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Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses
Published on: July 14, 2014
Quantitative analysis of the spatial distance between autophagy-related membrane structures and the endoplasmic
Yang Shi1, Kuninori Suzuki1,2,3
1Department of Integrated Biosciences, Graduate School of Frontier Sciences, the University of Tokyo, Kashiwa, Chiba, Japan.
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.
