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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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Extractability of the transport proteins Can1 and Gap1 by Triton X-100. Membranes were isolated from exponentially growing cells as described in Materials and methods. Aliquots corresponding to 50 μg of membrane protein were treated with increasing concentrations of Triton X-100. The nonsolubilized proteins were resolved by SDS-PAGE and detected by specific antibodies on Western blots. The figure is representative of three independent experiments. WT, wild type.
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fig3: Extractability of the transport proteins Can1 and Gap1 by Triton X-100. Membranes were isolated from exponentially growing cells as described in Materials and methods. Aliquots corresponding to 50 μg of membrane protein were treated with increasing concentrations of Triton X-100. The nonsolubilized proteins were resolved by SDS-PAGE and detected by specific antibodies on Western blots. The figure is representative of three independent experiments. WT, wild type.

Mentions: Gene ontology term analysis revealed that proteins involved in vesicle-mediated transport (9/28 strains showing altered compartmentation [32%]; background frequency of 4.9%; p-value of 4.7 × 10−4) and lipid biosynthesis (8/28 [27%]; background of 1.5%; p-value of 5.0 × 10−7) were significantly overrepresented among the genes detected in the screen. This strongly suggests that lipids and the lipid composition of the plasma membrane play a major role in lateral compartmentation. To test whether the immediate lipid milieu of Can1 is changed in the mutants exhibiting an altered distribution, we checked whether Can1 is more accessible to increasing concentrations of Triton X-100. As shown in Fig. 3, Can1-GFP solubilized with lower concentrations of detergent in the mutants as compared with the wild type. This agrees with the behavior of Can1 after treating the cells with uncouplers; the protein disperses (Grossmann et al., 2007), and, at the same time, it is more efficiently extractable by Triton X-100 (Fig. 3). Thus, the transporters appear to be recruited to a preexisting core MCC compartment with a specific lipid composition. As a control, we tested the Triton X-100 extractability of Gap1, a protein that is homogeneously distributed in wild-type cells (Lauwers et al., 2007). The data show that there is no difference in the extractability between the wild-type and the Nce102 and Pil1 deletion mutants (Fig. 3).


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)

Extractability of the transport proteins Can1 and Gap1 by Triton X-100. Membranes were isolated from exponentially growing cells as described in Materials and methods. Aliquots corresponding to 50 μg of membrane protein were treated with increasing concentrations of Triton X-100. The nonsolubilized proteins were resolved by SDS-PAGE and detected by specific antibodies on Western blots. The figure is representative of three independent experiments. WT, wild type.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2600745&req=5

fig3: Extractability of the transport proteins Can1 and Gap1 by Triton X-100. Membranes were isolated from exponentially growing cells as described in Materials and methods. Aliquots corresponding to 50 μg of membrane protein were treated with increasing concentrations of Triton X-100. The nonsolubilized proteins were resolved by SDS-PAGE and detected by specific antibodies on Western blots. The figure is representative of three independent experiments. WT, wild type.
Mentions: Gene ontology term analysis revealed that proteins involved in vesicle-mediated transport (9/28 strains showing altered compartmentation [32%]; background frequency of 4.9%; p-value of 4.7 × 10−4) and lipid biosynthesis (8/28 [27%]; background of 1.5%; p-value of 5.0 × 10−7) were significantly overrepresented among the genes detected in the screen. This strongly suggests that lipids and the lipid composition of the plasma membrane play a major role in lateral compartmentation. To test whether the immediate lipid milieu of Can1 is changed in the mutants exhibiting an altered distribution, we checked whether Can1 is more accessible to increasing concentrations of Triton X-100. As shown in Fig. 3, Can1-GFP solubilized with lower concentrations of detergent in the mutants as compared with the wild type. This agrees with the behavior of Can1 after treating the cells with uncouplers; the protein disperses (Grossmann et al., 2007), and, at the same time, it is more efficiently extractable by Triton X-100 (Fig. 3). Thus, the transporters appear to be recruited to a preexisting core MCC compartment with a specific lipid composition. As a control, we tested the Triton X-100 extractability of Gap1, a protein that is homogeneously distributed in wild-type cells (Lauwers et al., 2007). The data show that there is no difference in the extractability between the wild-type and the Nce102 and Pil1 deletion mutants (Fig. 3).

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