Eisosome proteins play an important structural role in organizing the yeast plasma membrane. As expected from their architectural function, eisosome proteins localize very stably and are extremely high in abundance. For comparison, copy numbers of main eisosome components per cell (115,000 for Pil1 and 104,000 for Lsp1) are much larger than those of tubulin (5,590) or actin (60,000; Norbeck and Blomberg, 1997
; Ghaemmaghami et al., 2003
). As a result of these properties, Pil1 and Lsp1 could be classified as part of a membrane cytoskeleton. Consistent with this idea, we show that Pil1 and Lsp1 self-assemble into a protein scaffold that binds and deforms membranes, providing a mechanism for how these proteins organize the plasma membrane into domains.
Similar to other BAR domain–containing proteins (such as F-BARs; Frost et al., 2008
), Pil1 and Lsp1 form higher-order complexes on membranes. In contrast to other BAR domain proteins, however, the assemblies formed by eisosome proteins are extremely stable in the absence of membranes. Even though we cannot pinpoint exact contacts as a result of limited resolution, our current model of eisosome architecture posits three distinct interactions: one to form the BAR dimer (interaction 1), a second end-to-end contact of BAR domain dimers to form thin filaments (interaction 2), and a third lateral interaction to form helices (interaction 3; ). The overall similarity and subtle differences of the assemblies formed by Pil1 and Lsp1 suggest that the strength of the different interactions may differ between the two proteins. For example, Lsp1 interaction 2 may have a lower affinity compared with Pil1, which would result in a requirement for combined binding energy from end-to-end interactions 2 and lateral interactions 3 to stabilize thick Lsp1 helices. Smaller assemblies, such as thin filaments, may be unstable and fall apart, explaining the absence of thin Lsp1 filaments and the increased pool of nonassembled Lsp1 in cells and biochemical assays. Immunolocalization of Pil1 and Lsp1 in EMs performed in this study (and Strádalová et al. 
) shows that both proteins are present in eisosomes covering membrane furrows, but how both proteins associate to form them and whether their different properties are used to modulate eisosome structure are yet unclear.
Figure 8. Model for the assembly of eisosomes on the plasma membrane. (A) The assembly of eisosomes can be separated conceptually into three steps: interactions of the proteins to form dimers (interaction 1), association of dimers to form thin filaments (interaction (more ...)
Interaction 3 is likely modulated by phosphorylation, as indicated by its sensitivity to phosphomimicking mutations of Pil1, which leads to formation of thin helices. Similarly, pil1ΔN formed only thin filaments, further suggesting that the N-terminal segment containing two of phosphorylation sites is required for interaction 3. These data may explain eisosome disassembly after overexpression of Pkh kinases, addition of myriocin, or other treatments that increase Pil1 phosphorylation (Walther et al., 2007
; Luo et al., 2008
; Fröhlich et al., 2009
Pil1 and Lsp1 are most likely targeted to the plasma membrane by efficient membrane binding (). We predict that, initially, dimers or thin filament pieces interact with membranes and assemble in vitro into a stable helix with a membrane tubule inside or in vivo into a furrowlike lattice (). Several lines of evidence suggest that Pil1 and Lsp1 interact with PI(4,5)P2: (a) Pil1 and Lsp1 tubulate liposomes containing low amounts of PI(4,5)P2; (b) fluorescence spectroscopy of NBD-labeled Pil1 yields a strong signal consistent with membrane binding when PI(4,5)P2-containing liposomes are present; (c) in sedimentation assays, Lsp1 interacts more strongly with PI(4,5)P2-containing liposomes than with those containing other types of charged lipids at the same concentration; (d) inactivation of Mss4, leading to PI(4,5)P2 depletion, has a strong effect on eisosome localization in vivo; (e) conversely, deletion of two PI(4,5)P2 phosphatases (SJL1 and SJL2) and increased PI(4,5)P2 levels lead to enlarged Pil1-GFP assemblies; and (f) PIL1 and SJL1 show highly similar genetic interaction profiles in independently generated E-MAP datasets.
