Quantitative analysis of autophagy-related protein stoichiometry by fluorescence microscopy

被引:88
作者
Geng, Jiefei [1 ,2 ,3 ]
Baba, Misuzu
Nair, Usha [1 ,2 ,3 ]
Klionsky, Daniel J. [1 ,2 ,3 ]
机构
[1] Univ Michigan, Inst Life Sci, Dept Mol Cellular & Dev Biol, Ann Arbor, MI 48109 USA
[2] Univ Michigan, Inst Life Sci, Dept Biol Chem, Ann Arbor, MI 48109 USA
[3] Japan Womens Univ, Fac Sci, Dept Chem & Biol Sci, Tokyo 1128681, Japan
关键词
D O I
10.1083/jcb.200711112
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
In yeast, similar to 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.
引用
收藏
页码:129 / 140
页数:12
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