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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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Nce102-GFP localization depends on sphingolipid levels. (a) Nce102-GFP was imaged under normal growth conditions (control), after addition of 5 µM myriocin for 1 h (myriocin), after sequential treatment with 5 µM myriocin for 1 h and 50 µM PHS for 15 min (myriocin→PHS), or after addition of 50 µM PHS for 15 min (PHS). Boxes indicate the area magnified in the bottom panels. (b) Fluorescence intensities of the area are shown plotted against xy image coordinates. (c) Nce102 redistribution is not dependent on new protein synthesis. Nce102-GFP cells were treated with myriocin or myriocin and PHS successively as in a after 10-min preincubation and continued presence of cycloheximide (CHX). (d) Nce102 partitions into detergent-resistant membranes dependent on sphingoid bases. Untreated Nce102-TAP–expressing cells and cells treated as in a were lysed in buffer containing 1% Triton X-100 and analyzed by gradient centrifugation and Western blotting against TAP (left). The same blots were probed with Pma1 antibodies (right). (e) pil1(4A)-GFP is resistant to disassembly after myriocin treatment. pil1(4A)-GFP–expressing cells were imaged after 1-h 5 µM myriocin incubation. (f) Redistribution of Nce102-GFP after myriocin treatment is independent of eisosome disassembly. Cells expressing pil1(4A) and Nce102-GFP were imaged before (left) and after 1 h treatment with 5 µM myriocin (right). (g and h) Sur7-mars does not behave like Nce102 after myriocin treatment. Wild-type Pil1 (g) or pil1(4A) (h) cells expressing Sur7-mars were treated with myriocin and imaged. wt, wild type. Bars, 5 µm.
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fig9: Nce102-GFP localization depends on sphingolipid levels. (a) Nce102-GFP was imaged under normal growth conditions (control), after addition of 5 µM myriocin for 1 h (myriocin), after sequential treatment with 5 µM myriocin for 1 h and 50 µM PHS for 15 min (myriocin→PHS), or after addition of 50 µM PHS for 15 min (PHS). Boxes indicate the area magnified in the bottom panels. (b) Fluorescence intensities of the area are shown plotted against xy image coordinates. (c) Nce102 redistribution is not dependent on new protein synthesis. Nce102-GFP cells were treated with myriocin or myriocin and PHS successively as in a after 10-min preincubation and continued presence of cycloheximide (CHX). (d) Nce102 partitions into detergent-resistant membranes dependent on sphingoid bases. Untreated Nce102-TAP–expressing cells and cells treated as in a were lysed in buffer containing 1% Triton X-100 and analyzed by gradient centrifugation and Western blotting against TAP (left). The same blots were probed with Pma1 antibodies (right). (e) pil1(4A)-GFP is resistant to disassembly after myriocin treatment. pil1(4A)-GFP–expressing cells were imaged after 1-h 5 µM myriocin incubation. (f) Redistribution of Nce102-GFP after myriocin treatment is independent of eisosome disassembly. Cells expressing pil1(4A) and Nce102-GFP were imaged before (left) and after 1 h treatment with 5 µM myriocin (right). (g and h) Sur7-mars does not behave like Nce102 after myriocin treatment. Wild-type Pil1 (g) or pil1(4A) (h) cells expressing Sur7-mars were treated with myriocin and imaged. wt, wild type. Bars, 5 µm.

Mentions: Because Nce102 negatively regulates Pkh kinases that localize to eisosomes, we next tested whether Nce102 distribution between MCCs at eisosomes and the rest of the plasma membrane is affected by sphingolipid levels. To this end, we determined Nce102-GFP localization after blocking sphingolipid synthesis. Strikingly, after a 1 h incubation of cells with myriocin, the punctate pattern of Nce102-GFP localization in MCCs disappeared, and the protein distributed diffusely across the plasma membrane (Fig. 9 a). Consistently, in intensity plots of surface images, myriocin-treated cells show a rather flat distribution of the Nce102-GFP signal, whereas control samples show many Nce102-GFP peaks corresponding to MCCs at eisosomes (Fig. 9 b). Relocalization of Nce102-GFP could be reversed by addition of exogenous PHS for a short time (15 min). In this case, the MCC pattern of Nce102-GFP localization reappeared, showing an even more pronounced pattern of Nce102-GFP foci than normal (Fig. 9 b).


