The sphingolipid long-chain base-Pkh1/2-Ypk1/2 signaling pathway regulates eisosome assembly and turnover.
Eisosomes are recently described fungal structures that play roles in the organization of the plasma membrane and endocytosis. Their major protein components are Pil1 and Lsp1, and previous studies showed that these proteins are phosphorylated by the sphingolipid long-chain base-activated Pkh1 and Pkh2 protein kinases in vitro. We show that Pkh1 and Pkh2 phosphorylate Pil1 and Lsp1 in vivo to produce species B, and that heat stress, which activates Pkh1 and Pkh2, generates a more highly phosphorylated species, C. Cells with low Pkh activity lack species B and C and contain abnormally organized eisosomes. To verify that Pil1 phosphorylation is essential for correct eisosome organization, phosphorylated serine and threonine residues were identified and changed to alanines. A variant Pil1 protein lacking five phosphorylation sites did not form eisosomes during log phase growth, indicating that phosphorylation is critical for eisosome organization. We also found that eisosomes are dynamic structures and disassemble when the Ypk protein kinases, which are activated by the sphingolipid-Pkh signaling pathway, are inactivated or when the sphingolipid signal is pharmacologically blocked with myriocin. We conclude that eisosome formation and turnover are regulated by the sphingolipid-Pkh1/2-Ypk1/2 signaling pathway. These data and previous data showing that endocytosis is regulated by the sphingolipid-Pkh1/2-Ypk1/2 signaling pathway suggest that Pkh1 and -2 respond to changes in membrane sphingolipids and transmit this information to eisosomes via Pil1 phosphorylation. Eisosomes then control endocytosis to align the composition and function of the plasma membrane to match demand.
Luo
Guangzuo
G
Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky 40536, USA.
Gruhler
Albrecht
A
Liu
Ying
Y
Jensen
Ole N
ON
Dickson
Robert C
RC
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D004705:Endocytosis; D005786:Gene Expression Regulation; D038362:Glycogen Synthase Kinase 3; D006639:Histidine / Q000737:chemistry; D008954:Models, Biological; D008969:Molecular Sequence Data; D011494:Protein Kinases / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013107:Sphingolipids / Q000378:metabolism*
D029701:Saccharomyces cerevisiae Proteins; D013107:Sphingolipids; D006639:Histidine; D011494:Protein Kinases; C060388:YPK2 protein, S cerevisiae; D011505:Protein-Tyrosine Kinases; D064413:3-Phosphoinositide-Dependent Protein Kinases; C118385:PKH1 protein, S cerevisiae; C118386:PKH2 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D038362:Glycogen Synthase Kinase 3; C068124:MCK1 protein, S cerevisiae