Large static protein assemblies that mark endocytic sites at the plasma membrane in Saccharomyces cerevisiae have now been identified by Walther, Brickner, Walter and colleagues. How endocytosis is initiated at particular plasma-membrane sites, and whether it is initiated at specific or random locations, has been unclear. The identification of these so-called 'eisosomes' has therefore provided important insights into these issues.
Writing in Nature, Walther et al. describe how, by examining a library of fluorescently tagged yeast proteins, they identified two closely related cytoplasmic proteins — Pil1 and Lsp1 — that colocalized to bright dots (the eisosomes) under the plasma membrane. The transmembrane protein Sur7 also localized to these structures, and by observing single cells using time-lapse movies, the authors showed that eisosomes are static structures.
Pil1 was found to be required for the proper localization of both Lsp1 and Sur7. By contrast, the loss of Lsp1 or Sur7 did not alter the distribution of the remaining two proteins. These data indicate that Pil1 is the fundamental component of eisosomes and that, despite their similarity, Pil1 and Lsp1 have different functions.
The overexpression of SUR7 has previously been shown to suppress the effects of deleting a gene for an endocytic effector. This led the authors to investigate whether eisosomes have a role in endocytosis and, indeed, they function in the endocytosis of lipid and protein cargoes. By monitoring the uptake of a fluorescent lipid marker of endocytosis (FM4-64) in cells that expressed fluorescently labelled Pil1, they saw that all of the FM4-64-labelled foci localized to eisosomes, although not all eisosomes contained FM4-64. A subset of eisosomes are therefore active at any one time. Furthermore, by following the uptake of FM4-64 and the fluorescently labelled hexose transporter Hxt2, they observed that this lipid and protein localized to eisosomes before being internalized. The loss of Pil1 led to the clustering of eisosome remnants and redirected endocytosis to these clusters, as would be expected if eisosomes mark endocytic sites.
The assembly of actin patches at endocytic sites promotes vesicle formation. Using a compound that blocks actin polymerization and inhibits endocytosis, the authors showed that FM4-64 localization to eisosomes is actin independent, but that its uptake from eisosomes is actin dependent. Actin patches formed at eisosomes, although they also formed at other plasma-membrane sites. This indicates that, similar to metazoan cells, yeast might have parallel endocytic pathways that have different molecular requirements.
So, eisosomes mark static sites of endocytosis and might "...regulate when and where endocytosis occurs." Although all the possible interactions have only been studied superficially so far, various genetic, biochemical and cytological interactions link the three known eisosome components to the endocytic machinery.
