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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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Model for Nce102 function in sphingolipid sensing. Nce102 (green) senses sphingolipid levels in the plasma membrane by distributing between the thick sphingolipid-rich MCC (blue) and the rest of the plasma membrane (gray) depending on sphingolipid levels. In the MCC, it inhibits Pkh kinases (red) that localize under this domain at eisosomes.
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fig10: Model for Nce102 function in sphingolipid sensing. Nce102 (green) senses sphingolipid levels in the plasma membrane by distributing between the thick sphingolipid-rich MCC (blue) and the rest of the plasma membrane (gray) depending on sphingolipid levels. In the MCC, it inhibits Pkh kinases (red) that localize under this domain at eisosomes.

Mentions: Our experiments revealed that Nce102 has fascinating properties, suggesting that it might act as part of a sphingolipid sensor. Most remarkable, its localization is dynamic and responsive to changes in sphingolipid levels: Nce102 partitions between two domains in the plasma membrane, MCCs overlying eisosomes and the rest of the plasma membrane. We show that the distribution between these two domains is controlled by sphingolipid availability. Nce102 localization to MCCs brings it into close contact with the underlying eisosomes and Pkh kinases (Fig. S5; Walther et al., 2007). Nce102 negatively regulates the activity of these kinases, and, in the presence of sphingolipids, blocks their downstream functions. Conversely, if sphingolipid synthesis is blocked, Nce102 redistributes away from Pkh kinases (Fig. S5), alleviating their inhibition. Therefore, we propose a model that Nce102 acts as part of a sphingolipid-sensing mechanism and that its distribution in the plasma membrane regulates Pkh kinases (Fig. 10). In the simplest hypothesis, Nce102 could simply accomplish repression of the kinases by regulated juxtaposition to them, which is a common scheme in kinase signaling. Based on filipin staining, MCCs were suggested as sites of increased ergosterol concentration in the plasma membrane (Grossmann et al., 2007), and because sterols preferentially interact with sphingolipids, it is likely that sphingolipids are also concentrated there, forming detergent-resistant, liquid-ordered membrane domains or lipid rafts (Simons and Ikonen, 1997; Malinska et al., 2003) where Nce102 was found previously (Bagnat et al., 2000). Thus, it is possible that Nce102 also reacts to ergosterol levels in the plasma membrane. However, we did not observe an effect of nonessential erg mutants or block of sterol synthesis on eisosomes or Nce102 localization (unpublished data). Consistent with our model, Nce102 localizes to eisosome remnants that also show increased filipin staining, likely reflecting increased concentration of ergosterol and possibly sphingolipids (Grossmann et al., 2007). Alternatively, filipin might preferentially report on free sterols not in complex with sphingolipids, and staining of MCCs could actually indicate a lower concentration of sphingolipids in this compartment. This view is supported by a recent observation that filipin staining increases if sphingolipid synthesis is blocked (Jin et al., 2008). A further alternative is that Nce102 could directly bind sphingolipids, changing its affinities to other proteins that help localize it to MCCs and/or switching its activity as a Pkh kinase inhibitor on and off. In either model, Nce102 leaves the MCC when sphingolipid levels there are low, releasing its inhibition of Pkh kinases that now phosphorylate Pil1 and other targets. This is consistent with our observation that Nce102 detergent solubility changes after inhibition of sphingoid base synthesis, indicating that it partitions between different membrane environments. Most likely, this change corresponds to the relocalization of Nce102 from MCCs to the remainder of the membrane observed by microscopy. The interpretation of this result, however, remains vague, as most proteins in the yeast plasma membrane differ only in the degree of their resistance to Triton X-100 detergent extraction. For example, the MCP marker Pma1 was also previously used as a marker for detergent-resistant lipid rafts (Bagnat et al., 2000; Lee et al., 2002; Malinska et al., 2003). The difference in Triton X-100 solubility between Nce102 and Pma1 after myriocin treatment might therefore indicate that these proteins differently partition into such lipid rafts or that the MCP is actually more complex and contains subdomains not easily resolved by light microscopy. Consistent with the later notion, TORC2 (Tor kinase complex 2) is localized at a distinct plasma membrane domain separate from MCC and MCP and is also partially detergent resistant (Aronova et al., 2007; Berchtold and Walther, 2009).


