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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.
Ever since its initial discovery, the green fluorescent protein (GFP) has substantially changed the research methodology of modern biology. By fusing GFP and its various derivatives to other interesting, but otherwise invisible, proteins, researchers can “watch” the positions, movements and interactions of tagged proteins in different conditions. The application of fluorescent protein methodologies is still expanding. Recently, Wu and Pollard reported that protein concentration can be directly measured by fluorescence microscopy in Schizosaccharomyces pombe.1 Inspired by their results, we applied similar methods in Saccharomyces cerevisiae to study the stoichiometry of Atg proteins during the vesicle formation process.2
This method is feasible and useful in the study of autophagy. First, most Atg proteins localize to a very restricted region, the phagophore assembly site (PAS, also known as the pre-autophagosomal structure).3,4 This property makes the quantification process much easier. Second, it is also important to know the stoichiometry of Atg proteins at this locus. The PAS is thought to be the organization center for the formation of the Cvt vesicle and autophagosome. Multiple lines of evidence have pointed out that the amount of some Atg proteins (e.g., Atg8 and Atg9) at the PAS is under precise regulation and related to the quantitative property of the vesicle formation process, such as vesicle size or vesicle number.5-7 However, traditional methods cannot provide information on the quantitative analysis of Atg proteins at a specific subcellular locus. In contrast, this fluorescence microscopy method can directly measure the protein amount in living cells and even in a time-course experiment.
In this protocol article, we describe the method to quantify the amount of Atg proteins at the PAS. Theoretically we can use this method to measure the protein amount at any subcellular localization. In our analysis of Atg proteins, the PAS is the clearest structure for fluorescence quantification. The method can be divided into two basic parts. The first part is to establish a standard curve showing the linear relationship between fluorescence intensity and protein amount; the second part is to use this standard curve to measure and calculate the protein amount at the PAS. In some cases, relative quantification provides sufficient information, for example, in asking how does the Atg8 concentration at the PAS change in different conditions, or what are the kinetics of Atg8 recruitment to the PAS? In this situation, after confirming the linear relationship between the protein amount and fluorescence intensity, it is possible to simply measure the relative intensity and convert it into relative protein amount without determining the absolute value.
Living colors A.v. monoclonal antibody JL-8 (Clontech, 632381).
This work was supported by Public Health Service grant GM53396 to D.J.K. from the National Institutes of Health.