PMC:4786408 / 10853-13497
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{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4786408","sourcedb":"PMC","sourceid":"4786408","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4786408","text":"E122D mutation of ATG5 impairs ATG12–ATG5 conjugation\nTo examine the effect of the ATG5E122D mutation on formation of the ATG12–ATG5-ATG16L1 complex, we expressed the recombinant human proteins in insect Hi5 cells and analyzed the complexes by affinity isolation. We could detect the ATG12–ATG5 complex when both wild-type proteins were co-expressed, but we could only detect a minimal amount of the ATG12–ATG5E122D complex (Figure 4A). Although overexpression of human ATG5WT in HEK293 cells or Drosophila tissues resulted in efficient covalent conjugation with overexpressed human ATG12 (Figure 4B and C) or endogenous Drosophila Atg12 (Figure 4D), mutant ATG5E122D was dramatically impaired in this process (Figure 4B, C and D). Interestingly, expression levels of ATG5WT and ATG5E122D monomers were comparable to each other, indicating that the mutation affects the conjugation process, rather than the stability of proteins. This was consistent with the structural location of ATG5 E122 adjacent to the surface that interacts with ATG12 (Figure 2B). To confirm that the mutation does not overtly alter the structure of ATG5 or binding to ATG16L1, we analyzed formation of the noncovalent ATG5-ATG16L1 complex using constructs containing a TEV protease site. Both wild-type and mutant ATG5 protein were efficiently co-precipitated with ATG16L1 (Figure 4E). Indeed, the co-crystal structure of a human ATG5E122D-ATG16L1 complex (Figure 2 and Table 1) superimposes well with the previously determined structure of the WT proteins (Figure 2D), with the major obvious difference being replacement of the side-chain (Figure 2E and F). Thus, it appears that the E122D mutation interferes with the ATG12–ATG5 conjugation process, but not with ATG5 folding or binding of ATG16L1.\n10.7554/eLife.12245.006Figure 4. E122D mutation interferes with formation of the ATG12–ATG5 conjugate.\n(A) Coomassie Blue-stained SDS-PAGE gel following glutathione affinity purification from lysates of Hi5 cells infected with baculoviruses expressing GST-ATG12 and either WT or E122D mutant ATG5. (B and C) HEK293 cells expressing the indicated proteins were analyzed by IB. (D) Drosophila whole bodies expressing the indicated transgenes under the control of Tub-Gal4 were analyzed by IB. (E) Lysates from Hi5 cells expressing the indicated proteins were subjected to His/Ni-NTA purification and subsequent TEV protease treatment. Proteins were analyzed by Coomassie Blue staining.\nDOI: http://dx.doi.org/10.7554/eLife.12245.006\n10.7554/eLife.12245.007Table 1. Crystallography data collection and refinement statistics.\nDOI: http://dx.doi.org/10.7554/eLife.12245.007","divisions":[{"label":"Title","span":{"begin":0,"end":53}},{"label":"Figure caption","span":{"begin":1776,"end":2506}},{"label":"Title","span":{"begin":1809,"end":1878}}],"tracks":[]}