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Plasma membrane microdomains regulate turnover of transport proteins in yeast.

Grossmann G, Malinsky J, Stahlschmidt W, Loibl M, Weig-Meckl I, Frommer WB, Opekarová M, Tanner W - J. Cell Biol. (2008)

Bottom Line: Deletion of Pil1, a component of eisosomes, or of Nce102, an integral membrane protein of MCC, results in the dissipation of all MCC markers.These deletion mutants also show accelerated endocytosis of MCC-resident permeases Can1 and Fur4.Addition of arginine to wild-type cells leads to a similar redistribution and increased turnover of Can1.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Biology and Plant Physiology, University of Regensburg, 93053 Regensburg, Germany.

ABSTRACT
In this study, we investigate whether the stable segregation of proteins and lipids within the yeast plasma membrane serves a particular biological function. We show that 21 proteins cluster within or associate with the ergosterol-rich membrane compartment of Can1 (MCC). However, proteins of the endocytic machinery are excluded from MCC. In a screen, we identified 28 genes affecting MCC appearance and found that genes involved in lipid biosynthesis and vesicle transport are significantly overrepresented. Deletion of Pil1, a component of eisosomes, or of Nce102, an integral membrane protein of MCC, results in the dissipation of all MCC markers. These deletion mutants also show accelerated endocytosis of MCC-resident permeases Can1 and Fur4. Our data suggest that release from MCC makes these proteins accessible to the endocytic machinery. Addition of arginine to wild-type cells leads to a similar redistribution and increased turnover of Can1. Thus, MCC represents a protective area within the plasma membrane to control turnover of transport proteins.

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Distribution of MCC markers in selected knockout strains. Distributions of HUP1-GFP, Can1-GFP, Sur7-GFP, and filipin-stained sterols were monitored in the library of single gene deletion strains (see Materials and methods). Examples of detected phenotypes (classification of phenotypes: wild type [WT]–like, −; weak, +; medium, ++; strong, +++) on tangential confocal sections (HUP1, Can1, and Sur7) or wide-field images (filipin; transversal sections) are presented. Note the relatively high background fluorescence intensity between MCC patches in cells expressing HUP1-GFP (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200806035/DC1; Grossmann et al., 2006). For a full dataset of all mutant phenotypes listed in Table II, see supplemental material. Bars, 5 μm.
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fig2: Distribution of MCC markers in selected knockout strains. Distributions of HUP1-GFP, Can1-GFP, Sur7-GFP, and filipin-stained sterols were monitored in the library of single gene deletion strains (see Materials and methods). Examples of detected phenotypes (classification of phenotypes: wild type [WT]–like, −; weak, +; medium, ++; strong, +++) on tangential confocal sections (HUP1, Can1, and Sur7) or wide-field images (filipin; transversal sections) are presented. Note the relatively high background fluorescence intensity between MCC patches in cells expressing HUP1-GFP (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200806035/DC1; Grossmann et al., 2006). For a full dataset of all mutant phenotypes listed in Table II, see supplemental material. Bars, 5 μm.

Mentions: To identify proteins involved in the plasma membrane compartmentation, we performed a visual genome-wide screen for deletion mutants that shows an alteration in or a complete loss of MCC compartmentation. The hexose/H+ symporter HUP1 of the unicellular alga Chlorella kessleri was selected for the genome-wide screen. When expressed in S. cerevisiae, HUP1 accumulates in MCC patches and serves as the most sensitive marker of MCC integrity. Moreover, when expressed in yeast under the control of alcohol dehydrogenase promoter, HUP1 is stably expressed under all growth conditions (Grossmann et al., 2006, 2007). The yeast strain collection of nonessential gene knockouts was transformed with the HUP1-GFP fusion (see Materials and methods). The transformation was successful in 91.3% (4,413/4,836) of mutants from the collection. The screen was performed by taking confocal images of the surface and a cross section of at least 30 cells each plus a differential interference contrast image. For 4,365 strains (98.9%), an analyzable GFP signal was obtained. An altered distribution of HUP1-GFP was detected in 28 strains (Table II). These strains were subsequently checked for the distributions of Can1-GFP, Sur7-GFP, and of the plasma membrane sterols by staining with filipin (Fig. 2 and Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200806035/DC1). A complete image dataset of the mutant phenotypes is available in the supplemental material.


