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J. Biol. Chem., Vol. 283, Issue 16, 10433-10444, April 18, 2008

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The Sphingolipid Long-chain Base-Pkh1/2-Ypk1/2 Signaling Pathway Regulates Eisosome Assembly and Turnover^*

Guangzuo Luo¹, Albrecht Gruhler¹², Ying Liu³, Ole N. Jensen⁴, and Robert C. Dickson⁵

From the Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky, 40536 and the Protein Research Group, Department of Biochemistry & Molecular Biology, University of Southern Denmark, Odense, Denmark

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.

Received for publication, December 6, 2007 , and in revised form, February 12, 2008.

^* This work was supported in part by Grant GM41302 from the National Institutes of Health (NIH) and by core facilities supported by a Grant P20-RR020171 from the National Center for Research Resources, a component of NIH. The LTQ-FT ICR-MS instrument was financed by a generous grant from the Danish Basic Research Foundation to the Center for Experimental Bioinformatics. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S7.

¹ Both authors contributed equally to this work.

² Novo Nordisk A/S, Biopharm. Research Unit, Novo Nordisk, Måløv DK-2760, Denmark.

³ Dept. of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001, China.

⁴ A Lundbeck Foundation Research Professor and the recipient of a Young Investigator Award from the Danish Research Councils. To whom correspondence may be addressed: Tel.: 45-6550-2368; Fax: 45-6550-2467; E-mail: jenseno{at}bmb.sdu.dk. ⁵ To whom correspondence may be addressed: Dept. of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY 40536. Tel.: 859-323-6052; Fax: 859-257-2283; E-mail: bobd{at}uky.edu.

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