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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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Nce102 is homogenously distributed in membranes of buds and shmoos. (A) Development of the membrane distribution of Nce102-GFP, Sur7-GFP, and Pil1-GFP in mother cells and buds of increasing size (I–IV). (B) Localization of the proteins examined in A and Can1-GFP in cells (genetic background: BY4741 MATa; OD600 = 0.25) treated with 30 μg/ml α factor for 2 h. 3D reconstructions of confocal z stacks are presented. Bars, 2 μm.
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fig5: Nce102 is homogenously distributed in membranes of buds and shmoos. (A) Development of the membrane distribution of Nce102-GFP, Sur7-GFP, and Pil1-GFP in mother cells and buds of increasing size (I–IV). (B) Localization of the proteins examined in A and Can1-GFP in cells (genetic background: BY4741 MATa; OD600 = 0.25) treated with 30 μg/ml α factor for 2 h. 3D reconstructions of confocal z stacks are presented. Bars, 2 μm.

Mentions: As shown in Fig. 1, Nce102 clearly localizes to the MCC of mother cells. However, in young buds, Nce102-GFP appears to be distributed homogenously (Fig. 5 A). Only after the bud diameter reaches about one third of the mother (Fig. 5 A, state III) do discernible patches become apparent. In contrast, Sur7-GFP and Pil1 are organized in patches in the buds from the time of their emergence (Fig. 5 A). One of the main differences between young bud and mother cell membranes is the rate of growth, which is determined by the arrival of secretory cargo. A similar situation exists in growing shmoos, the mating projections induced by mating factors. In agreement with this similarity between shmoos and buds, we observed a homogeneous distribution of Nce102-GFP and patches of Sur7-GFP and Pil1-GFP in shmoos (Fig. 5 B). Interestingly, Can1-GFP, which is hard to visualize in young buds, can be seen in shmoos, and its distribution correlates with that of Nce102-GFP. These data implicate an interdependence of Can1 and Nce102. However, these two proteins can be separated from each other by membrane depolarization. As reported previously, Can1 patches can be dissipated by the addition of FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), a potent protonophore (Grossmann et al., 2007). The addition of FCCP to cells expressing Can1-GFP and Nce102-mRFP causes dissipation of the Can1 patches only, whereas Nce102 remains in the MCC (Fig. 6). This indicates that the interaction between Nce102 and Can1 is mainly stabilized by electrostatic forces. Thus, Nce102 behaves similarly to Sur7, which has previously been shown to not respond to uncouplers either (Grossmann et al., 2007).


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)

Nce102 is homogenously distributed in membranes of buds and shmoos. (A) Development of the membrane distribution of Nce102-GFP, Sur7-GFP, and Pil1-GFP in mother cells and buds of increasing size (I–IV). (B) Localization of the proteins examined in A and Can1-GFP in cells (genetic background: BY4741 MATa; OD600 = 0.25) treated with 30 μg/ml α factor for 2 h. 3D reconstructions of confocal z stacks are presented. Bars, 2 μm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2600745&req=5

fig5: Nce102 is homogenously distributed in membranes of buds and shmoos. (A) Development of the membrane distribution of Nce102-GFP, Sur7-GFP, and Pil1-GFP in mother cells and buds of increasing size (I–IV). (B) Localization of the proteins examined in A and Can1-GFP in cells (genetic background: BY4741 MATa; OD600 = 0.25) treated with 30 μg/ml α factor for 2 h. 3D reconstructions of confocal z stacks are presented. Bars, 2 μm.
Mentions: As shown in Fig. 1, Nce102 clearly localizes to the MCC of mother cells. However, in young buds, Nce102-GFP appears to be distributed homogenously (Fig. 5 A). Only after the bud diameter reaches about one third of the mother (Fig. 5 A, state III) do discernible patches become apparent. In contrast, Sur7-GFP and Pil1 are organized in patches in the buds from the time of their emergence (Fig. 5 A). One of the main differences between young bud and mother cell membranes is the rate of growth, which is determined by the arrival of secretory cargo. A similar situation exists in growing shmoos, the mating projections induced by mating factors. In agreement with this similarity between shmoos and buds, we observed a homogeneous distribution of Nce102-GFP and patches of Sur7-GFP and Pil1-GFP in shmoos (Fig. 5 B). Interestingly, Can1-GFP, which is hard to visualize in young buds, can be seen in shmoos, and its distribution correlates with that of Nce102-GFP. These data implicate an interdependence of Can1 and Nce102. However, these two proteins can be separated from each other by membrane depolarization. As reported previously, Can1 patches can be dissipated by the addition of FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), a potent protonophore (Grossmann et al., 2007). The addition of FCCP to cells expressing Can1-GFP and Nce102-mRFP causes dissipation of the Can1 patches only, whereas Nce102 remains in the MCC (Fig. 6). This indicates that the interaction between Nce102 and Can1 is mainly stabilized by electrostatic forces. Thus, Nce102 behaves similarly to Sur7, which has previously been shown to not respond to uncouplers either (Grossmann et al., 2007).

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