To ensure the fidelity of protein transport in the secretory transport pathway, eukaryotic cells employ sophisticated protein sorting machineries to recognize specific cargo proteins to allow them to be efficiently packaged into transport vesicles and deliver to their correct destinations. To deepen our understanding of the underlying molecular mechanisms, it is important to develop a robust approach to systematically reveal cargo proteins that depend on a specific cargo sorting machinery to be incorporated into vesicles. Here, I used an in vitro assay that reconstitutes packaging of human cargo proteins into vesicles to quantify cargo capture. Quantitative mass spectrometry analyses of the isolated vesicles revealed novel cytosolic proteins that are associated with vesicles in...[ Read more ]

To ensure the fidelity of protein transport in the secretory transport pathway, eukaryotic cells employ sophisticated protein sorting machineries to recognize specific cargo proteins to allow them to be efficiently packaged into transport vesicles and deliver to their correct destinations. To deepen our understanding of the underlying molecular mechanisms, it is important to develop a robust approach to systematically reveal cargo proteins that depend on a specific cargo sorting machinery to be incorporated into vesicles. Here, I used an in vitro assay that reconstitutes packaging of human cargo proteins into vesicles to quantify cargo capture. Quantitative mass spectrometry analyses of the isolated vesicles revealed novel cytosolic proteins that are associated with vesicles in a GTP-dependent manner. Functional analysis indicates that two of them, FAM84B and PRRC1, regulate anterograde transport of newly synthesized epidermal growth factor receptor (EGFR) from the ER to the Golgi. Comparing control cells with cells depletion of the cargo receptors, ERGIC53 and Surf4, specific cargo proteins were identified that depend on each of these two cargo receptors to be packaged into vesicles. I also reveal various specific clients of other three cargo receptors, ERGIC1, LMAN2 and TMED10. Moreover, I found that delivery of a set of secreted proteins were regulated by cytosolic cargo adaptor AP-1. These results indicate that the vesicle formation assay coupled with quantitative mass spectrometry analysis is a robust tool to reveal novel cytosolic proteins that associated with vesicle membranes to regulate anterograde trafficking and to reveal cargo proteins that depends on a specific factor to be packaged into transport vesicles.