Biophysical characterization of SARAH domain-mediated multimerization of Hippo pathway complexes in Drosophila

Area of Science:

• Cell Biology
• Molecular Biology
• Biophysics

Background:

• The Hippo pathway is crucial for controlling cell proliferation, growth, and stem cell maintenance.
• Core components like Hippo kinase, Salvador, and dRassF interact via conserved SARAH domains.
• These interactions are critical for regulating Hippo pathway signaling, but the precise mechanisms remain unclear.

Purpose of the Study:

• To biophysically characterize SARAH domain-mediated complexes involving Hippo, Salvador, and dRassF.
• To elucidate how these distinct complexes modulate Hippo kinase activity and overall pathway function.
• To understand the mechanistic basis of differential regulation within the Hippo pathway.

Main Methods:

• Purification of SARAH domains from *Drosophila melanogaster*.
• Unbiased pulldown assays to identify protein interactions.
• Native mass spectrometry (MS) to determine complex stoichiometry and solution behavior.
• Stability assays to quantify the strength of SARAH domain interactions.

Main Results:

• Isolated SARAH domains recapitulate cellular assemblies, with Hippo acting as a universal binding partner.
• Salvador SARAH domain homodimerization was observed and found to be conserved in mammalian homologs.
• All characterized SARAH domain complexes exist as dimers in solution and exhibit varying stabilities.
• Complex formation distinctly modulates Hippo kinase activity: homodimerization of Hippo activates it, heterodimerization with Salvador enhances activity, while complexation with dRassF inhibits it.

Conclusions:

• SARAH domain interactions are sufficient to form functional regulatory complexes within the Hippo pathway.
• The identity, stoichiometry, and stability of these dimeric complexes provide mechanistic insights into Hippo pathway regulation.
• Differential complex formation by SARAH domains explains how Hippo pathway activity is precisely controlled.