MARCKS Effector Domain, a reversible lipid ligand, illuminates late stages of membrane fusion

Area of Science:

• Cell Biology
• Membrane Trafficking
• Protein-Lipid Interactions

Background:

• The HOPS (Homotypic Vacuolar Protein Sorting) complex is essential for yeast vacuolar membrane tethering and fusion.
• SNARE proteins mediate membrane fusion, requiring precise assembly and regulation.
• Sec17 (yeast NSF) and Sec18 (yeast SNAPs) are critical for disassembling SNARE complexes and driving fusion.

Purpose of the Study:

• To investigate the distinct stages of yeast vacuolar fusion.
• To elucidate the role of the HOPS complex in SNARE assembly and fusion progression.
• To characterize the mechanism of fusion blockade by the MARCKS Effector Domain (MED) peptide.

Main Methods:

• Utilized reconstituted yeast vacuolar fusion assays.
• Employed the MED peptide as a tool to probe late-stage fusion.
• Investigated the effects of salt extraction, calmodulin inactivation, and Sec17/Sec18 addition on MED-blocked fusion.
• Assessed SNARE complex assembly and antibody sensitivity.

Main Results:

• MED peptide reversibly blocks fusion at a late stage, dependent on SNARE zippering and Sec17/Sec18 activity.
• HOPS remains engaged with SNAREs during zippering, facilitating Sec17/Sec18-driven fusion even with partial zippering.
• A synthetic tether can replace HOPS when SNAREs are fully zippered, but requires Sec17/Sec18 to overcome the MED block.

Conclusions:

• MED peptide provides a novel way to dissect distinct steps in vacuolar fusion, including tethering, SNARE assembly, and Sec17/Sec18 engagement.
• HOPS plays a continuous role throughout SNARE zippering, distinct from its initial tethering function.
• Efficient vacuolar fusion requires coordinated action of SNARE zippering, HOPS, and the Sec17/Sec18 machinery.