Vacuole fusion is driven by SNARE proteins that require activation-or priming-by the AAA+ protein Sec18 (NSF) before they can bring membranes together and trigger the merger of two bilayers into a continuous membrane. Sec18 resides on vacuoles prior to engaging inactive cis-SNARE complexes through its interaction with the regulatory lipid phosphatidic acid (PA). Binding PA causes Sec18 to undergo large conformational changes that keeps it bound to the membrane, thus precluding its interactions with SNAREs. Such conformational changes can be measured by various biochemical and biophysical assays. The conversion of PA to diacylglycerol by the PA phosphatase Pah1 releases Sec18 from the membrane-bound pool and promotes its transfer to SNARE complexes allowing priming to occur. Here we describe four spectroscopy-based methods to distinguish conformational changes from alterations in secondary structure during PA binding. These methods only require purified protein and short chain soluble lipids, making the methods rapid and affordable ways to screen the effects of specific protein-lipid interactions. The assays described in this chapter include 1-anilino-8-naphthalenesulfonate (ANS) spectroscopy and intrinsic tryptophan fluorescence to detect exposure of hydrophobic regions; differential scanning fluorometry to measure changes in protein stability across a temperature gradient; and circular dichroism to examine changes in secondary structure.

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Journal Article

Authors

Fratti R, Calderin JD, Starr ML

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