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. Author manuscript; available in PMC: 2024 Apr 17.
Published in final edited form as: Nat Aging. 2024 Jan 4;4(2):198–212. doi: 10.1038/s43587-023-00548-1

Fig. 6 |. The WD40 domain of ATG-16.2 is dispensable for autophagosome formation but required for exophergenesis.

Fig. 6 |

a, Schematic of ATG-16.2 rescue constructs. Full-length ATG-16.2 protein includes ATG5-interacting motif, coiled-coil domain (CC) and WD40 domain. ATG-16.2ΔWD40 is a truncated ATG-16.2 protein containing amino acids 1–234. ATG-16.2(Phe394Ala, Ile509Met) protein contains a point mutation of phenylalanine to alanine in position 349 and in isoleucine to methionine in position 509 in the WD40 domain. See Extended Data Fig. 6a for the primary structure of ATG-16.2. b, AlphaFold model of ATG-16.2 (UniProt Q09406). The N-terminal region of ATG-16.2 is predicted to contain a helical structure with a conserved ATG5-interacting motif and a CC domain. The C terminus contains a seven-bladed WD40 beta-propeller domain. Phe394 (F) is located on blade 4 on the linker between β-sheets B-C, and Ile509 (I) is located on the linker between blade 6–7 before β-sheet A (enlarged). The enlarged surface model reveal indicates that Phe394 is surface exposed, whereas Iso509 is not. c, Mean GFP::LGG-1/ATG8-positive punctae in day 1 WT (n = 32), atg-16.2(ok3224) (n = 33) and atg-16.2(ok3224) mutants transgenically expressing full-length atg-16.2 (n = 32), atg-16.2 lacking the WD40 domain (ΔWD40) (n = 30) or atg-16.2(Phe394Ala, Ile509Met) (F394A, I509M) (n = 31) from the neuronal rgef-1 promoter. Error bars are the s.d. over three independent experiments, ****P < 0.0001, by one-way ANOVA by Dunnett’s multiple-comparisons test. d, Mean percentage of day 2 WT (n = 258 animals), atg-16.2(ok3224) (n = 271 animals) and atg-16.2(ok3224) animals expressing full-length atg-16.2 (n = 214 animals), atg-16.2 lacking the WD40 domain (ΔWD40) (n = 275 animals) or atg-16.2(Phe394Ala, Ile509Met) (F394A, I509M (n = 212 animals) from the neuronal rgef-1 promoter with ALMR exophers. Error bars are the s.d. of n = 6 experiments, ****P < 0.0001; ***P = 0.002, NS P = 0.30, P = 0.77 by two-sided Cochran–Mantel–Haenszel test.