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Published online July 14, 2008
doi:10.1083/jcb.200711112
The Journal of Cell Biology, Vol. 182, No. 1, 129-140
The Rockefeller University Press, 0021-9525 $30.00
© 2008 Geng et al.
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Article

 Quantitative analysis of autophagy-related protein stoichiometry by fluorescence microscopy

Jiefei Geng1, Misuzu Baba2, Usha Nair1, and Daniel J. Klionsky1

1 Life Sciences Institute and Departments of Molecular, Cellular, and Developmental Biology and Biological Chemistry, University of Michigan, Ann Arbor, MI 48109
2 Department of Chemical and Biological Sciences, Faculty of Science, Japan Women's University, Mejirodai, Tokyo 112-8681, Japan

Correspondence to Daniel J. Klionsky: klionsky{at}umich.edu

In yeast, ~31 autophagy-related (Atg) proteins have been identified. Most of them reside at the phagophore assembly site (PAS), although the function of the PAS mostly remains unclear. One reason for the latter is the lack of stoichiometric information regarding the Atg proteins at this site. We report the application of fluorescence microscopy to study the amount of Atg proteins at the PAS. We find that an increase in the amount of Atg11 at the PAS enhances the recruitment of Atg8 and Atg9 to this site and facilitates the formation of more cytoplasm-to-vacuole targeting vesicles. In response to autophagy induction, the amount of most Atg proteins remains unchanged at the PAS, whereas we see an enhanced recruitment of Atg8 and 9 at this site. During autophagy, the amount of Atg8 at the PAS showed a periodic change, indicating the formation of autophagosomes. Application of this method and further analysis will provide more insight into the functions of Atg proteins.

Abbreviations used in this paper: Ape1, aminopeptidase I; Atg, autophagy related; CC, coiled coil; CEN, centromeric; Cvt, cytoplasm-to-vacuole targeting; IEM, immuno-EM; PAS, phagophore assembly site; prApe1, precursor Ape1; TEM, transmission EM.

© 2008 Geng et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).


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