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Interaction of the deubiquitinating enzyme Ubp2 and the e3 ligase Rsp5 is required for transporter/receptor sorting in the multivesicular body pathway.

Lam MH, Urban-Grimal D, Bugnicourt A, Greenblatt JF, Haguenauer-Tsapis R, Emili A - PLoS ONE (2009)

Bottom Line: We sought to elucidate the possible involvement of the S. cerevisiae deubiquitinating enzyme Ubp2 in transporter and receptor trafficking after we (this study) and others established that affinity purified Ubp2 interacts stably with the E3 ubiquitin ligase Rsp5 and the (ubiquitin associated) UBA domain containing protein Rup1.Moreover, the defect was absent in conditions where recycling was absent, implicating Ubp2 in sorting at the multivesicular body.Taken together, our data suggest a previously overlooked role for Ubp2 as a positive regulator of Rsp5-mediated membrane protein trafficking subsequent to endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Banting and Best Department of Medical Research, Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT
Protein ubiquitination is essential for many events linked to intracellular protein trafficking. We sought to elucidate the possible involvement of the S. cerevisiae deubiquitinating enzyme Ubp2 in transporter and receptor trafficking after we (this study) and others established that affinity purified Ubp2 interacts stably with the E3 ubiquitin ligase Rsp5 and the (ubiquitin associated) UBA domain containing protein Rup1. UBP2 interacts genetically with RSP5, while Rup1 facilitates the tethering of Ubp2 to Rsp5 via a PPPSY motif. Using the uracil permease Fur4 as a model reporter system, we establish a role for Ubp2 in membrane protein turnover. Similar to hypomorphic rsp5 alleles, cells deleted for UBP2 exhibited a temporal stabilization of Fur4 at the plasma membrane, indicative of perturbed protein trafficking. This defect was ubiquitin dependent, as a Fur4 N-terminal ubiquitin fusion construct bypassed the block and restored sorting in the mutant. Moreover, the defect was absent in conditions where recycling was absent, implicating Ubp2 in sorting at the multivesicular body. Taken together, our data suggest a previously overlooked role for Ubp2 as a positive regulator of Rsp5-mediated membrane protein trafficking subsequent to endocytosis.

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Related in: MedlinePlus

UBP2 is not necessary for cycloheximide (CHX) triggered internalization of Fur4.(A) pFUR4-GFP bearing cells were grown in raffinose overnight. Galactose was added for 2 hours to induce synthesis, and glucose was then added for 10 min to chase Fur4-GFP to the plasma membrane. CHX (0.1mg/ml) was added and GFP signal examined by fluorescence microscopy and Nomarski optics. Time refers to the time after addition of CHX, with 0 min as pre-induction. (B) Uracil uptake was measured at different times after the addition of CHX in WT and ubp2Δ strains. Results are expressed as a percentage of the initial uracil uptake, and plotted on a log scale. (C) Fur4 ubiquitin profile at the plasma membrane is unchanged in ubp2Δ mutants. pFUR4 was transformed into WT, ubp2Δ, rup1Δ, and rsp5-1, and cells, which were grown in raffinose overnight. Expression from pFUR4 was induced for 90 min with galactose before adding glucose for 15min to chase Fur4 to the plasma membrane. Total protein extracts (lysate) and enriched membrane fractions were collected and analyzed by Western blotting to visualize Fur4 and Fur4-ubiquitin conjugates. 3-phosphoglycerate kinase (PGK) and porin, a mitochondrial membrane protein, were used as loading controls.
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pone-0004259-g006: UBP2 is not necessary for cycloheximide (CHX) triggered internalization of Fur4.(A) pFUR4-GFP bearing cells were grown in raffinose overnight. Galactose was added for 2 hours to induce synthesis, and glucose was then added for 10 min to chase Fur4-GFP to the plasma membrane. CHX (0.1mg/ml) was added and GFP signal examined by fluorescence microscopy and Nomarski optics. Time refers to the time after addition of CHX, with 0 min as pre-induction. (B) Uracil uptake was measured at different times after the addition of CHX in WT and ubp2Δ strains. Results are expressed as a percentage of the initial uracil uptake, and plotted on a log scale. (C) Fur4 ubiquitin profile at the plasma membrane is unchanged in ubp2Δ mutants. pFUR4 was transformed into WT, ubp2Δ, rup1Δ, and rsp5-1, and cells, which were grown in raffinose overnight. Expression from pFUR4 was induced for 90 min with galactose before adding glucose for 15min to chase Fur4 to the plasma membrane. Total protein extracts (lysate) and enriched membrane fractions were collected and analyzed by Western blotting to visualize Fur4 and Fur4-ubiquitin conjugates. 3-phosphoglycerate kinase (PGK) and porin, a mitochondrial membrane protein, were used as loading controls.

