µm. (F) Line scan analysis for the region highlighted by the yellow dash line in E. (G) Vps13–GFP localization at the ER–endosome contact site. Vps13–GFP, DsRed–HDEL (ER), and Mup1–BFP (endosome) expressing cells were stimulated with methionine. Scale bar: 1 µm. (H) Live cell-imaging analysis of the ER (GFP-HDEL) and endosome (mCherry–Vps21). Scale bar: 500 nm. (I) The diameter of the ER contact with the endosome. The membrane was designed ER by the observation of its bound ribosomes, dimensions, and staining by high-pressure freezing and electron tomography. ER diameters per 100-nm interval starting at the endosome contact along the 500 nm length of ER were determined. (J and K) Mup1–GFP processing in vps13 mutants after methionine stimulation. Cell lysates were analyzed by immunoblotting using anti-GFP and anti-G6PDH antibodies. (L) Western blotting analysis of Vps13 expression. Cell lysates were analyzed by immunoblotting using anti-HA and anti-Pgk1 antibodies. Source data are available for this figure: SourceData FS2.