How can self-assembly of Pil1 and Lsp1 promote formation or stabilization of curved membranes, such as tubules and furrows? Two mechanisms for BAR domain–induced membrane bending are currently considered (Kozlov et al., 2010
): protein scaffolding of the membrane and insertion of a wedge into one leaflet of the lipid bilayer. Both of these mechanisms are used by other BAR domain–containing proteins (Peter et al., 2004
). Our reconstructions of Lsp1 with and without bound membranes show very similar structures, suggesting a scaffold mechanism. In addition, a part of the protein at the concave surface of the Pil1 or Lsp1 coat may be inserted as a wedge in one leaflet of the bilayer, for example, represented by the part of Lsp1 observed close to the membrane surface. Consistent with this notion, we found that a membrane-facing N-terminal segment of Pil1 or Lsp1 is required for efficient membrane binding of the proteins in addition to a positive patch of amino acids on the concave surface of the BAR domain. As a consequence of the insertion of a membrane wedge, the order of the outer membrane leaflet could be disordered, leading to absence of resolved density in this region and thus potentially explaining the gap apparent in our reconstructions between the lipid layer to the protein scaffold.
Many of these considerations are based on similarity between the models of Pil1 and Lsp1 assemblies in vitro and the structure of eisosomes, forming membrane furrows in yeast (Strádalová et al., 2009
). This interpretation is supported by (a) a very similar structure for recombinant Pil1 and Lsp1 assemblies as for purified eisosomes isolated from yeast cells, (b) alterations of the in vitro structure caused by phosphomimicking mutations in Pil1, consistent with the phenotype of these mutations in yeast, and (c) the striated pattern of eisosomes on plasma membrane furrows or the cytoplasmic side of the plasma membrane, which resembles the pattern of thick helices formed by the recombinant proteins. Despite the overall close resemblance of the structures, there are at least two important differences. First, eisosomes contain both Pil1 and Lsp1 proteins. Thus, in vivo, the building blocks of the lattice could be Pil1 and Lsp1 heterodimers or a mixture of both types of homodimers, rather than a single species of homodimers present in vitro, and the different properties of the two proteins could be used to modulate the assembly. Second, whereas the in vitro filaments are closed cylinders coating a membrane tubule, eisosomes in vivo coat a membrane furrow, which likely resembles a half-cylinder. Attachment of the membrane to the cell wall and the large turgor pressure could prevent the closure of the lattice to a helix similar to the ones seen in vitro. Alternatively, a transition phase of specific lipid or protein composition at the eisosome boundary could prevent the closure of the tubules. It remains possible that the furrows are closed to a tube or otherwise remodeled as a result of the rearrangement of the proteins under some conditions. Such remodeling may be supported by flexibility of Lsp1 BAR domain tips and arrangement of subunits, reflected in tube diameter variability, observed in vitro. Interestingly, during uptake of the membrane dye FM4-64, some but not all eisosomes are labeled by bright dye-containing foci, indicating that the plasma membrane has a different structure at those sites.
From our work, several intriguing similarities between eisosomes and endophilin/amphyphysin BAR domain proteins emerge. Both protein families consist of BAR domains, can assemble into a scaffold on membranes, are connected to PI(4,5)P2
-rich membranes, and function with synaptojanin proteins (Itoh et al., 2005
). Additionally, both sets of proteins were linked to endocytosis, but their deletions have mild defects on protein uptake in most systems (Schuske et al., 2003
; Verstreken et al., 2003
; Walther et al., 2006
; Grossmann et al., 2008
; Brach et al., 2011
). Endophilin recruits synaptojanin to endocytic sites through an SH3 domain (Schuske et al., 2003
). Neither Pil1 nor Lsp1 contains such a domain. However, it was recently reported that the membrane-bending activity of endophilin particularly is important for many functions of the protein in Caenorhabditis elegans
(Bai et al., 2010
), and we now find that eisosome proteins also bend membranes. Based on these considerations and the similarity of interaction profiles between pil1Δ
, it is possible that both genes participate in the same process, e.g., in PI(4,5)P2
turnover. Interestingly, membrane curvature, for example, caused by interaction with endophilin, aids synaptojanin activity (Chang-Ileto et al., 2011
In summary, formation of an eisosome protein scaffold can mechanistically explain how the yeast plasma membrane is organized in domains of distinct composition, in particular for the MCC. We posit that membrane binding and assembly by Pil1 and Lsp1 will create a specific environment in the overlaying MCC, which is locally curved and may have increased PI(4,5)P2 concentration as a result of the presence of many binding sites for this lipid. This special environment then drives formation of the MCC domain.