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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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Can1 is released from MCC patches before endocytosis. (A) Can1-GFP was localized in the wild-type (WT), nce102Δ, and pil1Δ cells before (top) and 90 min after the addition of 5 mM arginine (middle). Arginine-induced loss of patchy Can1-GFP pattern on the surface confocal sections (left) and the amount of the internalized protein on transversal sections could be easily followed. Note the significantly more intensive vacuolar staining in the mutants lacking the MCC patches as compared with wild type. The whole experiment is documented in Fig. S3 (available at http://www.jcb.org/cgi/content/full/jcb.200806035/DC1). (B) Extractability of Can1-GFP in Triton X-100 was detected in the membranes of wild-type cells before and 10 min after the addition of 5 mM arginine. Anti-GFP antibody was used for the detection of the protein on Western blots. Bar, 5 μm.
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fig8: Can1 is released from MCC patches before endocytosis. (A) Can1-GFP was localized in the wild-type (WT), nce102Δ, and pil1Δ cells before (top) and 90 min after the addition of 5 mM arginine (middle). Arginine-induced loss of patchy Can1-GFP pattern on the surface confocal sections (left) and the amount of the internalized protein on transversal sections could be easily followed. Note the significantly more intensive vacuolar staining in the mutants lacking the MCC patches as compared with wild type. The whole experiment is documented in Fig. S3 (available at http://www.jcb.org/cgi/content/full/jcb.200806035/DC1). (B) Extractability of Can1-GFP in Triton X-100 was detected in the membranes of wild-type cells before and 10 min after the addition of 5 mM arginine. Anti-GFP antibody was used for the detection of the protein on Western blots. Bar, 5 μm.

Mentions: Similar although more rapid effects were observed when the fluorescence of cells expressing Can1-GFP was followed after the addition of 5 mM arginine (Fig. 8 A and Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200806035/DC1). Also, Can1-GFP was internalized faster in the two mutants as compared with the wild-type cells. After 90 min of incubation, stronger vacuolar staining is apparent in the mutants as compared with the wild type. Furthermore, we observed that in the wild-type cells, the Can1-GFP pattern becomes more dispersed upon the addition of arginine (Fig. 8 A, WT surface). In the presence of its abundant substrate, the transporter's distribution is similar to that observed in the mutants from our screen (i.e., Can1 is dissipated from the patches to the surrounding area). Accordingly, the Triton X-100 extractability of Can1-GFP from membranes of cells treated with 5 mM arginine for 10 min is changed (Fig. 8 B) and is comparable with that found in the membranes from the two mutants (Fig. 3). However, the addition of cycloheximide does not cause a spreading of Can1-GFP (unpublished data). Induced spreading of a transport protein was observed previously for HUP1-GFP; patchy accumulation of this transporter was more distinct in a low glucose medium (Grossmann et al., 2006).


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)

Can1 is released from MCC patches before endocytosis. (A) Can1-GFP was localized in the wild-type (WT), nce102Δ, and pil1Δ cells before (top) and 90 min after the addition of 5 mM arginine (middle). Arginine-induced loss of patchy Can1-GFP pattern on the surface confocal sections (left) and the amount of the internalized protein on transversal sections could be easily followed. Note the significantly more intensive vacuolar staining in the mutants lacking the MCC patches as compared with wild type. The whole experiment is documented in Fig. S3 (available at http://www.jcb.org/cgi/content/full/jcb.200806035/DC1). (B) Extractability of Can1-GFP in Triton X-100 was detected in the membranes of wild-type cells before and 10 min after the addition of 5 mM arginine. Anti-GFP antibody was used for the detection of the protein on Western blots. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Can1 is released from MCC patches before endocytosis. (A) Can1-GFP was localized in the wild-type (WT), nce102Δ, and pil1Δ cells before (top) and 90 min after the addition of 5 mM arginine (middle). Arginine-induced loss of patchy Can1-GFP pattern on the surface confocal sections (left) and the amount of the internalized protein on transversal sections could be easily followed. Note the significantly more intensive vacuolar staining in the mutants lacking the MCC patches as compared with wild type. The whole experiment is documented in Fig. S3 (available at http://www.jcb.org/cgi/content/full/jcb.200806035/DC1). (B) Extractability of Can1-GFP in Triton X-100 was detected in the membranes of wild-type cells before and 10 min after the addition of 5 mM arginine. Anti-GFP antibody was used for the detection of the protein on Western blots. Bar, 5 μm.
Mentions: Similar although more rapid effects were observed when the fluorescence of cells expressing Can1-GFP was followed after the addition of 5 mM arginine (Fig. 8 A and Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200806035/DC1). Also, Can1-GFP was internalized faster in the two mutants as compared with the wild-type cells. After 90 min of incubation, stronger vacuolar staining is apparent in the mutants as compared with the wild type. Furthermore, we observed that in the wild-type cells, the Can1-GFP pattern becomes more dispersed upon the addition of arginine (Fig. 8 A, WT surface). In the presence of its abundant substrate, the transporter's distribution is similar to that observed in the mutants from our screen (i.e., Can1 is dissipated from the patches to the surrounding area). Accordingly, the Triton X-100 extractability of Can1-GFP from membranes of cells treated with 5 mM arginine for 10 min is changed (Fig. 8 B) and is comparable with that found in the membranes from the two mutants (Fig. 3). However, the addition of cycloheximide does not cause a spreading of Can1-GFP (unpublished data). Induced spreading of a transport protein was observed previously for HUP1-GFP; patchy accumulation of this transporter was more distinct in a low glucose medium (Grossmann et al., 2006).

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