Identification of mEAK-7 as a human V-ATPase regulator via cryo-EM data mining

Area of Science:

• Cell Biology
• Structural Biology
• Biochemistry

Background:

• Vacuolar-type adenosine triphosphatases (V-ATPases) are crucial proton pumps in cellular organelles.
• V-ATPases also play significant roles as signaling hubs within cells.
• Identifying endogenous V-ATPase binding partners is key to understanding its multifaceted functions.

Purpose of the Study:

• To identify endogenous binding partners of human V-ATPases.
• To elucidate the structural basis of V-ATPase interactions with its binding partners.
• To investigate the functional implications of these interactions in cellular signaling.

Main Methods:

• Cryo-electron microscopy (Cryo-EM) for high-resolution structural analysis of human V-ATPases.
• Extensive particle classification and focused refinement to resolve V-ATPase states.
• Artificial intelligence (AI) modeling to interpret additional densities in the cryo-EM maps.

Main Results:

• A novel binding partner, mEAK-7, was identified associated with human V-ATPases in state 2.
• The structure revealed mEAK-7 interacts with V-ATPase subunits A, B, D, and E.
• The observed V-ATPase particles with mEAK-7 density were approximately 17% of the dataset, resolved at 3.3 Å.

Conclusions:

• mEAK-7 is an endogenous binding partner of V-ATPases in state 2.
• mEAK-7 binding may regulate V-ATPase proton pump activity or localization.
• The V-ATPase-mEAK-7 complex is proposed to mediate mechanistic target of rapamycin (mTOR) signaling pathways.