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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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Nonphosphorylatable Pil1 is resistant to Δnce102 . (a and b) Representative top and mid sections of wild-type (wt; left) or Δnce102 cells (right) expressing Pil1-GFP (a) or nonphosphorylatable pil1(4A) fused to GFP (b) are shown. Bars, 5 µm.
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fig4: Nonphosphorylatable Pil1 is resistant to Δnce102 . (a and b) Representative top and mid sections of wild-type (wt; left) or Δnce102 cells (right) expressing Pil1-GFP (a) or nonphosphorylatable pil1(4A) fused to GFP (b) are shown. Bars, 5 µm.

Mentions: Therefore, we tested genetically whether Nce102 acts on eisosomes by altering Pil1 phosphorylation that could then indirectly modulate eisosome assembly. If Δnce102 effect is mediated by phosphorylation, we expect that a pil1(4A)-GFP mutant in which four residues that are required for the effect of Pkh kinases on eisosome assembly are changed to alanine (S45A, S59A, S230A, and T233A; Walther et al., 2007) blocks the effect of NCE102 deletion. However, if Nce102's effect on eisosomes is independent of Pil1 phosphorylation state, we expect to see similar effects of Δnce102 on wild-type Pil1 and pil1(4A). Indeed, pil1(4A)-GFP localization was indistinguishable between Δnce102 and wild-type cells, showing slightly more eisosome pil1(4A) assembly at the plasma membrane compared with wild-type Pil1 (Fig. 4 b). Therefore, pil1(4A) is epistatic to Δnce102. Because Pil1 needs to get phosphorylated on residues mutated in pil1(4A) to develop the Δnce102 eisosome phenotype, we conclude that Nce102 acts upstream of Pil1 phosphorylation.


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)

Nonphosphorylatable Pil1 is resistant to Δnce102 . (a and b) Representative top and mid sections of wild-type (wt; left) or Δnce102 cells (right) expressing Pil1-GFP (a) or nonphosphorylatable pil1(4A) fused to GFP (b) are shown. Bars, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: Nonphosphorylatable Pil1 is resistant to Δnce102 . (a and b) Representative top and mid sections of wild-type (wt; left) or Δnce102 cells (right) expressing Pil1-GFP (a) or nonphosphorylatable pil1(4A) fused to GFP (b) are shown. Bars, 5 µm.
Mentions: Therefore, we tested genetically whether Nce102 acts on eisosomes by altering Pil1 phosphorylation that could then indirectly modulate eisosome assembly. If Δnce102 effect is mediated by phosphorylation, we expect that a pil1(4A)-GFP mutant in which four residues that are required for the effect of Pkh kinases on eisosome assembly are changed to alanine (S45A, S59A, S230A, and T233A; Walther et al., 2007) blocks the effect of NCE102 deletion. However, if Nce102's effect on eisosomes is independent of Pil1 phosphorylation state, we expect to see similar effects of Δnce102 on wild-type Pil1 and pil1(4A). Indeed, pil1(4A)-GFP localization was indistinguishable between Δnce102 and wild-type cells, showing slightly more eisosome pil1(4A) assembly at the plasma membrane compared with wild-type Pil1 (Fig. 4 b). Therefore, pil1(4A) is epistatic to Δnce102. Because Pil1 needs to get phosphorylated on residues mutated in pil1(4A) to develop the Δnce102 eisosome phenotype, we conclude that Nce102 acts upstream of Pil1 phosphorylation.

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