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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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Sites of classical endocytosis do not colocalize with MCC. (B and C) The plasma membrane distributions of Rvs161 (B) and Ede1 (C), markers of late and early endocytic steps, respectively, were tested for colocalization with the MCC marker Sur7. For comparison, localization of the MCC resident Nce102 was analyzed (A). Tangential confocal sections showing the cell surface are presented. Because of a high mobility of Rvs161 patches, a maximum intensity projection of 36 frames (5 s per frame) instead of a single frame is shown in B. In this arrangement, a higher number of Rvs161 patches could be localized toward the stable Sur7 pattern at the same time. The rate of colocalization was quantified by fluorescence intensity profiles (top diagrams and arrows in merge) and 2D scatter plots of the whole full resolution images (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200806035/DC1). For easy orientation in the scatter plots, real pixel colors were used. Note the diagonal orientation of the Nce102-derived scatter plot demonstrating the colocalization of red and green fluorescence signals and a clear separation of red and green pixels in the two other cases. Examples of Sur7 patches adjacent to endocytic sites are highlighted (arrowheads). Bar, 5 μm.
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fig9: Sites of classical endocytosis do not colocalize with MCC. (B and C) The plasma membrane distributions of Rvs161 (B) and Ede1 (C), markers of late and early endocytic steps, respectively, were tested for colocalization with the MCC marker Sur7. For comparison, localization of the MCC resident Nce102 was analyzed (A). Tangential confocal sections showing the cell surface are presented. Because of a high mobility of Rvs161 patches, a maximum intensity projection of 36 frames (5 s per frame) instead of a single frame is shown in B. In this arrangement, a higher number of Rvs161 patches could be localized toward the stable Sur7 pattern at the same time. The rate of colocalization was quantified by fluorescence intensity profiles (top diagrams and arrows in merge) and 2D scatter plots of the whole full resolution images (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200806035/DC1). For easy orientation in the scatter plots, real pixel colors were used. Note the diagonal orientation of the Nce102-derived scatter plot demonstrating the colocalization of red and green fluorescence signals and a clear separation of red and green pixels in the two other cases. Examples of Sur7 patches adjacent to endocytic sites are highlighted (arrowheads). Bar, 5 μm.

Mentions: We monitored the colocalization of Rvs161-GFP and Sur7-mRFP for 180 s (36 frames; one frame per 5 s). Being aware of the thickness of a confocal section (∼700 nm), artifactual overlaps of the fluorescence signals were avoided by analyzing tangential confocal sections that revealed the cell surface. Fig. 9 B shows a merged image of all time frames for Rvs161-GFP compared with Sur7-mRFP. The overlap with MCC patches is minimal (Fig. 9 B and Fig. S4 A, available at http://www.jcb.org/cgi/content/full/jcb.200806035/DC1). Similarly, mutually exclusive localization of MCC patches and generally accepted early endocytosis markers Ede1 (Fig. 9 C and Fig. S4 B) and Sla2 (not depicted) were observed. This strongly supports the observation that Can1 is more rapidly degraded when it is not confined to MCC, as is the case in the pil1Δ and nce102Δ mutants. This is also consistent with the observation that in comparison to basal turnover in the presence of cycloheximide, Can1 is internalized and degraded considerably faster in response to the addition of 5 mM arginine followed by the rapid spreading of permease (Fig. 8 B).


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)

Sites of classical endocytosis do not colocalize with MCC. (B and C) The plasma membrane distributions of Rvs161 (B) and Ede1 (C), markers of late and early endocytic steps, respectively, were tested for colocalization with the MCC marker Sur7. For comparison, localization of the MCC resident Nce102 was analyzed (A). Tangential confocal sections showing the cell surface are presented. Because of a high mobility of Rvs161 patches, a maximum intensity projection of 36 frames (5 s per frame) instead of a single frame is shown in B. In this arrangement, a higher number of Rvs161 patches could be localized toward the stable Sur7 pattern at the same time. The rate of colocalization was quantified by fluorescence intensity profiles (top diagrams and arrows in merge) and 2D scatter plots of the whole full resolution images (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200806035/DC1). For easy orientation in the scatter plots, real pixel colors were used. Note the diagonal orientation of the Nce102-derived scatter plot demonstrating the colocalization of red and green fluorescence signals and a clear separation of red and green pixels in the two other cases. Examples of Sur7 patches adjacent to endocytic sites are highlighted (arrowheads). Bar, 5 μm.
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fig9: Sites of classical endocytosis do not colocalize with MCC. (B and C) The plasma membrane distributions of Rvs161 (B) and Ede1 (C), markers of late and early endocytic steps, respectively, were tested for colocalization with the MCC marker Sur7. For comparison, localization of the MCC resident Nce102 was analyzed (A). Tangential confocal sections showing the cell surface are presented. Because of a high mobility of Rvs161 patches, a maximum intensity projection of 36 frames (5 s per frame) instead of a single frame is shown in B. In this arrangement, a higher number of Rvs161 patches could be localized toward the stable Sur7 pattern at the same time. The rate of colocalization was quantified by fluorescence intensity profiles (top diagrams and arrows in merge) and 2D scatter plots of the whole full resolution images (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200806035/DC1). For easy orientation in the scatter plots, real pixel colors were used. Note the diagonal orientation of the Nce102-derived scatter plot demonstrating the colocalization of red and green fluorescence signals and a clear separation of red and green pixels in the two other cases. Examples of Sur7 patches adjacent to endocytic sites are highlighted (arrowheads). Bar, 5 μm.
Mentions: We monitored the colocalization of Rvs161-GFP and Sur7-mRFP for 180 s (36 frames; one frame per 5 s). Being aware of the thickness of a confocal section (∼700 nm), artifactual overlaps of the fluorescence signals were avoided by analyzing tangential confocal sections that revealed the cell surface. Fig. 9 B shows a merged image of all time frames for Rvs161-GFP compared with Sur7-mRFP. The overlap with MCC patches is minimal (Fig. 9 B and Fig. S4 A, available at http://www.jcb.org/cgi/content/full/jcb.200806035/DC1). Similarly, mutually exclusive localization of MCC patches and generally accepted early endocytosis markers Ede1 (Fig. 9 C and Fig. S4 B) and Sla2 (not depicted) were observed. This strongly supports the observation that Can1 is more rapidly degraded when it is not confined to MCC, as is the case in the pil1Δ and nce102Δ mutants. This is also consistent with the observation that in comparison to basal turnover in the presence of cycloheximide, Can1 is internalized and degraded considerably faster in response to the addition of 5 mM arginine followed by the rapid spreading of permease (Fig. 8 B).

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