Fig. 2

CRY constructs carrying specific mutations of cysteine and the C-terminus tail differentially affect TIM stability. (A) Mutating a series of cysteine residues reveals that the C523S mutation in CRY destabilizes TIM in the dark (top and middle panels). While most constructs carrying mutated residues in the CRY C-terminus have partial effects on TIM stability in the dark, E530Q and F534L have dramatic effects on light-dependent degradation of TIM (middle and bottom panel). Quantification of TIM and loading control band b-GAL is shown in (B). Black filled bars denote darkness, and open bars denote light conditions. Error bars denote standard error of mean, n = 3. Asterisk denotes significant difference between dark and light (p < 0.05 by Student’s t-test); different upper-case letters denote significant difference between wildtype and mutant constructs in dark, whereas different lower-case letters denote significant difference between wildtype and mutant constructs in light (one-way ANOVA, post hoc Tukey test, p < 0.05);. (C,D) Effect of C523S, E530Q and F534L on TIM levels in dark is not blocked by a mitochondrial complex III inhibitor antimycin A. A representative Western blot is shown in (C) and quantification of TIM and loading control band b-GAL is shown in (D). Error bar denotes standard error of mean, n = 3. Asterisk denotes significant difference by Student’s t-test, p < 0.05. Full-length blots are presented in Supplemental Figure S4 and S10.