Determining Atg protein stoichiometry at the phagophore assembly site  by fluorescence microscopy.
In eukaryotic cells, autophagy is a lysosomal/ vacuolar degradative pathway necessary for the turnover of different macromolecules. Autophagy is under precise regulation, not only qualitatively but also quantitatively, and excess or reduced levels of autophagy may lead to various human diseases. In yeast, genetic screens led to the identification of more than 30 autophagy-related (ATG) genes, and most of the gene products reside at the phagophore assembly site (PAS). However, our attempt to understand the quantitative properties of autophagy is usually hampered, because traditional methods of analysis cannot provide stoichiometric information. We have recently used a fluorescence microscopy-based method to study the stoichiometry of Atg proteins at the PAS, trying to explain the mechanism of how the vesicle formation process is precisely regulated. This article describes a practical guide on this method. Its application and further analysis will improve our understanding of the quantitative properties of autophagy.
Life Sciences Institute and Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, USA.
Jiefei
J
Geng
Daniel J
DJ
Klionsky
D048868:Adaptor Proteins, Signal Transducing / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D001343:Autophagy / Q000502:physiology; D002478:Cells, Cultured; D019411:Clinical Laboratory Techniques; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D010588:Phagosomes / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D025841:Small Ubiquitin-Related Modifier Proteins / Q000032:analysis* / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000032:analysis* / Q000378:metabolism; D015003:Yeasts
D048868:Adaptor Proteins, Signal Transducing; D025841:Small Ubiquitin-Related Modifier Proteins; D033921:Vesicular Transport Proteins