Vps4 disassembles an ESCRT-III filament by global unfolding and processive translocation

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• AAA+ ATPases are essential molecular machines that disassemble protein complexes.
• Vps4 is a key AAA+ ATPase involved in ESCRT-III disassembly, vital for viral budding and cellular processes.
• Understanding the mechanism of complex disassembly is crucial for various biological pathways.

Purpose of the Study:

• To investigate the mechanism by which Vps4 disassembles ESCRT-III filaments.
• To determine if Vps4 unfolds substrates locally or globally during disassembly.
• To compare the disassembly mechanism of Vps4 with other known unfoldases.

Main Methods:

• Hydrogen/deuterium exchange mass spectrometry (HDX-MS) was used to monitor protein unfolding.
• Saccharomyces cerevisiae Vps4 was used to disassemble a chimeric Vps24-2 ESCRT-III filament.
• Site-directed cysteine mutagenesis and cross-linking were employed to map protein interactions.

Main Results:

• Vps4 completely unfolds ESCRT-III substrates on a timescale consistent with disassembly.
• The unfoldase ClpX exhibited a similar unfolding pattern, suggesting a common mechanism.
• Cross-linking experiments supported a model where Vps4 unfolds and threads substrates through its central pore.

Conclusions:

• Vps4 disassembles ESCRT-III by completely unfolding the substrate.
• This unfolding mechanism is conserved among AAA+ ATPases like Vps4 and ClpX.
• The findings provide critical insights into the molecular machinery of protein complex disassembly.