Reconstituted Proteoliposome Fusion Mediated by Yeast SNARE-Family Proteins
Membrane fusion mediated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-family proteins is an essential process for intracellular membrane trafficking in all eukaryotic cells, which delivers proteins and lipids to their appropriate subcellular membrane compartments such as organelles and plasma membrane. The molecular basis of SNARE-mediated membrane fusion has been revealed by studying fusion of reconstituted proteoliposomes bearing purified SNARE-family proteins and chemically defined lipid species. This chapter describes the detailed experimental protocols for (1) purification of recombinant SNARE-family and SM (Sec1/Munc18-family) proteins in the yeast Saccharomyces cerevisiae; (2) preparation of reconstituted proteoliposomes bearing purified yeast SNARE proteins; and (3) developing an assay to monitor lipid mixing between reconstituted SNARE-bearing proteoliposomes. Lipid mixing assays for reconstituted SNARE-bearing proteoliposomes are useful for evaluating the intrinsic capacity of SNARE-family proteins to directly catalyze membrane fusion and to determine the specificity of membrane fusion.
Key wordsMembrane reconstitution Membrane trafficking Membrane fusion SNARE protein Liposome Proteoliposome Lipid mixing assay Saccharomyces cerevisiae
This work was supported by the Program to Disseminate Tenure Tracking System from the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT) and Grants-in-Aid for Scientific Research from MEXT to J.M.