Plasma membrane microdomains regulate turnover of transport proteins in yeast.

Grossmann G, Malinsky J, Stahlschmidt W, Loibl M, Weig-Meckl I, Frommer WB, Opekarová M, Tanner W - J. Cell Biol. (2008)

Distribution of MCC markers in selected knockout strains. Distributions of HUP1-GFP, Can1-GFP, Sur7-GFP, and filipin-stained sterols were monitored in the library of single gene deletion strains (see Materials and methods). Examples of detected phenotypes (classification of phenotypes: wild type [WT]–like, −; weak, +; medium, ++; strong, +++) on tangential confocal sections (HUP1, Can1, and Sur7) or wide-field images (filipin; transversal sections) are presented. Note the relatively high background fluorescence intensity between MCC patches in cells expressing HUP1-GFP (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200806035/DC1; Grossmann et al., 2006). For a full dataset of all mutant phenotypes listed in Table II, see supplemental material. Bars, 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2600745&req=5

fig2: Distribution of MCC markers in selected knockout strains. Distributions of HUP1-GFP, Can1-GFP, Sur7-GFP, and filipin-stained sterols were monitored in the library of single gene deletion strains (see Materials and methods). Examples of detected phenotypes (classification of phenotypes: wild type [WT]–like, −; weak, +; medium, ++; strong, +++) on tangential confocal sections (HUP1, Can1, and Sur7) or wide-field images (filipin; transversal sections) are presented. Note the relatively high background fluorescence intensity between MCC patches in cells expressing HUP1-GFP (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200806035/DC1; Grossmann et al., 2006). For a full dataset of all mutant phenotypes listed in Table II, see supplemental material. Bars, 5 μm.
Mentions: To identify proteins involved in the plasma membrane compartmentation, we performed a visual genome-wide screen for deletion mutants that shows an alteration in or a complete loss of MCC compartmentation. The hexose/H+ symporter HUP1 of the unicellular alga Chlorella kessleri was selected for the genome-wide screen. When expressed in S. cerevisiae, HUP1 accumulates in MCC patches and serves as the most sensitive marker of MCC integrity. Moreover, when expressed in yeast under the control of alcohol dehydrogenase promoter, HUP1 is stably expressed under all growth conditions (Grossmann et al., 2006, 2007). The yeast strain collection of nonessential gene knockouts was transformed with the HUP1-GFP fusion (see Materials and methods). The transformation was successful in 91.3% (4,413/4,836) of mutants from the collection. The screen was performed by taking confocal images of the surface and a cross section of at least 30 cells each plus a differential interference contrast image. For 4,365 strains (98.9%), an analyzable GFP signal was obtained. An altered distribution of HUP1-GFP was detected in 28 strains (Table II). These strains were subsequently checked for the distributions of Can1-GFP, Sur7-GFP, and of the plasma membrane sterols by staining with filipin (Fig. 2 and Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200806035/DC1). A complete image dataset of the mutant phenotypes is available in the supplemental material.

Bottom Line: Deletion of Pil1, a component of eisosomes, or of Nce102, an integral membrane protein of MCC, results in the dissipation of all MCC markers.These deletion mutants also show accelerated endocytosis of MCC-resident permeases Can1 and Fur4.Addition of arginine to wild-type cells leads to a similar redistribution and increased turnover of Can1.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Biology and Plant Physiology, University of Regensburg, 93053 Regensburg, Germany.

ABSTRACT
In this study, we investigate whether the stable segregation of proteins and lipids within the yeast plasma membrane serves a particular biological function. We show that 21 proteins cluster within or associate with the ergosterol-rich membrane compartment of Can1 (MCC). However, proteins of the endocytic machinery are excluded from MCC. In a screen, we identified 28 genes affecting MCC appearance and found that genes involved in lipid biosynthesis and vesicle transport are significantly overrepresented. Deletion of Pil1, a component of eisosomes, or of Nce102, an integral membrane protein of MCC, results in the dissipation of all MCC markers. These deletion mutants also show accelerated endocytosis of MCC-resident permeases Can1 and Fur4. Our data suggest that release from MCC makes these proteins accessible to the endocytic machinery. Addition of arginine to wild-type cells leads to a similar redistribution and increased turnover of Can1. Thus, MCC represents a protective area within the plasma membrane to control turnover of transport proteins.

Show MeSH
Related in: MedlinePlus