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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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Pil1 phosphorylation is increased in Δnce102 cells. (a) Pil1-TAP was purified from SILAC-labeled wild-type and Δnce102 cells, mixed in equal amounts, resolved by SDS-PAGE, and two resulting Pil1 bands were separately analyzed by MS. The contaminant Nsr1 was present in a 1:1 ratio from both strains in each band. Pil1 was1.3-fold enriched in the upper band (red) and decreased to a ratio 0.6 in the lower band (black). (b) Pil1-TAP purified from SILAC-labeled wild-type and Δnce102 cells was analyzed by MS. Unphosphorylated Pil1 peptide amino acids 222–248 is shown in black, and the T233 phosphopeptide is shown in red. (c) Pil1-TAP purified from SILAC-labeled untreated cells or treated with myriocin for 1 h was analyzed as in b. Representative data of three experiments are shown. Ratio H/L denotes the mean ratio of the abundance of heavy- versus light-labeled peptides.
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fig5: Pil1 phosphorylation is increased in Δnce102 cells. (a) Pil1-TAP was purified from SILAC-labeled wild-type and Δnce102 cells, mixed in equal amounts, resolved by SDS-PAGE, and two resulting Pil1 bands were separately analyzed by MS. The contaminant Nsr1 was present in a 1:1 ratio from both strains in each band. Pil1 was1.3-fold enriched in the upper band (red) and decreased to a ratio 0.6 in the lower band (black). (b) Pil1-TAP purified from SILAC-labeled wild-type and Δnce102 cells was analyzed by MS. Unphosphorylated Pil1 peptide amino acids 222–248 is shown in black, and the T233 phosphopeptide is shown in red. (c) Pil1-TAP purified from SILAC-labeled untreated cells or treated with myriocin for 1 h was analyzed as in b. Representative data of three experiments are shown. Ratio H/L denotes the mean ratio of the abundance of heavy- versus light-labeled peptides.

Mentions: To substantiate these findings, we determined whether the phosphorylation state of Pil1 is altered in Δnce102 cells. To this end, we purified Pil1 fused to a tandem affinity purification (TAP) tag from wild-type and metabolically heavy lysine-labeled Δnce102 cells (stable isotope labeling by amino acids in cell culture [SILAC]; Fig. S2 a; Ong et al., 2002) and analyzed it by two strategies. First, we mixed proteins of Pil1-TAP eluates 1:1 and separated them by SDS-PAGE, resulting in a clearly visible doublet of Pil1, where the upper band represents phosphorylated Pil1 (Walther et al., 2007). Separate analysis of the two bands by liquid chromatography (LC) mass spectrometry (MS)/MS revealed that contaminant proteins present in both bands have a 1:1 ratio of protein from wild-type and Δnce102 sample, as determined by comparing mean peptide peak intensities. In contrast, Pil1 from Δnce102 was 30% enriched in the upper phospho-Pil1 band (1.28 ratio, Pil1 heavy vs. light) and correspondingly decreased in the lower band (0.59 ratio, Pil1 heavy vs. light; Fig. 5 a; and Fig. S2, c and d).


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)

Pil1 phosphorylation is increased in Δnce102 cells. (a) Pil1-TAP was purified from SILAC-labeled wild-type and Δnce102 cells, mixed in equal amounts, resolved by SDS-PAGE, and two resulting Pil1 bands were separately analyzed by MS. The contaminant Nsr1 was present in a 1:1 ratio from both strains in each band. Pil1 was1.3-fold enriched in the upper band (red) and decreased to a ratio 0.6 in the lower band (black). (b) Pil1-TAP purified from SILAC-labeled wild-type and Δnce102 cells was analyzed by MS. Unphosphorylated Pil1 peptide amino acids 222–248 is shown in black, and the T233 phosphopeptide is shown in red. (c) Pil1-TAP purified from SILAC-labeled untreated cells or treated with myriocin for 1 h was analyzed as in b. Representative data of three experiments are shown. Ratio H/L denotes the mean ratio of the abundance of heavy- versus light-labeled peptides.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig5: Pil1 phosphorylation is increased in Δnce102 cells. (a) Pil1-TAP was purified from SILAC-labeled wild-type and Δnce102 cells, mixed in equal amounts, resolved by SDS-PAGE, and two resulting Pil1 bands were separately analyzed by MS. The contaminant Nsr1 was present in a 1:1 ratio from both strains in each band. Pil1 was1.3-fold enriched in the upper band (red) and decreased to a ratio 0.6 in the lower band (black). (b) Pil1-TAP purified from SILAC-labeled wild-type and Δnce102 cells was analyzed by MS. Unphosphorylated Pil1 peptide amino acids 222–248 is shown in black, and the T233 phosphopeptide is shown in red. (c) Pil1-TAP purified from SILAC-labeled untreated cells or treated with myriocin for 1 h was analyzed as in b. Representative data of three experiments are shown. Ratio H/L denotes the mean ratio of the abundance of heavy- versus light-labeled peptides.
Mentions: To substantiate these findings, we determined whether the phosphorylation state of Pil1 is altered in Δnce102 cells. To this end, we purified Pil1 fused to a tandem affinity purification (TAP) tag from wild-type and metabolically heavy lysine-labeled Δnce102 cells (stable isotope labeling by amino acids in cell culture [SILAC]; Fig. S2 a; Ong et al., 2002) and analyzed it by two strategies. First, we mixed proteins of Pil1-TAP eluates 1:1 and separated them by SDS-PAGE, resulting in a clearly visible doublet of Pil1, where the upper band represents phosphorylated Pil1 (Walther et al., 2007). Separate analysis of the two bands by liquid chromatography (LC) mass spectrometry (MS)/MS revealed that contaminant proteins present in both bands have a 1:1 ratio of protein from wild-type and Δnce102 sample, as determined by comparing mean peptide peak intensities. In contrast, Pil1 from Δnce102 was 30% enriched in the upper phospho-Pil1 band (1.28 ratio, Pil1 heavy vs. light) and correspondingly decreased in the lower band (0.59 ratio, Pil1 heavy vs. light; Fig. 5 a; and Fig. S2, c and d).

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