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ADP ribosylation factor 6 binding to phosphatidylinositol 4,5-bisphosphate-containing vesicles creates defects in the
1Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, New York 14853, USA.
Abstract:
The effects of binding of myristoylated ADP ribosylation factor 6 (myr-ARF6), an activator of phospholipase D (PLD), to a model membrane were investigated using an electron spin resonance (ESR) labeling technique. Initial studies were conducted in vesicles composed of 1-palmitoyl-2-oleoyl phosphatidylethanolamine, dipalmitoylphosphatidylcholine, phosphatidylinositol 4,5-biphosphate (PIP(2)), and cholesterol. Recombinant ARF6 binding significantly enhances defects in both the headgroup and acyl-chain regions of the membrane, which are revealed by the emergence of sharp components in the spectra from a headgroup label, 1,2-dipalmitoylphosphatidyl-2,2,6,6-tetramethyl-1-piperidinyloxy-choline (DPPTC), and a chain label, 10PC, after myr-ARF6 binding. Binding of non-myristoylated ARF6 (non-ARF6) shows markedly reduced effects. Interestingly, no change in spectra from DPPTC was observed upon myr-ARF6 binding when PIP(2) in the vesicles was replaced by other negatively charged lipids, including phosphatidylinositol, phosphatidylserine, and phosphatidylglycerol, even when normalized for charge. The production of the sharp peak appears to be a specific event, because another GTP binding protein, CDC42, which binds PIP(2) and activates PLD, fails to induce changes in vesicle structure. These results suggest a previously unappreciated role for ARF in mediating a protein/lipid interaction that produces defects in lipid bilayers. This function may serve as an initial event in destabilizing membrane structure for subsequent membrane fusion or biogenesis of vesicles.
Insights
Myristoylated ARF6 binding to model membranes creates defects, particularly with PIP(2), suggesting a role in membrane destabilization for fusion or vesicle formation.
Area of Science:
- Biochemistry
- Cell Biology
- Membrane Biophysics
Background:
- ADP ribosylation factor 6 (ARF6) activates phospholipase D (PLD).
- Protein-lipid interactions are crucial for membrane dynamics.
- Phosphatidylinositol 4,5-biphosphate (PIP(2)) plays a key role in cellular signaling and membrane trafficking.
Purpose of the Study:
- To investigate the effects of myristoylated ARF6 (myr-ARF6) binding on model membrane structure.
- To elucidate the role of PIP(2) in ARF6-induced membrane alterations.
- To understand the mechanism of ARF6 in mediating protein-lipid interactions.
Main Methods:
- Electron spin resonance (ESR) labeling technique.
- Utilized model membranes composed of specific lipids (PE, PC, PIP(2), cholesterol).
- Compared binding effects of myristoylated ARF6, non-myristoylated ARF6, and CDC42.
Main Results:
- myr-ARF6 binding significantly enhanced membrane defects in both headgroup and acyl-chain regions.
- Specific lipid interactions, particularly with PIP(2), were crucial for observed spectral changes.
- Non-myristoylated ARF6 and CDC42 showed markedly reduced or no effects, indicating specificity.
Conclusions:
- ARF6 binding induces specific defects in lipid bilayers, mediated by PIP(2).
- This protein-lipid interaction may initiate membrane destabilization for fusion or vesicle biogenesis.
- ARF6 plays a previously unappreciated role in regulating membrane structure and dynamics.