FAR8 Antibody

Definition and Purpose of FAR8 Antibody

The FAR8 antibody targets Far8, a core component of the yeast Far complex (Far3-7-8-9-10-11), which regulates mitophagy by dephosphorylating the mitophagy receptor Atg32 . This antibody is essential for immunoprecipitation (IP) and Western blot (WB) assays to study Far8’s interactions and localization.

Protein Interaction Mapping

• Co-immunoprecipitation Assays:
  The FAR8 antibody identifies interactions between Far8 and other Far complex proteins (Far3, Far7, Far9, Far11) and phosphatase Ppg1 . For example:

  • Far8 co-immunoprecipitates with Far11 only in the presence of Ppg1 (Figure 3J) .

  • Disruption of PPG1 abolishes Far8-Far11 interaction, highlighting Ppg1’s scaffolding role .

Subcellular Localization Studies

• Far8’s mitochondrial/ER localization is confirmed using GFP-tagged Far9 mutants (Tom5TA for mitochondria, Cyb5TA for ER) .

Mechanistic Insights into Mitophagy

• FAR8 antibody detects phosphorylation states of Atg32, a mitophagy receptor. Dephosphorylation by the Far complex inhibits mitophagy, while dissociation activates it .

Regulation of Atg32 Phosphorylation

• The Far8-Atg32 fusion protein remains unphosphorylated unless Ppg1 is deleted, demonstrating direct regulatory interplay .

Assembly of the Far Complex

• Core Interactions:

  • Far8 binds Far11 only with Ppg1 present .

  • Far3, Far7, and Far9 are required for Far8-Atg32 interaction .

• Catalytic Dependency:
  Ppg1’s phosphatase activity is essential for Far8-Far11 binding (Figure 4F) .

Mechanistic Model of FAR8 Function

1. Complex Assembly:
   Ppg1 recruits Far11, forming a subcomplex that binds Far8 and other Far proteins .

2. Atg32 Binding:
   The Far complex directly interacts with phosphorylated Atg32 via Far8’s N-terminal region (residues 151–250) .

3. Mitophagy Regulation:
   Association with Atg32 inhibits phosphorylation, while dissociation (triggered by stress) activates mitophagy .

Experimental Validation

• GST Pull-Down Assays:
  Recombinant His6-Far8 binds GST-Atg32(N250), confirming direct interaction .

• Localization Disruption:
  In ppg1Δ cells, Far11-GFP becomes cytoplasmic, indicating Ppg1’s role in Far complex membrane anchoring .