Mentions: After two hours of galactose induction of Fur4-GFP followed by glucose, CHX was added to wildtype and ubp2Δ cells. The fate of Fur4-GFP was then followed by fluorescence imaging and uracil uptake measurement (Figure 6, A and B). Plasma membrane fluorescence disappeared in a similar way in wildtype and ubp2Δ cells (Figure 6A), and the loss of uracil uptake occurred with identical rate in both type of cells (Figure 6B). Endocytic internalization of Fur4 is thus normal in ubp2Δ cells. In agreement with the observation of a normal rate of internalization, the pattern of ubiquitination (regularly spaced Fur4 species evidenced above the main Fur4-GFP signal) on Western blots of plasma membrane enriched fractions was identical in wildtype and ubp2Δ cells (Figure 6C). This is in comparison to rsp5-1 cells, which showed a distinct lack of Fur4-ubiquitin conjugates as expected. This data implies that Ubp2 is likely not involved in the maintenance or deubiquitination of Fur4 at the plasma membrane.


Interaction of the deubiquitinating enzyme Ubp2 and the e3 ligase Rsp5 is required for transporter/receptor sorting in the multivesicular body pathway.

Lam MH, Urban-Grimal D, Bugnicourt A, Greenblatt JF, Haguenauer-Tsapis R, Emili A - PLoS ONE (2009)

UBP2 is not necessary for cycloheximide (CHX) triggered internalization of Fur4.(A) pFUR4-GFP bearing cells were grown in raffinose overnight. Galactose was added for 2 hours to induce synthesis, and glucose was then added for 10 min to chase Fur4-GFP to the plasma membrane. CHX (0.1mg/ml) was added and GFP signal examined by fluorescence microscopy and Nomarski optics. Time refers to the time after addition of CHX, with 0 min as pre-induction. (B) Uracil uptake was measured at different times after the addition of CHX in WT and ubp2Δ strains. Results are expressed as a percentage of the initial uracil uptake, and plotted on a log scale. (C) Fur4 ubiquitin profile at the plasma membrane is unchanged in ubp2Δ mutants. pFUR4 was transformed into WT, ubp2Δ, rup1Δ, and rsp5-1, and cells, which were grown in raffinose overnight. Expression from pFUR4 was induced for 90 min with galactose before adding glucose for 15min to chase Fur4 to the plasma membrane. Total protein extracts (lysate) and enriched membrane fractions were collected and analyzed by Western blotting to visualize Fur4 and Fur4-ubiquitin conjugates. 3-phosphoglycerate kinase (PGK) and porin, a mitochondrial membrane protein, were used as loading controls.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2626285&req=5

