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A genome-wide screen for genes affecting eisosomes reveals Nce102 function in sphingolipid signaling.

Fröhlich F, Moreira K, Aguilar PS, Hubner NC, Mann M, Walter P, Walther TC - J. Cell Biol. (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Organelle Architecture and Dynamics, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.

ABSTRACT
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.

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Overexpression of Nce102 suppresses eisosomes disassembly after sphingolipid synthesis block. Nce102-mars (right) controlled by the Gal promoter was either not expressed in raffinose-containing medium or induced in galactose-containing medium in Pil1-GFP (left) cells, which were incubated for 1 h with 5 µM myriocin as indicated. Bar, 5 µm.
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fig8: Overexpression of Nce102 suppresses eisosomes disassembly after sphingolipid synthesis block. Nce102-mars (right) controlled by the Gal promoter was either not expressed in raffinose-containing medium or induced in galactose-containing medium in Pil1-GFP (left) cells, which were incubated for 1 h with 5 µM myriocin as indicated. Bar, 5 µm.

Mentions: We previously showed that Pil1 hyperphosphorylation caused by decreased sphingolipid synthesis causes eisosome disassembly (Walther et al., 2007). If our hypothesis is true and Nce102 is an inhibitor of Pil1 phosphorylation acting downstream of sphingolipids, we predict that overexpression of Nce102 will rescue eisosome disassembly when sphingolipid synthesis is decreased. To test this, we expressed Nce102-mars from the inducible Gal promoter in cells that have Pil1-GFP–marked eisosomes. When these cells are grown on raffinose, Nce102-mars is not expressed, and eisosomes appear normal, as these cells also express endogenous Nce102 (Fig. 8). When these cells grow on galactose, Nce102-mars is overexpressed (Fig. 8). Strikingly, when we blocked sphingolipid synthesis in these cells by treating them with myriocin, the normal effect of disassembling eisosomes apparent in control cells was completely blocked (Fig. 8). This shows that increasing Nce102 levels blocks the effect of inhibiting sphingolipid synthesis on eisosomes.


A genome-wide screen for genes affecting eisosomes reveals Nce102 function in sphingolipid signaling.

Fröhlich F, Moreira K, Aguilar PS, Hubner NC, Mann M, Walter P, Walther TC - J. Cell Biol. (2009)

Overexpression of Nce102 suppresses eisosomes disassembly after sphingolipid synthesis block. Nce102-mars (right) controlled by the Gal promoter was either not expressed in raffinose-containing medium or induced in galactose-containing medium in Pil1-GFP (left) cells, which were incubated for 1 h with 5 µM myriocin as indicated. Bar, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2712959&req=5

fig8: Overexpression of Nce102 suppresses eisosomes disassembly after sphingolipid synthesis block. Nce102-mars (right) controlled by the Gal promoter was either not expressed in raffinose-containing medium or induced in galactose-containing medium in Pil1-GFP (left) cells, which were incubated for 1 h with 5 µM myriocin as indicated. Bar, 5 µm.
Mentions: We previously showed that Pil1 hyperphosphorylation caused by decreased sphingolipid synthesis causes eisosome disassembly (Walther et al., 2007). If our hypothesis is true and Nce102 is an inhibitor of Pil1 phosphorylation acting downstream of sphingolipids, we predict that overexpression of Nce102 will rescue eisosome disassembly when sphingolipid synthesis is decreased. To test this, we expressed Nce102-mars from the inducible Gal promoter in cells that have Pil1-GFP–marked eisosomes. When these cells are grown on raffinose, Nce102-mars is not expressed, and eisosomes appear normal, as these cells also express endogenous Nce102 (Fig. 8). When these cells grow on galactose, Nce102-mars is overexpressed (Fig. 8). Strikingly, when we blocked sphingolipid synthesis in these cells by treating them with myriocin, the normal effect of disassembling eisosomes apparent in control cells was completely blocked (Fig. 8). This shows that increasing Nce102 levels blocks the effect of inhibiting sphingolipid synthesis on eisosomes.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Organelle Architecture and Dynamics, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.

ABSTRACT
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.

Show MeSH