P4-ATPase control over phosphoinositide membrane asymmetry and neomycin resistance

Area of Science:

• Cell Biology
• Biochemistry
• Molecular Biology

Background:

• Neomycin, an aminoglycoside antibiotic, exhibits potent antibacterial effects but is limited by nephrotoxicity and ototoxicity.
• The precise mechanism of neomycin uptake into eukaryotic cells is not fully elucidated.
• The phospholipid flippase Neo1 in yeast is crucial for neomycin resistance and maintaining membrane asymmetry.

Purpose of the Study:

• To investigate the role of Neo1 and phosphatidylinositol-4-phosphate (PI4P) in neomycin sensitivity.
• To elucidate the transport pathway of PI4P and its implications for aminoglycoside uptake.
• To understand the mechanisms of phosphoinositide homeostasis and extracellular phosphoinositide signaling.

Main Methods:

• Genetic analysis of Neo1 mutations in yeast to assess neomycin sensitivity.
• Knockdown of ATP9A in human cells to evaluate its role in extracellular PI4P exposure.
• Cryo-electron microscopy (Cryo-EM) to visualize PI4P binding to Neo1.
• Investigating nonvesicular transport (Osh6) and vesicular transport (COPII) of PI4P.

Main Results:

• Mutations in Neo1 lead to neomycin hypersensitivity by exposing PI4P on the cell surface.
• Human ATP9A knockdown also results in extracellular PI4P exposure and correlates with neomycin sensitivity.
• PI4P transport involves the endoplasmic reticulum, Golgi, and nonvesicular/vesicular transport pathways.
• Cryo-EM confirmed PI4P binding within Neo1's translocation pathway.
• Loss of Neo1 function in the Golgi causes PI4P secretion, acting as a neomycin receptor.

Conclusions:

• Extracellular PI4P acts as a receptor for neomycin, facilitating its endocytic uptake and influencing aminoglycoside sensitivity.
• The study reveals novel mechanisms for phosphoinositide homeostasis and PI4P transport.
• Findings highlight the role of extracellular phosphoinositides in cellular signaling and drug uptake.