Phosphatidic Acid Sequesters Sec18p from cis-SNARE Complexes to Inhibit Priming

Area of Science:

• Cell biology
• Membrane trafficking
• Protein-lipid interactions

Background:

• Yeast vacuole fusion is essential for cellular function.
• Sec18p/N-ethylmaleimide sensitive factor (NSF) and Sec17p/α-SNAP mediate cis-SNARE complex activation.
• Phosphatidic acid (PA) phosphatase activity, converting PA to diacylglycerol (DAG), regulates Sec18p association with SNAREs.

Purpose of the Study:

• To investigate the hypothesis that Sec18p associates with PA-rich membrane microdomains before transferring to cis-SNARE complexes.
• To elucidate the role of PA and its conversion to DAG in regulating Sec18p recruitment for vacuole fusion.

Main Methods:

• Liposome binding assays using PA and DAG containing liposomes to assess Sec18p binding.
• Utilizing a specific PA-binding domain to block Sec18p interaction with PA and isolated vacuoles.
• Assessing the effect of excess PA on vacuole priming activity.
• Analyzing Sec18p association with cis-SNAREs in yeast vacuoles lacking Pah1p and complemented vacuoles.

Main Results:

• Sec18p demonstrated a fivefold greater preference for binding to PA-liposomes compared to DAG-liposomes.
• A specific PA-binding domain inhibited Sec18p binding to PA-liposomes and displaced endogenous Sec18p from vacuoles.
• Excess PA addition blocked vacuole priming activity, mimicking PA phosphatase inhibition.
• Yeast lacking Pah1p exhibited reduced Sec18p association with cis-SNAREs, which was restored by PAH1 complementation.

Conclusions:

• The conversion of PA to DAG by PA phosphatase facilitates Sec18p recruitment to cis-SNARE complexes.
• Pah1p-mediated regulation of PA concentration is critical for controlling SNARE priming by Sec18p during yeast vacuole fusion.