pone-0004259-g006: UBP2 is not necessary for cycloheximide (CHX) triggered internalization of Fur4.(A) pFUR4-GFP bearing cells were grown in raffinose overnight. Galactose was added for 2 hours to induce synthesis, and glucose was then added for 10 min to chase Fur4-GFP to the plasma membrane. CHX (0.1mg/ml) was added and GFP signal examined by fluorescence microscopy and Nomarski optics. Time refers to the time after addition of CHX, with 0 min as pre-induction. (B) Uracil uptake was measured at different times after the addition of CHX in WT and ubp2Δ strains. Results are expressed as a percentage of the initial uracil uptake, and plotted on a log scale. (C) Fur4 ubiquitin profile at the plasma membrane is unchanged in ubp2Δ mutants. pFUR4 was transformed into WT, ubp2Δ, rup1Δ, and rsp5-1, and cells, which were grown in raffinose overnight. Expression from pFUR4 was induced for 90 min with galactose before adding glucose for 15min to chase Fur4 to the plasma membrane. Total protein extracts (lysate) and enriched membrane fractions were collected and analyzed by Western blotting to visualize Fur4 and Fur4-ubiquitin conjugates. 3-phosphoglycerate kinase (PGK) and porin, a mitochondrial membrane protein, were used as loading controls.
Mentions: After two hours of galactose induction of Fur4-GFP followed by glucose, CHX was added to wildtype and ubp2Δ cells. The fate of Fur4-GFP was then followed by fluorescence imaging and uracil uptake measurement (Figure 6, A and B). Plasma membrane fluorescence disappeared in a similar way in wildtype and ubp2Δ cells (Figure 6A), and the loss of uracil uptake occurred with identical rate in both type of cells (Figure 6B). Endocytic internalization of Fur4 is thus normal in ubp2Δ cells. In agreement with the observation of a normal rate of internalization, the pattern of ubiquitination (regularly spaced Fur4 species evidenced above the main Fur4-GFP signal) on Western blots of plasma membrane enriched fractions was identical in wildtype and ubp2Δ cells (Figure 6C). This is in comparison to rsp5-1 cells, which showed a distinct lack of Fur4-ubiquitin conjugates as expected. This data implies that Ubp2 is likely not involved in the maintenance or deubiquitination of Fur4 at the plasma membrane.

Bottom Line: We sought to elucidate the possible involvement of the S. cerevisiae deubiquitinating enzyme Ubp2 in transporter and receptor trafficking after we (this study) and others established that affinity purified Ubp2 interacts stably with the E3 ubiquitin ligase Rsp5 and the (ubiquitin associated) UBA domain containing protein Rup1.Moreover, the defect was absent in conditions where recycling was absent, implicating Ubp2 in sorting at the multivesicular body.Taken together, our data suggest a previously overlooked role for Ubp2 as a positive regulator of Rsp5-mediated membrane protein trafficking subsequent to endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Banting and Best Department of Medical Research, Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT
Protein ubiquitination is essential for many events linked to intracellular protein trafficking. We sought to elucidate the possible involvement of the S. cerevisiae deubiquitinating enzyme Ubp2 in transporter and receptor trafficking after we (this study) and others established that affinity purified Ubp2 interacts stably with the E3 ubiquitin ligase Rsp5 and the (ubiquitin associated) UBA domain containing protein Rup1. UBP2 interacts genetically with RSP5, while Rup1 facilitates the tethering of Ubp2 to Rsp5 via a PPPSY motif. Using the uracil permease Fur4 as a model reporter system, we establish a role for Ubp2 in membrane protein turnover. Similar to hypomorphic rsp5 alleles, cells deleted for UBP2 exhibited a temporal stabilization of Fur4 at the plasma membrane, indicative of perturbed protein trafficking. This defect was ubiquitin dependent, as a Fur4 N-terminal ubiquitin fusion construct bypassed the block and restored sorting in the mutant. Moreover, the defect was absent in conditions where recycling was absent, implicating Ubp2 in sorting at the multivesicular body. Taken together, our data suggest a previously overlooked role for Ubp2 as a positive regulator of Rsp5-mediated membrane protein trafficking subsequent to endocytosis.

Show MeSH
Related in: MedlinePlus