Tethering guides fusion-competent trans-SNARE assembly

Area of Science:

• Cell biology
• Membrane trafficking
• Protein-protein interactions

Background:

• Soluble N-ethylmaleimide-sensitive factor receptor (R-SNAREs), Q-SNAREs, and Sec1/Munc18 (SM)-family proteins are vital for membrane fusion in cellular transport.
• The yeast vacuolar tethering/SM complex, homotypic fusion and vacuole protein sorting (HOPS), significantly enhances fusion between R-SNARE and 3Q-SNARE-bearing membranes.

Purpose of the Study:

• To investigate the role of phosphoinositide-binding proteins (GST-PX and GST-FYVE) in supporting membrane fusion.
• To determine if tethering alone can drive fusion or if SNARE complex assembly and function are also modulated.

Main Methods:

• Utilized recombinant dimeric proteins (GST-PX, GST-FYVE) that bind phosphoinositides to tether proteoliposomes.
• Assessed fusion support with and without SNAREs, and in the presence of disassembly factors (Sec17, Sec18, ATP) and the HOPS complex.

Main Results:

• GST-PX and GST-FYVE successfully tethered membranes by binding phosphoinositides, supporting proteoliposome fusion independently of SNAREs.
• GST-PX-mediated tethering promoted the assembly of new, active SNARE complexes, rather than activating pre-formed inactive ones.
• While GST-PX supported fusion, the HOPS complex was essential when SNAREs were disassembled (using Sec17, Sec18, ATP), indicating tethering alone is insufficient in such conditions.

Conclusions:

• Membrane tethering by phosphoinositide-binding proteins can initiate fusion by facilitating new SNARE complex assembly.
• The HOPS complex plays a critical role in membrane fusion when SNAREs require disassembly and reassembly, highlighting a distinct mechanism from simple tethering.
• A model is proposed where tethering orients SNARE domains to enable parallel, active assembly for efficient membrane fusion.