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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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Autores principales: Skruzny, Michal, Pohl, Emma, Gnoth, Sandina, Malengo, Gabriele, Sourjik, Victor
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7218409/
https://www.ncbi.nlm.nih.gov/pubmed/32400111
http://dx.doi.org/10.15252/msb.20199009
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author Skruzny, Michal
Pohl, Emma
Gnoth, Sandina
Malengo, Gabriele
Sourjik, Victor
author_facet Skruzny, Michal
Pohl, Emma
Gnoth, Sandina
Malengo, Gabriele
Sourjik, Victor
author_sort Skruzny, Michal
collection PubMed
description 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.
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spelling pubmed-72184092020-05-13 The protein architecture of the endocytic coat analyzed by FRET microscopy Skruzny, Michal Pohl, Emma Gnoth, Sandina Malengo, Gabriele Sourjik, Victor Mol Syst Biol Articles 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. John Wiley and Sons Inc. 2020-05-13 /pmc/articles/PMC7218409/ /pubmed/32400111 http://dx.doi.org/10.15252/msb.20199009 Text en © 2020 The Authors. Published under the terms of the CC BY 4.0 license This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
spellingShingle Articles
Skruzny, Michal
Pohl, Emma
Gnoth, Sandina
Malengo, Gabriele
Sourjik, Victor
The protein architecture of the endocytic coat analyzed by FRET microscopy
title The protein architecture of the endocytic coat analyzed by FRET microscopy
title_full The protein architecture of the endocytic coat analyzed by FRET microscopy
title_fullStr The protein architecture of the endocytic coat analyzed by FRET microscopy
title_full_unstemmed The protein architecture of the endocytic coat analyzed by FRET microscopy
title_short The protein architecture of the endocytic coat analyzed by FRET microscopy
title_sort protein architecture of the endocytic coat analyzed by fret microscopy
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7218409/
https://www.ncbi.nlm.nih.gov/pubmed/32400111
http://dx.doi.org/10.15252/msb.20199009
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