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)

Model for Nce102 function in sphingolipid sensing. Nce102 (green) senses sphingolipid levels in the plasma membrane by distributing between the thick sphingolipid-rich MCC (blue) and the rest of the plasma membrane (gray) depending on sphingolipid levels. In the MCC, it inhibits Pkh kinases (red) that localize under this domain at eisosomes.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig10: Model for Nce102 function in sphingolipid sensing. Nce102 (green) senses sphingolipid levels in the plasma membrane by distributing between the thick sphingolipid-rich MCC (blue) and the rest of the plasma membrane (gray) depending on sphingolipid levels. In the MCC, it inhibits Pkh kinases (red) that localize under this domain at eisosomes.
Mentions: Our experiments revealed that Nce102 has fascinating properties, suggesting that it might act as part of a sphingolipid sensor. Most remarkable, its localization is dynamic and responsive to changes in sphingolipid levels: Nce102 partitions between two domains in the plasma membrane, MCCs overlying eisosomes and the rest of the plasma membrane. We show that the distribution between these two domains is controlled by sphingolipid availability. Nce102 localization to MCCs brings it into close contact with the underlying eisosomes and Pkh kinases (Fig. S5; Walther et al., 2007). Nce102 negatively regulates the activity of these kinases, and, in the presence of sphingolipids, blocks their downstream functions. Conversely, if sphingolipid synthesis is blocked, Nce102 redistributes away from Pkh kinases (Fig. S5), alleviating their inhibition. Therefore, we propose a model that Nce102 acts as part of a sphingolipid-sensing mechanism and that its distribution in the plasma membrane regulates Pkh kinases (Fig. 10). In the simplest hypothesis, Nce102 could simply accomplish repression of the kinases by regulated juxtaposition to them, which is a common scheme in kinase signaling. Based on filipin staining, MCCs were suggested as sites of increased ergosterol concentration in the plasma membrane (Grossmann et al., 2007), and because sterols preferentially interact with sphingolipids, it is likely that sphingolipids are also concentrated there, forming detergent-resistant, liquid-ordered membrane domains or lipid rafts (Simons and Ikonen, 1997; Malinska et al., 2003) where Nce102 was found previously (Bagnat et al., 2000). Thus, it is possible that Nce102 also reacts to ergosterol levels in the plasma membrane. However, we did not observe an effect of nonessential erg mutants or block of sterol synthesis on eisosomes or Nce102 localization (unpublished data). Consistent with our model, Nce102 localizes to eisosome remnants that also show increased filipin staining, likely reflecting increased concentration of ergosterol and possibly sphingolipids (Grossmann et al., 2007). Alternatively, filipin might preferentially report on free sterols not in complex with sphingolipids, and staining of MCCs could actually indicate a lower concentration of sphingolipids in this compartment. This view is supported by a recent observation that filipin staining increases if sphingolipid synthesis is blocked (Jin et al., 2008). A further alternative is that Nce102 could directly bind sphingolipids, changing its affinities to other proteins that help localize it to MCCs and/or switching its activity as a Pkh kinase inhibitor on and off. In either model, Nce102 leaves the MCC when sphingolipid levels there are low, releasing its inhibition of Pkh kinases that now phosphorylate Pil1 and other targets. This is consistent with our observation that Nce102 detergent solubility changes after inhibition of sphingoid base synthesis, indicating that it partitions between different membrane environments. Most likely, this change corresponds to the relocalization of Nce102 from MCCs to the remainder of the membrane observed by microscopy. The interpretation of this result, however, remains vague, as most proteins in the yeast plasma membrane differ only in the degree of their resistance to Triton X-100 detergent extraction. For example, the MCP marker Pma1 was also previously used as a marker for detergent-resistant lipid rafts (Bagnat et al., 2000; Lee et al., 2002; Malinska et al., 2003). The difference in Triton X-100 solubility between Nce102 and Pma1 after myriocin treatment might therefore indicate that these proteins differently partition into such lipid rafts or that the MCP is actually more complex and contains subdomains not easily resolved by light microscopy. Consistent with the later notion, TORC2 (Tor kinase complex 2) is localized at a distinct plasma membrane domain separate from MCC and MCP and is also partially detergent resistant (Aronova et al., 2007; Berchtold and Walther, 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.

Show MeSH