Visualization of multiple localizations of GLUT4 by fluorescent probes of PYP-tag with designed unnatural warhead.

Within a cell, multiple copies of the same protein coexist in different pathways and behave differently. Being able to individually analyze the constant actions of proteins in a cell is crucial to know the pathways through which they pass and which physiological functions they are deeply involved in. However, until now, it has been difficult to distinguish protein copies with distinct translocation properties by fluorescence labeling with different colors in living cells. In this study, we have created an unnatural ligand with an unprecedented protein-tag labeling property in living cells and overcome the above-mentioned problem. Of special interest is that some fluorescent probes with the ligand can selectively and efficiently label intracellular proteins without binding to cell-surface proteins, even if the proteins are present on the cell membrane. We also developed a cell-membrane impermeable fluorescent probe that selectively labels cell-surface proteins without labeling of intracellular proteins. These localization-selective properties enabled us to visually discriminate two kinetically distinct glucose transporter 4 (GLUT4) molecules that show different multiple subcellular localization and translocation dynamics in live cells. Taking advantage of the probes, we revealed that