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Eisosomes mark static sites of endocytosis

Abstract

Endocytosis functions to recycle plasma membrane components, to regulate cell-surface expression of signalling receptors and to internalize nutrients in all eukaryotic cells. Internalization of proteins, lipids and other cargo can occur by one of several pathways that have different, but often overlapping, molecular requirements1,2,3,4,5. To mediate endocytosis, effectors assemble transiently underneath the plasma membrane, carry out the mechanics of membrane deformation, cargo selection and vesicle internalization, and then disassemble. The mechanism by which endocytosis initiates at particular locations on the plasma membrane has remained unknown. Sites of endocytosis might be formed randomly, induced by stochastic protein and/or lipid clustering. Alternatively, endocytosis might initiate at specific locations. Here we describe large immobile protein assemblies at the plasma membrane in the yeast Saccharomyces cerevisiae that mark endocytic sites. These structures, termed eisosomes (from the Greek ‘eis’, meaning into or portal, and ‘soma’, meaning body), are composed primarily of two cytoplasmic proteins, Pil1 and Lsp1. A plasma membrane protein, Sur7, localizes to eisosomes. These structures colocalize with sites of protein and lipid endocytosis, and their components genetically interact with known endocytic effectors. Loss of Pil1 leads to clustering of eisosome remnants and redirects endocytosis and endocytic effector proteins to these clusters.

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Figure 1: Eisosomes are large protein assemblies at the cell cortex.
Figure 2: Pil1 is required for Lsp1 and Sur7 localization.
Figure 3: Eisosomes colocalize with sites of endocytosis.
Figure 4: Endocytic actin patches form at eisosomes.

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Acknowledgements

We thank R. Kelly, G. Davis, M. von Zastrow, S. Emr, D. Drubin and members of the Drubin and Walter laboratories for discussion and comments; N. Davis for the Ste3–HA plasmid; and D. Brickner for technical assistance. T.C.W. was supported by the Human Frontier Science Program, J.H.B. by the Helen Hay Whitney Foundation, P.S.A. by the Damon Runyon Cancer Research Fund and Fundación Antorchas, and S.B. by the UCSF Herbert H. Boyer Fund. This work was supported in part by a grant from the NIH and by a Sandler Opportunity Fund Award (UCSF). P.W. is an Investigator of the Howard Hughes Medical Institute.

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Correspondence to Tobias C. Walther.

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Supplementary information

Supplementary Figure 1

Quantification of eisosomes per cell. Eisosomes were counted in image stacks acquired from Pil1-GFP expressing cells. The number of eisosomes per cell is shown.

Supplementary Figure 2

Quantification of large invaginations in pil1Δ and pil1Δlsp1Δ cells.

Supplementary Figure 3

Lsp1 and Pil1 have opposing effects on endocytosis.

Supplementary Figure 4

Long chain base levels are unchanged in pil1Δ lsp1Δ, pil1Δ lsp1Δ strains.

Supplementary Figure 5

pil1Δ genetically interacts with endocytic effector genes.

Supplementary Table 1

Interactions of endocytic actin effectors.

Supplementary Notes

This file contains Supplementary Figure Legends, Supplementary Movie Legends and additional references.

Supplementary Movie 1

Localization of Pil1.

Supplementary Movie 2

Abp1 hotspots at clustered eisosomes.

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Walther, T., Brickner, J., Aguilar, P. et al. Eisosomes mark static sites of endocytosis. Nature 439, 998–1003 (2006). https://doi.org/10.1038/nature04472

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