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The presence and abundance of proteins in the plasma membrane (PM) is regulated by exocytosis and endocytosis. Exocytosis is required for the maintenance of cellular homeostasis following stimulation, whereas endocytosis is important for the reuse and degradation of PM and receptor proteins. Through the specific labeling of proteins on the PM, the process of endocytosis can be tracked and monitored in living cells. Several genetic tags and site-specific labeling approaches that involve the coupling of small organic molecules to tag-fused proteins through either self-labeling or enzymatic ligation have been developed for this purpose (Chen et al., 2005; George et al., 2004; Hoffmann et al., 2010; Keppler et al., 2003; Los et al., 2008; Uttamapinant et al., 2010). However, these methods require a long time for an efficient labeling. Techniques with slow labeling rates are not suitable for live cell imaging. This is especially true for tracking proteins that are rapidly cycled to and from the PM in neurons because the integrity and function of these proteins require extremely fast membrane trafficking. A typical neuron in the human brain fires action potentials at 10 Hz, causing the fusion of several hundred synaptic vesicles every second (Sudhof, 2004). Accordingly, the rapid endocytosis of extensive transmembrane proteins following their exocytosis is required for the local regeneration of synaptic vesicles at the synaptic terminals. At present, fast endocytosis cannot be effectively traced using the available chemical probe-based labeling methods. Sun et al. have recently reported a fast-labeling variant of the SNAP-tag, termed SNAPf, which displays up to a tenfold increase in its reactivity towards benzylguanine substrates (Sun et al., 2011). The time required for 50% labeling of SNAPf (t1/2) is 11 to 34 s for different SNAP-surface BG dyes (Sun et al., 2011). However, the widespread use of SNAPf remains limited by the background fluorescence from unreacted or non-specifically bound substrates. Moreover, a thorough wash step is required to reduce the fluorescence signals of unreacted dyes for the vast majority of chemical labeling processes, including SNAPf-tag labeling; this requirement restricts certain labeling applications, such as the real-time imaging of the endocytosis of membrane proteins. To overcome this difficulty, Sun et al. developed fluorogenic SNAP-tag probes with low background fluorescence that become highly fluorescent only upon reaction with their target proteins (Sun et al., 2011). However, the incorporation of intramolecular quenchers greatly reduces the reactivity of the fluorogenic substrates for both SNAP-tag and SNAPf-tag (Sun et al., 2011), resulting in a slow labeling rate, with t1/2 ranging from 3 to 18 min for SNAPf-tag (Sun et al., 2011). Thus, a strong need still remains for an efficient labeling method that can combine both a fast labeling rate (within tens of seconds) and high specificity with real-time detection and high-contrast imaging. To achieve this objective, we developed a strategy to label cell surface proteins very quickly and with high specificity in living cells. The technique uses purified FKBP-fused fluorescent proteins (FPs) that binds to AP21967, a rapamycin analogue that has a high binding affinity to the FRB mutant (T2098L) but not to native FRB (Banaszynski et al., 2006). We termed this approach LAPREP, which stands for labeling PM proteins with recombinant FKBP-tagged fluorescent proteins. The labeling scheme is shown in Fig. 1A. We chose VAMP2, an important V-SNARE protein for vesicle fusion, as a target protein and fused the FRB (T2098L) domain to the C-terminus of VAMP2. FRB (T2098L) faces the extracellular region when it is at the PM, allowing contact between FKBP and FRB. To discriminate between VAMP2-expressing and VAMP2-non-expressing cells, we fused a blue FP, TagBFP, to the region immediately preceding FRB. The purified chimeric protein mKate2-FKBP, which we generated by fusing FKBP with mKate2, was mixed with AP21967 and added to HEK293 cells expressing VAMP2-TagBFP-FRB (T2098L). We found that mKate2FKBP-AP21967 effectively labeled VAMP2-FRB (T2098L) in cells expressing the target protein marked by TagBFP but not in cells without blue fluorescence, suggesting the high specificity of the labeling (Fig. 1B). The specific binding is dependent on AP21967, given that without AP21967, no VAMP2-TagRFP-FRB was labeled on the PM (Fig. 1C). Moreover, the binding of mKate2-FKBP to VAMP2-TagRFPFRB can be partially inhibited by the co-incubation of the

The specific and rapid labeling of cell surface proteins with recombinant FKBP-fused fluorescent proteins