A Dual-Color Fluorescence-Based Ratiometric Assay to Measure Endocytic Trafficking of Surface Proteins

Many membrane proteins on the cell surface are constantly internalized from, and re-delivered to, the plasma membrane. This endocytic cycling, which relies on accurate SNARE-mediated fusion of vesicles containing cargo proteins, is highly important for the function of many proteins such as signaling receptors. While the SNARE proteins that mediate fusion during specific events, such as neurotransmitter and hormone release, in mammalian cells has been heavily studied, the SNARE proteins that mediate surface delivery of specific cargo such as the receptors for these released factors are still not known. A primary barrier to defining core fusion components has been the lack of a quantitative and accessible assay to measure endocytic cycling of cargo proteins as well as SNAREs themselves. Here, we describe an internally controlled dual-color ratiometric fluorescence-based assay to monitor and measure the internalization and recycling of a pool of pre-existing surface cargo proteins, avoiding the confounding effects of other pathways and accounting for cell-to-cell variation in protein expression. This quantitative method will be useful for mechanistically investigating the SNARE and related fusion proteins that mediate surface delivery of specific cargo.