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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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Degradation of MCC transporters is accelerated in mutants affected in the domain formation. (A and B) Exponentially growing cultures of wild-type (WT), nce102Δ, and pil1Δ cells expressing Can1-GFP (A) and Fur4-GFP (B) were treated with cycloheximide. At the given time points, total membranes were isolated from the culture aliquots (see Materials and methods). The membrane proteins were resolved by SDS-PAGE, and Can1-GFP and Fur4-GFP were detected by anti-GFP antibody on Western blots. 2.5 μg of the total protein was loaded into each lane. Black lines indicate that intervening lanes have been spliced out.
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fig7: Degradation of MCC transporters is accelerated in mutants affected in the domain formation. (A and B) Exponentially growing cultures of wild-type (WT), nce102Δ, and pil1Δ cells expressing Can1-GFP (A) and Fur4-GFP (B) were treated with cycloheximide. At the given time points, total membranes were isolated from the culture aliquots (see Materials and methods). The membrane proteins were resolved by SDS-PAGE, and Can1-GFP and Fur4-GFP were detected by anti-GFP antibody on Western blots. 2.5 μg of the total protein was loaded into each lane. Black lines indicate that intervening lanes have been spliced out.

Mentions: We checked whether the compartmentation has any effect on protein stability and/or its turnover. Walther et al. (2006) reported a defect in endocytosis of the mating factor receptor Ste3 in the pil1Δ mutant, which was taken as an indication of the role of Pil1 in endocytosis. Because of the fact that Pil1 is located in close vicinity to the patches containing Can1, we also tested whether the endocytosis of arginine permease is dependent on the presence of Pil1, which would point to a more general role of Pil1 in this process. The expression of CAN1 is highly dependent on the growth phase, and its gene product is subjected to continuous turnover. At high extracellular arginine concentration, the permease is endocytosed and subsequently targeted to the vacuole (Opekarová et al., 1998). Its endocytosis follows the classical clathrin–actin-mediated pathway because Can1 turnover is severely inhibited in the sla2/end4 mutant at the nonpermissive temperature (unpublished data). Can1-GFP turnover was monitored in wild-type, nce102Δ, and pil1Δ cells in the presence of cycloheximide. The basal turnover of Can1-GFP was faster in the two mutants (Fig. 7 A); i.e., when it was not localized in the patches of the MCC compartment. This result has also been obtained with another proton symporter accumulated in MCC, the uracil permease Fur4 (Fig. 7 B; Dupre and Haguenauer-Tsapis, 2003; Bultynck et al., 2006). Like Can1, Fur4 is also homogenously distributed in the plasma membrane of nce102Δ and pil1Δ mutants (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200806035/DC1).


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)

Degradation of MCC transporters is accelerated in mutants affected in the domain formation. (A and B) Exponentially growing cultures of wild-type (WT), nce102Δ, and pil1Δ cells expressing Can1-GFP (A) and Fur4-GFP (B) were treated with cycloheximide. At the given time points, total membranes were isolated from the culture aliquots (see Materials and methods). The membrane proteins were resolved by SDS-PAGE, and Can1-GFP and Fur4-GFP were detected by anti-GFP antibody on Western blots. 2.5 μg of the total protein was loaded into each lane. Black lines indicate that intervening lanes have been spliced out.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Degradation of MCC transporters is accelerated in mutants affected in the domain formation. (A and B) Exponentially growing cultures of wild-type (WT), nce102Δ, and pil1Δ cells expressing Can1-GFP (A) and Fur4-GFP (B) were treated with cycloheximide. At the given time points, total membranes were isolated from the culture aliquots (see Materials and methods). The membrane proteins were resolved by SDS-PAGE, and Can1-GFP and Fur4-GFP were detected by anti-GFP antibody on Western blots. 2.5 μg of the total protein was loaded into each lane. Black lines indicate that intervening lanes have been spliced out.
Mentions: We checked whether the compartmentation has any effect on protein stability and/or its turnover. Walther et al. (2006) reported a defect in endocytosis of the mating factor receptor Ste3 in the pil1Δ mutant, which was taken as an indication of the role of Pil1 in endocytosis. Because of the fact that Pil1 is located in close vicinity to the patches containing Can1, we also tested whether the endocytosis of arginine permease is dependent on the presence of Pil1, which would point to a more general role of Pil1 in this process. The expression of CAN1 is highly dependent on the growth phase, and its gene product is subjected to continuous turnover. At high extracellular arginine concentration, the permease is endocytosed and subsequently targeted to the vacuole (Opekarová et al., 1998). Its endocytosis follows the classical clathrin–actin-mediated pathway because Can1 turnover is severely inhibited in the sla2/end4 mutant at the nonpermissive temperature (unpublished data). Can1-GFP turnover was monitored in wild-type, nce102Δ, and pil1Δ cells in the presence of cycloheximide. The basal turnover of Can1-GFP was faster in the two mutants (Fig. 7 A); i.e., when it was not localized in the patches of the MCC compartment. This result has also been obtained with another proton symporter accumulated in MCC, the uracil permease Fur4 (Fig. 7 B; Dupre and Haguenauer-Tsapis, 2003; Bultynck et al., 2006). Like Can1, Fur4 is also homogenously distributed in the plasma membrane of nce102Δ and pil1Δ mutants (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200806035/DC1).

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