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)

Nce102-GFP localization depends on sphingolipid levels. (a) Nce102-GFP was imaged under normal growth conditions (control), after addition of 5 µM myriocin for 1 h (myriocin), after sequential treatment with 5 µM myriocin for 1 h and 50 µM PHS for 15 min (myriocin→PHS), or after addition of 50 µM PHS for 15 min (PHS). Boxes indicate the area magnified in the bottom panels. (b) Fluorescence intensities of the area are shown plotted against xy image coordinates. (c) Nce102 redistribution is not dependent on new protein synthesis. Nce102-GFP cells were treated with myriocin or myriocin and PHS successively as in a after 10-min preincubation and continued presence of cycloheximide (CHX). (d) Nce102 partitions into detergent-resistant membranes dependent on sphingoid bases. Untreated Nce102-TAP–expressing cells and cells treated as in a were lysed in buffer containing 1% Triton X-100 and analyzed by gradient centrifugation and Western blotting against TAP (left). The same blots were probed with Pma1 antibodies (right). (e) pil1(4A)-GFP is resistant to disassembly after myriocin treatment. pil1(4A)-GFP–expressing cells were imaged after 1-h 5 µM myriocin incubation. (f) Redistribution of Nce102-GFP after myriocin treatment is independent of eisosome disassembly. Cells expressing pil1(4A) and Nce102-GFP were imaged before (left) and after 1 h treatment with 5 µM myriocin (right). (g and h) Sur7-mars does not behave like Nce102 after myriocin treatment. Wild-type Pil1 (g) or pil1(4A) (h) cells expressing Sur7-mars were treated with myriocin and imaged. wt, wild type. Bars, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig9: Nce102-GFP localization depends on sphingolipid levels. (a) Nce102-GFP was imaged under normal growth conditions (control), after addition of 5 µM myriocin for 1 h (myriocin), after sequential treatment with 5 µM myriocin for 1 h and 50 µM PHS for 15 min (myriocin→PHS), or after addition of 50 µM PHS for 15 min (PHS). Boxes indicate the area magnified in the bottom panels. (b) Fluorescence intensities of the area are shown plotted against xy image coordinates. (c) Nce102 redistribution is not dependent on new protein synthesis. Nce102-GFP cells were treated with myriocin or myriocin and PHS successively as in a after 10-min preincubation and continued presence of cycloheximide (CHX). (d) Nce102 partitions into detergent-resistant membranes dependent on sphingoid bases. Untreated Nce102-TAP–expressing cells and cells treated as in a were lysed in buffer containing 1% Triton X-100 and analyzed by gradient centrifugation and Western blotting against TAP (left). The same blots were probed with Pma1 antibodies (right). (e) pil1(4A)-GFP is resistant to disassembly after myriocin treatment. pil1(4A)-GFP–expressing cells were imaged after 1-h 5 µM myriocin incubation. (f) Redistribution of Nce102-GFP after myriocin treatment is independent of eisosome disassembly. Cells expressing pil1(4A) and Nce102-GFP were imaged before (left) and after 1 h treatment with 5 µM myriocin (right). (g and h) Sur7-mars does not behave like Nce102 after myriocin treatment. Wild-type Pil1 (g) or pil1(4A) (h) cells expressing Sur7-mars were treated with myriocin and imaged. wt, wild type. Bars, 5 µm.
Mentions: Because Nce102 negatively regulates Pkh kinases that localize to eisosomes, we next tested whether Nce102 distribution between MCCs at eisosomes and the rest of the plasma membrane is affected by sphingolipid levels. To this end, we determined Nce102-GFP localization after blocking sphingolipid synthesis. Strikingly, after a 1 h incubation of cells with myriocin, the punctate pattern of Nce102-GFP localization in MCCs disappeared, and the protein distributed diffusely across the plasma membrane (Fig. 9 a). Consistently, in intensity plots of surface images, myriocin-treated cells show a rather flat distribution of the Nce102-GFP signal, whereas control samples show many Nce102-GFP peaks corresponding to MCCs at eisosomes (Fig. 9 b). Relocalization of Nce102-GFP could be reversed by addition of exogenous PHS for a short time (15 min). In this case, the MCC pattern of Nce102-GFP localization reappeared, showing an even more pronounced pattern of Nce102-GFP foci than normal (Fig. 9 b).

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