Quantitative analysis of the spatial distance between autophagy-related membrane structures and the endoplasmic reticulum in <i>Saccharomyces cerevisiae</i>

Macroautophagy/autophagy is the process by which cells degrade their cytoplasmic proteins or organelles in vacuoles to maintain cellular homeostasis under severe environmental conditions. In the yeast Saccharomyces cerevisiae, autophagy-related (Atg) proteins essential for autophagosome formation accumulate near the vacuole to form the dot-shaped phagophore assembly site/pre-autophagosomal structure (PAS). The PAS then generates the phagophore/isolation membrane (PG), which expands to become a closed double-membrane autophagosome. Hereinafter, we refer to the PAS, PG, and autophagosome as autophagy-related structures (ARSs). During autophagosome formation, Atg2 is responsible for tethering the ARS to the endoplasmic reticulum (ER) via ER exit sites (ERESs), and for transferring phospholipids from the ER to ARSs. Therefore, ARS and the ER are spatially close in the presence of Atg2 but are separated in its absence. Because the contact of an ARS with the ER must be established at the earliest stage of autophagosome formation, it is important to know whether the ARS is tethered to the ER. In this study, we developed a rapid and objective method to estimate tethering of the ARS to the ER by measuring the distance between the ARS and ERES under fluorescence microscopy, and found that tethering of the ARS to the ER was lost without Atg1. This method might be useful to predict the tethering activity of Atg2. Abbreviation: ARS, autophagy-related structure; Dautas, automated measurement of the distance between autophagy-related structures and ER exit sites analysis system; ERES, endoplasmic reticulum exit site; PAS, phagophore assembly site/pre-autophagosomal structure; PCR, polymerase chain reaction; PG, phagophore/isolation membrane; prApe1, precursor of vacuolar aminopeptidase I; Qautas, quantitative autophagy-related structure analysis system; SD/CA; synthetic dextrose plus casamino acid medium; WT, wild-type</p

Yeast strains used in this study.

<p>Yeast strains used in this study.</p

Autophagic activity of Atg8^G116 <i>atg4</i>Δ cells overexpressing Atg8/Atg8^G116.

<p><i>ATG8</i>^G116<i>atg4</i>Δ (GYS608) cells expressing indicated proteins were grown in SDCA medium to mid-log phase, and then treated with rapamycin for 2 h. Western blot analysis was performed with anti-Atg8 and anti-Ape1 antisera. Short- and long-exposed images were shown. Slower-migrating bands in the upper and middle panels correspond to unlipidated Atg8, and faster-migrating bands correspond to Atg8-PE. Slower-migrating bands in the lower panel correspond to the precursor form of Ape1 (prApe1), and faster-migrating bands correspond to the mature Ape1 (mApe1). ox indicates overexpression.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

Lengths of Atg1 or Atg16-labeled IMs in delipidation-mutant cells.

<p>(A) Atg1-mNG Atg8^G116 <i>atg4</i>Δ (YOC5469) and Atg16-mNG Atg8^G116 <i>atg4</i>Δ (YOC5470) cells carrying an empty or Atg4-expressing plasmid were grown to mid-log phase in SDCA medium containing CuSO₄, and then treated with rapamycin for 1 h. Scale bar: 2 μm. (B) Lengths of IMs were measured. Error bars indicate standard deviations. **<i>P</i> < 0.01 (two-tailed Student’s <i>t</i>-test).</p

Staining of autophagy-related structures with octadecyl rhodamine B.

<p>(A) Dpm1-GFP expressing cells (YCK445) harboring the pYEX-BX[prApe1] plasmid were grown in SDCA medium containing CuSO₄ and stained with octadecyl rhodamine B (R18). After the cells were washed with fresh medium, they were observed by fluorescence microscopy. (B) Wild-type cells harboring pRS314[GFP-Atg8] and pYEX-BX[prApe1] plasmids were grown in SDCA medium containing CuSO₄ and stained with R18. After the cells were washed with fresh medium, they were treated with rapamycin for 3 h. (C) Wild-type cells harboring pRS314[GFP-Atg8] and pYEX-BX[prApe1] plasmids were grown in SDCA medium containing CuSO₄ and stained with FM 4–64. After the cells were incubated with fresh medium without dye for 30 min, they were treated with rapamycin for 3 h. (D) mNG-Atg8 <i>atg4</i>Δ (YOC5272) and mNG-Atg8^G116 <i>atg4</i>Δ (YOC5330) cells carrying an empty or Atg4-expressing plasmid and mNG-Atg8^G116 <i>atg4</i>Δ <i>atg2</i>Δ (YOC5331) cells carrying an Atg4-expressing plasmid were grown to mid-log phase in SDCA medium containing CuSO₄, and then stained with R18. After the cells were washed with fresh medium, they were treated with rapamycin for 1 h. Scale bar: 2 μm. (E) Frequencies of mNG-positive structures stained with R18. Error bars indicate standard deviations. N.S., not significant. **<i>P</i> < 0.01 (two-tailed Student’s <i>t</i>-test). At least 40 cells were counted for each experiment (n = 4).</p

