The protein and lipid composition of eukaryotic plasma membranes is highly dynamic and regulated according to need. The sphingolipid-responsive Pkh kinases are candidates for mediating parts of this regulation, as they affect a diverse set of plasma membrane functions, such as cortical actin patch organization, efficient endocytosis, and eisosome assembly. Eisosomes are large protein complexes underlying the plasma membrane and help to sort a group of membrane proteins into distinct domains. In this study, we identify Nce102 in a genome-wide screen for genes involved in eisosome organization and Pkh kinase signaling. Nce102 accumulates in membrane domains at eisosomes where Pkh kinases also localize. The relative abundance of Nce102 in these domains compared with the rest of the plasma membrane is dynamically regulated by sphingolipids. Furthermore, Nce102 inhibits Pkh kinase signaling and is required for plasma membrane organization. Therefore, Nce102 might act as a sensor of sphingolipids that regulates plasma membrane function.

Eisosomes help sequester a subgroup of plasma membrane proteins into discrete membrane domains that colocalize with sites of endocytosis. Here we show that the major eisosome component Pil1 in vivo is a target of the long-chain base (LCB, the biosynthetic precursors to sphingolipids)-signaling pathway mediated by the Pkh-kinases. Eisosomes disassemble if Pil1 is hyperphosphorylated (i) upon overexpression of Pkh-kinases, (ii) upon reducing LCB concentrations by inhibiting serine-palmitoyl transferase in lcb1-mutant cells or by poisoning the enzyme with myriocin, and (iii) upon mimicking hyperphosphorylation in pil1-mutant cells. Conversely, more Pil1 assembles into eisosomes if Pil1 is hypophosphorylated (i) upon reducing Pkh-kinase activity in pkh1 pkh2-mutant cells, (ii) upon activating Pkh-kinases by addition of LCBs, and (iii) upon mimicking hypophosphorylation in pil1-mutant cells. The resulting enlarged eisosomes show altered organization. Other data suggest that Pkh signaling and sphingolipids are important for endocytosis. Taken together with our previous results that link eisosomes to endocytosis, these observations suggest that Pkh-kinase signaling relayed to Pil1 may help regulate endocytic events to modulate the organization of the plasma membrane.

The molecular composition of plasma membranes is constantly remodeled by endocytosis and exocytosis. Eisosomes are large cytoplasmic protein assemblies that localize to specialized domains on the yeast plasma membrane. They are of uniform size and immobile, and their disruption leads to large aberrant plasma membrane invaginations and endocytic defects. It is unknown how eisosomes are formed or inherited and what governs their size, distribution, and location. Here we show that eisosomes are formed de novo in the bud of dividing cells. They colonize newly formed membrane at a fixed density in a polarized wave proceeding from the bud neck to the bud tip and become anchored at the site of their formation. Pil1, one of the two main eisosome subunits, emerges as the central regulator of eisosome biogenesis that determines both size and location of eisosomes. Lowering Pil1 expression leads to normal-sized eisosomes at a reduced density, suggesting that eisosomes must be of a minimal size. Conversely, raising Pil1 expression leads to larger eisosomes at a fixed density, suggesting that under these conditions eisosome nucleation sites are limiting. Pil1 expression is regulated by the cell cycle, which synchronizes eisosome formation with plasma membrane growth. Our results establish a first framework of the molecular principles that define eisosome assembly and distribution.

Seg1 establishes a platform for the assembly of eisosomes and is important for determining their length.