The protein architecture of the endocytic coat analyzed by FRET microscopy.

Endocytosis is a fundamental cellular trafficking pathway, which requires an organized assembly of the multiprotein endocytic coat to pull the plasma membrane into the cell. Although the protein composition of the endocytic coat is known, its functional architecture is not well understood. Here, we determine the nanoscale organization of the endocytic coat by FRET microscopy in yeast Saccharomyces cerevisiae. We assessed pairwise proximities of 18 conserved coat-associated proteins and used clathrin subunits and protein truncations as molecular rulers to obtain a high-resolution protein map of the coat. Furthermore, we followed rearrangements of coat proteins during membrane invagination and their binding dynamics at the endocytic site. We show that the endocytic coat proteins are not confined inside the clathrin lattice, but form distinct functional layers above and below the lattice. Importantly, key endocytic proteins transverse the clathrin lattice deeply into the cytoplasm connecting thus the membrane and cytoplasmic parts of the coat. We propose that this design enables an efficient and regulated function of the endocytic coat during endocytic vesicle formation.

FRET Microscopy in Yeast.

Förster resonance energy transfer (FRET) microscopy is a powerful fluorescence microscopy method to study the nanoscale organization of multiprotein assemblies in vivo. Moreover, many biochemical and biophysical processes can be followed by employing sophisticated FRET biosensors directly in living cells. Here, we summarize existing FRET experiments and biosensors applied in yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, two important models of fundamental biomedical research and efficient platforms for analyses of bioactive molecules. We aim to provide a practical guide on suitable FRET techniques, fluorescent proteins, and experimental setups available for successful FRET experiments in yeasts.