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...

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Published in:Molecular systems biology Vol. 16; no. 5; pp. e9009 - n/a
Main Authors: Skruzny, Michal, Pohl, Emma, Gnoth, Sandina, Malengo, Gabriele, Sourjik, Victor
Format: Journal Article
Language:English
Published: London Nature Publishing Group UK 01-05-2020
EMBO Press
John Wiley and Sons Inc
Springer Nature
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Summary: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. Synopsis FRET‐based protein proximity mapping reveals the nanoscale organization of the conserved endocytic coat: Coat proteins form several functional layers above and below the clathrin lattice with key endocytic factors transversing through it. A high‐resolution protein map of the yeast endocytic coat is obtained by FRET proximity screen of 18 conserved coat‐associated proteins. Endocytic coat proteins are not confined inside, but localize on both sides of the clathrin lattice. Key coat proteins transverse the clathrin lattice to connect the endocytic coat with the cytoplasm. The protein architecture of the endocytic coat has mechanistic and regulatory implications for its function during endocytosis. Graphical Abstract FRET‐based protein proximity mapping reveals the nanoscale organization of the conserved endocytic coat: Coat proteins form several functional layers above and below the clathrin lattice with key endocytic factors transversing through it.
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ISSN:1744-4292
1744-4292
DOI:10.15252/msb.20199009