Hypothesis

We hypothesize that the hierarchical selectivity of autophagy is governed by a kinetic competition for limited ATG8‑LC3/GABARAP pools, where the oligomerization state and post‑translational modifications of cargo receptors set their binding affinity and thus determine which organelle or protein aggregate is sequestered first. When this triage is skewed—by chronic activation of a single receptor pathway or by age‑related decline in ATG8 lipidation—cells experience a zero‑sum trade‑off that preferentially sustains the favored pathway at the expense of others, leading to organelle‑specific dysfunction that underlies age‑related decline.

Core Mechanism

• Cargo receptors such as p62/OPTN (mitophagy/aggrephagy) and FAM134B/CCPG1 (ER‑phagy) share a common ATG8‑interacting motif (AIM).
• Phosphorylation of the AIM region (e.g., TBK1‑dependent phosphorylation of p62 Ser403) increases ATG8 binding affinity, while dephosphorylation reduces it.
• Oligomerization of receptors creates avidity effects that further amplify differences in effective affinity.
• Under conditions that favor one pathway (e.g., Urolithin A‑induced mitophagy via AMPK activation), the phosphorylated state of mitophagy receptors rises, outcompeting ER‑phagy receptors for the finite ATG8 pool.
• This competition does not alter overall autophagic flux but redistributes it, causing a relative suppression of the non‑favored pathway despite unchanged expression levels of its receptors.

Testable Predictions

1. Selective flux shift – Increasing mitophagy receptor phosphorylation will decrease ER‑phagy flux without changing total LC3‑II turnover.
2. Reciprocal effect – Enhancing ER‑phagy receptor phosphorylation (e.g., via AMPK‑dependent phosphorylation of FAM134B Ser304) will reduce mitophagy flux and increase mitochondrial ROS.
3. Age‑related bias – In aged skeletal muscle, basal phosphorylation of mitophagy receptors is elevated relative to ER‑phagy receptors, correlating with lower ER‑phagy markers and higher ER stress.
4. Rescue by affinity modulation – Expressing a phospho‑deficient mutant of p62 (S403A) in aged muscle will restore ER‑phagy flux and alleviate ER stress, even in the presence of Urolithin A.

Experimental Approach

• In vivo models – Use young and aged mice treated with Urolithin A or vehicle.
• Genetic tools – AAV‑mediated expression of WT or phospho‑mutant (S403A) p62 and phospho‑mimetic (S304D) FAM134B in muscle.
• Flux assays – mt‑Keima for mitophagy, ER‑GFP‑LC3 for ER‑phagy, coupled with lysosomal inhibition (chloroquine) to measure flux.
• Readouts – Western blot for phosphorylated receptors, LC3‑II/p62, ER stress markers (BiP, CHOP), mitochondrial ROS (MitoSOX), and functional assays (grip strength, endurance treadmill).
• Statistical test – Two‑way ANOVA (age × genotype) with post‑hoc comparisons; a significant interaction indicating that phospho‑mutant p62 rescues ER‑phagy flux only in aged mice would support the hypothesis; absence of such interaction would falsify the zero‑sum competition model.

By focusing on the competitive binding dynamics of ATG8 rather than the capacity of autophagy itself, this hypothesis reframes age‑related decline as a mismatched allocation of a limited degradative resource, offering a precise point of intervention—tuning receptor affinity to rebalance the autophagic hierarchy.