Abstract
Impairment of autophagic degradation of the ubiquitin- and LC3-binding protein "p62" leads to the formation of cytoplasmic inclusion bodies. However, little is known about the sorting mechanism of p62 to autophagic degradation. Here we identified a motif of murine p62 consisting of 11 amino acids (Ser334-Ser344) containing conserved acidic and hydrophobic residues across species, as an LC3 recognition sequence (LRS). The crystal structure of the LC3-LRS complex at 1.56 angstroms resolution revealed interaction of Trp340 and Leu343 of p62 with different hydrophobic pockets on the ubiquitin fold of LC3. In vivo analyses demonstrated that p62 mutants lacking LC3 binding ability accumulated without entrapping into autophagosomes in the cytoplasm and subsequently formed ubiquitin-positive inclusion bodies as in autophagy-deficient cells. These results demonstrate that the intracellular level of p62 is tightly regulated by autophagy through the direct interaction of LC3 with p62 and reveal that selective turnover of p62 via autophagy controls inclusion body formation.
Highlights
Genetic and molecular studies in the yeast Saccharomyces cerevisiae have identified 18 ATG essential for autophagosome formation [1]
Little is known molecularly about how p62 is sorted into autophagosomes. In addition to this issue, we showed that homeostatic levels of p62 control ubiquitin-positive inclusion body formation at the cytoplasm in hepatocytes and neurons of autophagy-deficient mice [21], it is still unclear whether inclusion formation is only because of deficiency in p62 turnover
light chain 3 (LC3)-interacting Region of p62—p62 is composed of three domains as follows; N-terminal Phox and Bem1 (PB1) domain, zinc finger domain (Zinc), and C-terminal ubiquitin-associated (UBA) domain [19, 20]
Summary
Plasmid Construction—Maltose-binding protein (MBP) fused p62 (MBP-p62), and its deletion mutant (MBP-p62M7) expression plasmids have been described previously [21]. The cell extracts were diluted 1:5 into TBS (20 mM Tris-HCl, pH 7.5, and 150 mM NaCl) and incubated with amylose resin (New England Biolabs) at 4 °C for 2 h. The MBP fusion protein-bound amylose resins were washed three times with TBS. The resulting immobilized amylose resins containing about 5 g of MBP fusion proteins were incubated with 15 g of LC3 family proteins (LC3, GABARAP, or GATE-16) in 50 l of TBS on ice for 1 h, and washed three times with 500 l of TBS. Crystallization and Data Collection—Crystals of LC3-LRS peptide complex were obtained at 15 °C by the hanging-drop vapor-diffusion method, with a mixture of 2.0 l of protein and the same volume of reservoir solution (0.1 M HEPES, pH 7.5, and 25% w/v polyethylene glycol 3,350). Accession Codes—Protein Data Bank: The coordinates and structure factors for LC3-LRS complex have been deposited under accession code 2ZJD
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
