Fig 1: Recruitment of autophagy machinery, including LC3B homologs, to stalled AVs is not sufficient to rescue the biogenesis defect.(A–E) Representative maximal projection micrographs of the distal neurites of fixed DRG neurons from aged mice. Stalled AVs are identified by colocalization of anti-ATG5 (green) and anti-ATG9 (blue). Antibodies to other autophagy components are visualized in red: ATG13 (A) or elongation complex components ATG12 (B), ATG7 (C), ATG16L1 (D), and ATG3 (E). Arrowheads denote colocalization state of AVs (stalled, filled arrowheads). Borders of magnifications of indicated puncta denote channel or colocalization state in merge. (F–I) Time series (channels merged) of Halo-ATG5, mAtg8s (mCh-GABARAP in F, mCh-GABARAPL2/GATE16 in G, mCh-GABARAPL1/GEC1 in H, and mScarlet-LC3A in I), and GFP-LC3B in the distal neurite of DRG neurons from aged mice depicting stalled AVs. Arrowheads denote colocalization of mAtg8s with stalled AVs. Magnified views of denoted puncta are shown below full micrograph; border color represents channel or colocalization state in merge. Time is indicated as time since stalled AV was first visible. (J) Quantification of the fraction of stalled AVs co-recruiting each LC3/GABARAP family member when individually overexpressed in DRGs from aged mice (mean ± SEM; n = 10 stalled AVs in three biological replicates for each mAtg8). ***p<0.001; ****p<0.0001 by two-tailed Fisher’s exact test. Scale bars, 2 µm.
Fig 2: NCOA4383–522 binding to (GST-GATE16)2 via peptide array.Related to Fig 2. (A) Binding of (GST-GATE16)2 to a peptide spot array composed of 20mers tiled across the NCOA4383–522 sequence, where each peptide is offset three amino acids. Peptides are arrayed N-to-C in rows (3) from left-to-right. Positive and negative controls at the right in the third row. (GST-GATE16)2 binding was detected using an HRP-conjugated anti-GST antibody. Sites of (GST-GATE16)2 binding are indicated by colored dots adjacent to the peptide array, with corresponding peptide sequence spans depicted. Positive control peptides arrayed left-to-right are noted with grey circles and were derived from p62/SQSTM (SGGDDDWTHLSS), ATG4B (EDEDFEILSL), and FYCO1 (DDAVFDIITDEELCQIQE), with known LIR motif underlined. Negative control peptide noted by light gray circle corresponded to a poly-His peptide (HHHHHHGSSHHHHHHGSSHH). (B) Far-Western as in (A), with GST replacing (GST-GATE16)2.
Fig 3: NCOA4383−522 binds directly to (GST-GATE16)2.(A) Schematic of NCOA4 domain architecture. The fragment of interest (NCOA4383−522) used in this study is noted. (B) SDS–PAGE gel of purified biotinylated AviTag-His6-NCOA4383−522-MBP (NCOA4383−522) and (GST-GATE16)2. (C) Far-Western blot of NCOA4383−522 binding to decreasing concentrations of (GST-GATE16)2, which was spotted on the membrane at the indicated concentration. Signal was detected by HRP-conjugated streptavidin directed to Avi-tagged NCOA4383−522, which was incubated on the membrane at a concentration of 0.1 μM. (D) Biolayer interferometry (BLI) trace of NCOA4383−522 binding to (GST-GATE16)2 where NCOA4383−522 was immobilized to the probe and was incubated with increasing concentrations (0, 78, 156, 313, and 625 nM, 1.25, 2.5, and 5 μM) of (GST-GATE16)2. Initialization of each incubation is noted by asterisks. Trace depicted is background-corrected for binding of (GST-GATE16)2 alone to the probe at each concentration.
Fig 4: Assessing the nonspecific association with NCOA4383–522 to (GST)2.Related to Fig 1. BLI response measurement of NCOA4383–522 binding to either (GST-GATE16)2 or to isolated (GST)2 where the NCOA4383–522 construct was immobilized to the probe and tested against increasing concentrations (0 and 625 nM; 1.25, 2.5, and 5 μM) of either construct. (GST-GATE16)2 curve reproduced from Fig 2B.
Fig 5: Assessment of nonspecific binding of NCOA4383–522 to (GST)2 in Far-Westerns.Related to Fig 1. (A) Far-Western blot of NCOA4383–522 binding to (GST-GATE16)2, which was spotted on the membrane at the indicated concentration. NCOA4383–522 incubated on the membrane at 0.1 μM. Signal detected by HRP-conjugated streptavidin. Two replicates are shown, with the lower replicate reproduced from Fig 1C. (B) Far-Western as in (A), with GST replacing (GST-GATE16)2.
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