Quaternary structures of Vac8 differentially regulate the Cvt and PMN pathways

Area of Science:

• Molecular Biology
• Cell Biology
• Structural Biology

Background:

• Armadillo (ARM) repeat proteins are structurally similar but functionally diverse.
• Vac8 (vacuole related 8) is a key protein in yeast autophagy, including piecemeal microautophagy of the nucleus (PMN) and cytoplasm-to-vacuole targeting (Cvt) pathways.
• Understanding how Vac8's structure dictates its diverse functions is crucial.

Purpose of the Study:

• To elucidate the structural basis of Vac8's multifunctional roles in autophagy.
• To investigate the molecular mechanisms by which Vac8 interacts with Atg13 and Nvj1.
• To understand how Vac8 adopts different quaternary structures for distinct cellular pathways.

Main Methods:

• X-ray crystallography to determine the structure of Vac8-Atg13 complex.
• Biochemical assays to analyze protein interactions and self-association.
• In vivo experiments in Saccharomyces cerevisiae to assess pathway regulation.
• Structural comparisons of Vac8-Atg13 and Vac8-Nvj1 complexes.

Main Results:

• The crystal structure of Vac8 bound to Atg13 was determined, revealing a 70-Å loop of Atg13 binding antiparallel to Vac8's ARM domain.
• The H1 helix of Vac8 intramolecularly regulates its self-association, essential for Cvt and PMN pathways.
• A Vac8 mutant lacking the H1 helix formed a heterotetramer with Atg13, adopting an extended superhelical structure specific to the Cvt pathway.
• Comparison with Vac8-Nvj1 structure showed Vac8 adopts different quaternary structures to regulate distinct pathways.

Conclusions:

• Vac8's H1 helix plays a critical role in regulating its self-association and function in autophagy.
• Vac8's ability to form different quaternary structures with distinct binding partners allows for differential regulation of closely related cellular pathways.
• This study provides a molecular understanding of how structural plasticity in ARM domain proteins enables functional diversity.