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. 2023 Aug 8;14:4765. doi: 10.1038/s41467-023-40530-4

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a Wild-type (wt) S. pombe Rop1 (spRop1) or the indicated mutants were expressed from the endogenous genomic locus. The cells were treated with DTT for three days (DTT 3d) and plated after serial dilution. The schemes show the domains of spRop1, with the APHs and putative AIM motif in gray and black, respectively, and deleted regions indicated. The W138A, V141A mutant is designed to abrogate the AIM motif. b ERphagy was tested by following the cleavage of a GFP-fusion of Yop1 (Yop1-GFP) in wt cells or cells expressing the indicated Rop1 mutants. Lysates from cells at stationary phase were analyzed by SDS-PAGE and immunoblotting with GFP antibodies. Blotting with GAPDH antibodies served as loading control. The right panel shows quantification of three independent experiments (means and SD). *** indicates significant differences with p-values < 0.001, calculated from two-tailed Student’s t tests. The exact p-values are listed in the Source Data file. c wt or mutant Rop1 used in (b) were FLAG-tagged. Lysates from logarithmically growing cells were analyzed by immunoblotting for FLAG. d Bulk autophagy was tested by following the relocalization of a mCherry fusion of the cytosolic protein Tdh1 (Tdh1-mCh) expressed at endogenous levels in wt cells or cells expressing the indicated Rop1 mutants. Stationary phase cells were analyzed by fluorescence microscopy. Scale bar, 10 µm. e Bulk autophagy was followed by the vacuolar cleavage of a GFP-fusion of the cytosolic protein Hsc1 (Hsc1-GFP) in the indicated strains grown to stationary phase. The right panel shows the percentage of Hsc1-GFP cleavage (means and SD). *p-value 0.07; *** significant differences with p-values < 0.001, calculated from two-tailed Student’s t tests. The exact p-values are listed in the Source Data file.