Localization of GFP-Atg8^G116 in <i>atg4</i>Δ cells lacking the scaffold complex for autophagy-related structures.

<p>(A) <i>atg4</i>Δ (GYS622), <i>atg4</i>Δ <i>atg11</i>Δ (YOC5270), <i>atg4</i>Δ <i>atg17</i>Δ (YOC5269), and <i>atg4</i>Δ <i>atg11</i>Δ <i>atg17</i>Δ (YOC5271) cells expressing GFP-Atg8^G116 and carrying an empty or Atg4-expressing plasmid were grown to mid-log phase in SDCA medium and treated with rapamycin for 1 h. Scale bar: 5 μm. (B) Number of GFP-Atg8^G116 puncta per cell. Error bars indicate standard deviations. N.S., not significant. **<i>P</i> < 0.01 (two-tailed Student’s <i>t</i>-test). At least 40 cells were counted for each experiment (n = 4).</p

IM expansion in delipidation-mutant cells.

<p>(A) mNeonGreen(mNG)-Atg8 <i>atg4</i>Δ (YOC5272) and mNG-Atg8^G116 <i>atg4</i>Δ (YOC5330) cells carrying an empty or Atg4-expressing plasmid, mNG-Atg8^G116 <i>atg4</i>Δ <i>atg2</i>Δ (YOC5331) cells carrying an Atg4-expressing plasmid, and mNG-Atg8^G116 <i>atg4</i>Δ (YOC5330) cells carrying an Atg8- or Atg8^G116-overexpressing (ox) plasmid were grown to mid-log phase in SDCA medium containing CuSO₄, and then treated with rapamycin for 1 h. Scale bar: 2 μm. (B) Lengths of IMs were measured. Error bars indicate standard deviations. **<i>P</i> < 0.01 (two-tailed Student’s <i>t</i>-test).</p

Autophagic activity of <i>ATG8</i>^G116 <i>atg4</i>Δ cells.

<p>(A) <i>ATG8 atg4</i>Δ (KVY13) or <i>ATG8</i>^G116 <i>atg4</i>Δ (GYS608) cells carrying an empty or Atg4-expressing plasmid were grown in SDCA medium to mid-log phase and treated with rapamycin for 2 h. Western blot analysis was performed using anti-Atg8 and anti-Ape1 antisera. Slower-migrating bands in the upper panel correspond to Atg8/Atg8^G116 (Unlipidated Atg8), and faster-migrating bands correspond to Atg8-PE. Slower-migrating bands in the lower panel correspond to the precursor form of Ape1 (prApe1), and faster-migrating bands correspond to the mature Ape1 (mApe1). (B) <i>ATG8 atg4</i>Δ (KVY13) or <i>ATG8</i>^G116 <i>atg4</i>Δ (GYS608) cells carrying the indicated plasmids were grown in SDCA medium to mid-log phase and treated with rapamycin for 2 h. Western blot analysis was performed with anti-GFP antibodies. Slower-migrating bands represent GFP-tagged Atg8 (GFP-Atg8), and faster-migrating bands represent cleaved GFP. (C) <i>ATG8</i>^G116 (YOC5308) and <i>ATG8</i>^G116 <i>atg4</i>Δ (YOC5309) cells were grown in SDCA medium to mid-log phase and shifted to SD(-N) medium and incubated for 4 h at 30°C. Then autophagic activity was measured by the alkaline phosphatase assay. Error bars indicate standard deviations. N.S. indicates not significant. *<i>P</i> < 0.05, **<i>P</i> < 0.01 (two-tailed Student’s <i>t</i>-test) (n = 3).</p

Primers used in this study.

<p>Primers used in this study.</p