An in vitro vesicle formation assay to analyze protein sorting in the secretory transport pathway

摘要: The fidelity of protein transport in the secretory pathway relies on accurate sorting proteins to their correct destination. To deepen our understanding underlying molecular mechanisms, it is important develop a robust approach systematically reveal cargo that depend specific machinery be efficiently packaged into vesicles. Here, we used an vitro assay reconstitutes packaging human vesicles quantify capture. Quantitative mass spectrometry analyses isolated revealed novel cytosolic are associated with vesicle membranes GTP-dependent manner or interact GTP-bound Sar1A membranes. Functional analysis indicates two them, FAM84B and PRRC1, regulate anterograde trafficking. Comparing control cells depleted receptors, SURF4 ERGIC53, clients each these export adaptors. Moreover, results indicate enriched contain transmembrane SNARE proteins. A protein, Vti1B, identified planar cell polarity Frizzled6, promotes vesicular release Frizzled6. Our formation combination quantitative spectrometric powerful tool trafficking protein.