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

ABSTRACT

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

KEYWORDS:

• Autophagy-related 2 (Atg2)
• endoplasmic reticulum exit sites (ERES)
• fluorescence microscopy
• morphological analysis
• phagophore
• yeast

Acknowledgements

We thank Dr. Eri Hirata (The University of Tokyo, Japan) for helpful advice on yeast experiments.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data of this study is available from the corresponding author (KS) upon reasonable request. The codes used in this study are shared on GitHub (https://github.com/kqpkqp/Dautas).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15548627.2024.2330033

Additional information

Funding

This work was supported by the Japan Science and Technology Agency [JST] SPRING [JPMJSP2108 to YS], Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan [20H05313, 21K19205, and 22H02569 to KS], and JST CREST [JP201032912 to KS].