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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

Ub-Fur4-GFP is correctly sorted at the MVB in ubp2Δ mutant cells.pFUR4-GFP or pUb-FUR4-GFP expression plasmids were transformed into WT and ubp2Δ cells. Strains were grown in sucrose, diluted (OD600 = 0.5) in media containing galactose and Fur4 synthesis induced for 4 hours. Transcription was stopped by adding glucose for 1 hour to chase Fur4 fusion reporters to the plasma membrane. Uracil (40 µg/ml) was then added to trigger internalization. GFP signal was viewed by fluorescence confocal microscopy both before (T0) and 60 min after uracil addition. (Note: cells in the left panel were also transformed with a control vector (YEp46Δ) and incubated with 0.1 mM copper sulphate, as the experiment was done in parallel to that shown in Figure 5.)
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pone-0004259-g007: Ub-Fur4-GFP is correctly sorted at the MVB in ubp2Δ mutant cells.pFUR4-GFP or pUb-FUR4-GFP expression plasmids were transformed into WT and ubp2Δ cells. Strains were grown in sucrose, diluted (OD600 = 0.5) in media containing galactose and Fur4 synthesis induced for 4 hours. Transcription was stopped by adding glucose for 1 hour to chase Fur4 fusion reporters to the plasma membrane. Uracil (40 µg/ml) was then added to trigger internalization. GFP signal was viewed by fluorescence confocal microscopy both before (T0) and 60 min after uracil addition. (Note: cells in the left panel were also transformed with a control vector (YEp46Δ) and incubated with 0.1 mM copper sulphate, as the experiment was done in parallel to that shown in Figure 5.)

Mentions: pFUR4-GFP and a plasmid overexpressing ubiquitin (+) or an empty plasmid (−) were transformed into cells. Strains were grown in sucrose, and diluted to OD600 = 0.5 in media containing galactose and 0.1 mM copper sulphate (to overexpress ubiquitin) and grown for 4 hours. Fur4 transcription was stopped by adding glucose for 1 hour to chase Fur4 to the plasma membrane. Uracil (40 µg/ml) was then added to induce Fur4 internalization. The GFP signal was viewed by fluorescence confocal microscopy before (−) and at 60 min after (+) uracil addition. Note that the left panel contains the same images as Figure 7, as the experiments were done in parallel.


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)

Ub-Fur4-GFP is correctly sorted at the MVB in ubp2Δ mutant cells.pFUR4-GFP or pUb-FUR4-GFP expression plasmids were transformed into WT and ubp2Δ cells. Strains were grown in sucrose, diluted (OD600 = 0.5) in media containing galactose and Fur4 synthesis induced for 4 hours. Transcription was stopped by adding glucose for 1 hour to chase Fur4 fusion reporters to the plasma membrane. Uracil (40 µg/ml) was then added to trigger internalization. GFP signal was viewed by fluorescence confocal microscopy both before (T0) and 60 min after uracil addition. (Note: cells in the left panel were also transformed with a control vector (YEp46Δ) and incubated with 0.1 mM copper sulphate, as the experiment was done in parallel to that shown in Figure 5.)
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2626285&req=5

pone-0004259-g007: Ub-Fur4-GFP is correctly sorted at the MVB in ubp2Δ mutant cells.pFUR4-GFP or pUb-FUR4-GFP expression plasmids were transformed into WT and ubp2Δ cells. Strains were grown in sucrose, diluted (OD600 = 0.5) in media containing galactose and Fur4 synthesis induced for 4 hours. Transcription was stopped by adding glucose for 1 hour to chase Fur4 fusion reporters to the plasma membrane. Uracil (40 µg/ml) was then added to trigger internalization. GFP signal was viewed by fluorescence confocal microscopy both before (T0) and 60 min after uracil addition. (Note: cells in the left panel were also transformed with a control vector (YEp46Δ) and incubated with 0.1 mM copper sulphate, as the experiment was done in parallel to that shown in Figure 5.)
Mentions: pFUR4-GFP and a plasmid overexpressing ubiquitin (+) or an empty plasmid (−) were transformed into cells. Strains were grown in sucrose, and diluted to OD600 = 0.5 in media containing galactose and 0.1 mM copper sulphate (to overexpress ubiquitin) and grown for 4 hours. Fur4 transcription was stopped by adding glucose for 1 hour to chase Fur4 to the plasma membrane. Uracil (40 µg/ml) was then added to induce Fur4 internalization. The GFP signal was viewed by fluorescence confocal microscopy before (−) and at 60 min after (+) uracil addition. Note that the left panel contains the same images as Figure 7, as the experiments were done in parallel.

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