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**14** 

*Japan* 

**Crystallographic Studies on** 

**Autophagy-Related Proteins** 

*1Institute of Microbial Chemistry, Tokyo* 

*2Tokyo Institute of Technology* 

*3Hokkaido University* 

Nobuo N. Noda1, Yoshinori Ohsumi2 and Fuyuhiko Inagaki3

Autophagy is an intracellular bulk degradation system conserved in most eukaryotes. Autophagy plays various physiological roles such as intracellular clearance, development, differentiation and intracellular immunity, and its dysfunction is related to severe diseases such as neurodegeneration and cancer (Mizushima, 2007; Mizushima et al., 2008). Upon induction of autophagy, isolation membranes appear in the cytoplasm and expand to enclose a portion of the cytoplasm to be double membrane structures, autophagosomes (Mizushima et al., 2011; Nakatogawa et al., 2009). In some cases, organelles such as mitochondria and peroxisomes, and even invading microbes are also enclosed. All the matters enclosed by an autophagosome are named cargoes. The autophagosome fuses with the lysosome in mammals and with the vacuole in yeast and plants and exposes its inner membrane containing cargoes to vacuolar/lysosomal hydrolases, which degrade cargoes. Using the budding yeast, *Saccharomyces cerevisiae*, as a model organism, many *ATG* genes required for autophagy, especially in the step of autophagosome formation, have been identified (Klionsky et al., 2003; Tsukada and Ohsumi, 1993). Most *ATG* genes are evolutionarily conserved among higher eukaryotes including mammals, suggesting that the basic molecular mechanisms of autophagy are also evolutionarily conserved. Of the 35 Atg proteins identified thus far, 18 have been shown as crucial for autophagosome formation (Nakatogawa et al., 2009). However, it remains to be elucidated how these Atg proteins mediate autophagosome formation. In order to establish the molecular functions of each Atg protein and unveil the mechanism of autophagy, we started comprehensive structural studies of Atg proteins using X-ray crystallography. In this chapter, we review X-ray crystallographic studies of Atg proteins focusing on experimental details. For a more detailed description of the structure and function of Atg proteins, please refer to other recent

reviews (Mizushima et al., 2011; Nakatogawa et al., 2009; Noda et al., 2009, 2010).

**2. X-ray crystallographic studies of Atg proteins involved in ubiquitin-like** 

Of the 18 Atg proteins essential for autophagosome formation, eight, namely, Atg3, Atg4, Atg5, Atg7, Atg8, Atg10, Atg12 and Atg16, constitute two ubiquitin-like conjugation

**1. Introduction** 

**conjugation reactions** 

studied by crystal structure analysis and engineering of chimers. *Extremophiles*, Vol.11, No.3, (May 2007), pp. 481-493, ISSN 1431-0651

