P4-ATPases control phosphoinositide membrane asymmetry and neomycin resistance

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Aminoglycoside antibiotics like neomycin possess potent antibacterial activity but are limited by nephrotoxicity and ototoxicity.
• The precise mechanisms governing neomycin uptake into eukaryotic cells are not fully elucidated.
• In budding yeast, the NEO1 gene product, a phospholipid flippase, is crucial for neomycin resistance and maintaining membrane asymmetry.

Purpose of the Study:

• To investigate the molecular basis of neomycin sensitivity in eukaryotic cells.
• To understand the role of phospholipid distribution in aminoglycoside antibiotic interaction.
• To explore the conserved mechanisms of phosphoinositide homeostasis and its link to neomycin sensitivity.

Main Methods:

• Genetic analysis of neomycin sensitivity in budding yeast, focusing on mutations in the NEO1 gene.
• Cryogenic electron microscopy (cryo-EM) to visualize the interaction between Neo1 and its substrates.
• Cellular assays in human cells using the orthologous ATP9A gene to assess conserved mechanisms.

Main Results:

• Mutations affecting Neo1 substrate recognition in yeast lead to neomycin hypersensitivity due to the exposure of phosphatidylinositol-4-phosphate (PI4P) on the plasma membrane's outer leaflet.
• Cryo-EM confirmed PI4P binding within the Neo1 translocation pathway, indicating a direct interaction.
• Neo1 activity at the Golgi apparatus is essential for preventing PI4P secretion to the cell surface; its deficiency, or that of its human ortholog ATP9A, results in PI4P exposure and neomycin sensitivity.

Conclusions:

• The study identifies exposed PI4P as a key factor mediating neomycin sensitivity in yeast and human cells.
• Conserved mechanisms involving phospholipid flippases like Neo1 and ATP9A are critical for maintaining phosphoinositide homeostasis and preventing aminoglycoside antibiotic entry.
• These findings have significant implications for understanding extracellular phosphoinositide signaling and developing safer aminoglycoside therapies.