Pkh-kinases control eisosome assembly and organization

Abstract

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.

Pil1 Controls Eisosome Biogenesis

“…Alternatively, however, Pil1 phosphorylation may primarily serve under physiological conditions to effect local structural rearrangements within an eisosome to regulate, for example, its activity to recruit endocytic effectors. As such, phosphorylation events may not globally affect assembly/disassembly properties as observed upon extreme hypo-and hyperphosphorylation observed under experimental conditions (Walther et al, 2007).…”

“…Moreover, Pil1 and Lsp1 are differentially phosphorylated in re-sponse to an increase of long-chain bases, which are the precursors of sphingolipids, by Pkh1/2, two kinases that localize to eisosomes (Zhang et al, 2004;Walther et al, 2007;Luo et al, 2008). Perturbation of the Pil1 phosphorylation status affects Pil1 assembly into eisosomes: specifically, eisosomes disassemble if Pil1 is hyperphosphorylated, and more Pil1 assembles into eisosomes if Pil1 is hypophosphorylated.…”

“…Pil1-GFP was detected by a chemifluorescence method (ECF) using an antibody against GFP (Invitrogen, A6455 rabbit polyclonal antibody against full-length GFP recognizing both the native and denatured protein) at 1:10,000 dilution, a rabbit polyclonal antibody against full-length Pil1 at 1:10,000 dilution (Walther et al, 2007), or a monoclonal mouse antibody against Pgk1 (Invitrogen, A6457) at 1:5000 dilution, followed by an alkaline phosphatase-labeled secondary antibody (Amersham Biosciences, Piscataway, NJ) diluted at 1:5000. The blots were incubated with an ECF Substrate, which when dephosphorylated fluoresces and were then scanned in a Typhoon 9400 (GE Healthcare, Waukesha, WI).…”

The lateral compartmentation of the yeast plasma membrane

“…Long chain bases also regulate the phosphorylation of the primary components of the eisosome, Pil1 (phosphorylation inhibited by long chain bases) and Lsp1 (long chain bases stimulate phosphorylation). Appearance of membrane-associated eisosomes drastically changes in response to Pil1 hyper-or hypophosphorylation [16,34]. Whether and how these changes in eisosome morphology influence the furrow-like invaginations of the plasma membrane (MCC) is not yet known.…”

Microcompartments within the yeast plasma membrane

“…Their de novo formation has been observed in dependence of the cell cycle in newly forming buds, occurring in waves toward the bud tip. The major components, Pil1 and Lsp1, are phosphorylated by a dual pair of phosphoinositide-dependent protein kinases, Pkh1 and Pkh2, which leads to the eisosome disassembly (Walther et al , 2007 ). In accordance with the lipid interaction partner of Pil1 and Lsp1, decreasing the levels of PI(4,5)P 2 results in the formation of fewer eisosomes at the plasma membrane, whereas increasing the levels elongate the membrane furrows (Karotki et al , 2011 ).…”