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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. 7 — a Scheme showing how a fusion of Atg2 to a GFP nanobody (Atg2-GBP) drags Yop1-GFP from the ER to the phagophore rim or to the tips of ER tubules. A similar strategy was used with Atg18b-GBP. b Atg2-GBP or Atg18b-GBP were tagged with mCherry and expressed together with Yop1-GFP or Sec63-GFP. The cells were imaged after 2 h of nitrogen starvation (-N 2 h). Arrowheads point to colocalization of the coexpressed proteins. Scale bar, 5 µm. c Wild-type (wt) S. pombe cells or cells expressing the indicated mutants at endogenous levels were treated with DTT for three days (DTT 3d) and plated after serial dilution. d wt or rop1∆ cells expressing the indicated fusion proteins were nitrogen-starved for 20 h and analyzed by TEM. Asterisks point to vacuoles containing electron-dense material, likely residual membranes of autophagosomes. V empty vacuole, M mitochondrion, N nucleus. Scale bars, 500 nm. e Quantification of the results in (d). Shown is the percentage of filled vacuoles per cell as individual measurements, 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. f Bulk autophagy was tested by vacuolar cleavage of a mCherry fusion of the cytosolic protein Tdh1 (Tdh1-mCh) in cells expressing Atg2-GBP or Atg18b-GBP together with GFP fusions of full-length Yop1, Yop1 carrying a deletion of the APH (Δ132-189), Yop1 carrying a deletion that retains the APH (Δ143-189), or Sec63. Lysates of cells grown to stationary phase were analyzed by blotting for mCherry. The lower panel shows a loading control in which proteins were stained with Revert 700 (Rev700). g Quantification of two experiments as in (f). h Two possible models for the function of Rop1 in autophagosome formation. Rop1 could stabilize the high membrane curvature of the phagophore rim or that of a specialized ER region and facilitate Atg2-mediated lipid flow from the ER to the rim of phagophores (arrows).