Structure-function relationship of Atg12, a ubiquitin-like modifier essential for autophagy.
Atg12, a post-translational modifier, is activated and conjugated to Atg5 by a ubiquitin-like conjugation system, though it has no obvious sequence homology to ubiquitin. The Atg12-Atg5 conjugate is essential for autophagy, an intracellular bulk degradation process. Here, we show that the carboxyl-terminal region of Atg12 that is predicted to fold into a ubiquitin-like structure is necessary and sufficient for both conjugation and autophagy, which indicates that the domain essential for autophagy resides in the ubiquitin-fold region. We further show that two hydrophobic residues within the ubiquitin-fold region are important for autophagy: mutation at Y149 affects conjugate formation catalyzed by Atg10, an E2-like enzyme, while mutation at F154 has no effect on Atg12-Atg5 conjugate formation but its hydrophobic nature is essential for autophagy. In response to the F154 mutation, Atg8-PE conjugation, the other ubiquitin-like conjugation in autophagy, is severely reduced and autophagosome formation fails. Gel filtration analysis suggests that F154 plays a critical role in the assembly of a functional Atg12-Atg5.Atg16 complex that is requisite for autophagosome formation.
Hanada
Takao
T
Division of Molecular Cell Biology, National Institute for Basic Biology, Okazaki, Japan.
Ohsumi
Yoshinori
Y
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001343:Autophagy / Q000502:physiology; D000071192:Autophagy-Related Protein 12; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D011485:Protein Binding; D017510:Protein Folding; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*
C491969:ATG12 protein, S cerevisiae; D000071192:Autophagy-Related Protein 12; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin