Mutation in ATG5 reduces autophagy and leads to ataxia with developmental delay
- University of Michigan, United States
- St Jude Children's Research Hospital, United States
- Howard Hughes Medical Institute, St. Jude Children's Research Hospital, United States
- Boğaziçi University, Turkey
- Hungarian Academy of Sciences, Hungary
- Eötvös Loránd University, Hungary
- Istanbul University, Turkey
Figures
Figure 1
Linkage analysis in consanguineous family with two siblings with ataxia, mental retardation and developmental delay maps defect to chromosomal interval containing ATG5.
Remote consanguinity was detected between parents of two previously described siblings having ataxia (Yapici and Eraksoy, 2005), illustrated here as third cousins. SNP and linkage results for …
Figure 2
The primary sequence of ATG5, including the mutant E122 residue, as well as the protein structure is highly conserved across eukaryotic species.
(A) Amino acid sequence alignment between ATG5 orthologs from human (HsaATG5), mouse (MmuAtg5), Drosophila melanogaster (DmeAtg5) and Saccharomyces cerevisiae (SceAtg5) was constructed at GenomeNet …
Figure 3
Cells from ataxia patients with ATG5E122D/E122D mutation exhibit autophagy defects.
(A) Decreased expression of ATG12–ATG5 conjugates in cells from ataxia patients with ATG5E122D/E122D mutation. ATG5 immunoblotting (IB) of ATG12–ATG5 conjugates of LCLs from individuals whose ATG5 …
Figure 4
E122D mutation interferes with formation of the ATG12–ATG5 conjugate.
(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 …
Figure 5
E141D mutation of yeast Atg5 attenuates autophagy.
(A-D) Yeast cells were grown in SMD to mid-log phase and nitrogen starved for the indicated times. (A) WLY176 atg5∆ yeast cells expressed GFP-Atg8 through its endogenous promoter and plasmid-based …
Figure 6
Ataxic phenotype of Atg5-null flies is suppressed by human ATG5WT but not by ATG5E122D.
(A) Genomic organization of the Atg5 locus and the Atg5-null mutant (Atg55cc5). Atg55cc5 mutants have a CRISPR-Cas9-mediated deletion in approximately 1.5 kb residues that eliminate more than 85% of …
Videos
Video 1
Climbing assay in 2 weeks-old wild-type flies (left) and Atg5-null flies (right).
https://doi.org/10.7554/eLife.12245.010
Video 2
Climbing assay in 2 weeks-old Atg5-null flies (left) and Atg5-null flies expressing ATG5WT (right).
https://doi.org/10.7554/eLife.12245.011
Video 3
Climbing assay in 2 weeks-old Atg5-null flies (left) and Atg5-null flies expressing ATG5E122D (right).
https://doi.org/10.7554/eLife.12245.012
Video 4
Climbing assay in 2 weeks-old Atg5-null flies expressing ATG5WT (left) or ATG5E122D (right).
https://doi.org/10.7554/eLife.12245.013Tables
Table 1
Crystallography data collection and refinement statistics.
| Data collection | |
|---|---|
| Beam line | APS 24-ID-C |
| Space group | C2 |
| Unit cell parameters | |
| a, b, c (Å) | 217.1, 84.5, 151.9 |
| α, β, γ (°) | 90, 133.8, 90 |
| Resolution (Å) (highest shell) | 50–3.0 (3.12-3.0) |
| Wavelength (Å) | 0.9792 |
| Number of measured reflections | 179,310 |
| Number of unique refections | 39,496 |
| Overall Rsym | 0.057 (0.645) |
| Completeness (%) | 98.9 (99.3) |
| Overall I/σI | 14.3 (2.3) |
| Multiplicity | 4.5 |
| Refinement | |
| Resolution (Å) | 50–3.0 |
| Rwork/Rfree | 0.198/0.244 |
| rmsd bond lengths (Å) | 0.008 |
| rmsd bond angles (°) | 0.994 |
| Number of protein atoms | 9403 |
| Ramachandran statistics | |
| Preferred (%) | 97.69 |
| Allowed (%) | 2.22 |
| Disallowed (%) | 0.09 |
