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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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Uracil-induced Fur4 sorting is perturbed in ubp2Δ mutant cells.A FUR4-GFP fusion reporter was transformed into cells, which were then grown in raffinose overnight. Galactose was added (OD600 = 0.6) for 2 hours to induce synthesis, followed by glucose for 10 min to chase Fur4-GFP to the plasma membrane, and then uracil (40 µg/ml) to trigger transporter internalization. (A) Fur4-GFP signal viewed by fluorescence microscopy at the indicated time points (min) after uracil addition. T0 indicates cells immediately before addition. (B) Fur4-GFP protein levels as measured by Western blotting. Total protein extracts were generated from cells harvested at the indicated time points following uracil addition. Fur4-GFP was probed with anti-GFP antibodies. 3-phosphoglycerate kinase (PGK) was used as a loading control. The asterisk indicates an unknown background band.
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pone-0004259-g004: Uracil-induced Fur4 sorting is perturbed in ubp2Δ mutant cells.A FUR4-GFP fusion reporter was transformed into cells, which were then grown in raffinose overnight. Galactose was added (OD600 = 0.6) for 2 hours to induce synthesis, followed by glucose for 10 min to chase Fur4-GFP to the plasma membrane, and then uracil (40 µg/ml) to trigger transporter internalization. (A) Fur4-GFP signal viewed by fluorescence microscopy at the indicated time points (min) after uracil addition. T0 indicates cells immediately before addition. (B) Fur4-GFP protein levels as measured by Western blotting. Total protein extracts were generated from cells harvested at the indicated time points following uracil addition. Fur4-GFP was probed with anti-GFP antibodies. 3-phosphoglycerate kinase (PGK) was used as a loading control. The asterisk indicates an unknown background band.

Mentions: In order to define whether the 5-FU sensitivity of ubp2Δ cells indeed comes from defects in trafficking, or merely from impaired RNA metabolism [40], we followed the fate of a GFP-tagged version of Fur4 in ubp2Δ and rup1Δ cells, using as control rsp5 mutant cells (Figure 4, A and B). A galactose-inducible version of Fur4-GFP was similarly targeted to the plasma membrane after galactose induction in all the strains. Glucose was then added to stop Fur4-GFP synthesis and chase to the plasma membrane any Fur4-GFP still in the secretory pathway. Uracil was then added to trigger Fur4-GFP endocytosis. Cells were harvested at various time points after the addition of uracil, and subjected to whole-cell imaging (Figure 4A). Total protein extracts were also prepared in parallel for Western blotting against Fur4-GFP in the case of wildtype and ubp2Δ cells (Figure 4B). Uracil triggered a progressive loss of plasma membrane GFP fluorescence in wildtype cells, a transient apparition of punctuate intracellular fluorescent dots (likely endosomes), followed by apparition of luminal vacuolar fluorescence (corresponding to free GFP, not immediately degraded by vacuolar proteases). Fur4-GFP displayed almost the same fate in rup1Δ cells. In contrast, in rsp5-1 cells, even at a permissive temperature, plasma membrane fluorescence was still detectable after two hours of uracil treatment, together with very faint vacuolar fluorescence. In ubp2Δ cells, the situation was intermediate between that observed in wildtype and rsp5 cells. Plasma membrane staining was still observed after 90 min of uracil treatment, and after two hours, both plasma membrane and vacuolar fluorescence were evidenced. In agreement with this observation, Fur4-GFP degradation was delayed in ubp2Δ compared to wildtype cells (Figure 4B).


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)

Uracil-induced Fur4 sorting is perturbed in ubp2Δ mutant cells.A FUR4-GFP fusion reporter was transformed into cells, which were then grown in raffinose overnight. Galactose was added (OD600 = 0.6) for 2 hours to induce synthesis, followed by glucose for 10 min to chase Fur4-GFP to the plasma membrane, and then uracil (40 µg/ml) to trigger transporter internalization. (A) Fur4-GFP signal viewed by fluorescence microscopy at the indicated time points (min) after uracil addition. T0 indicates cells immediately before addition. (B) Fur4-GFP protein levels as measured by Western blotting. Total protein extracts were generated from cells harvested at the indicated time points following uracil addition. Fur4-GFP was probed with anti-GFP antibodies. 3-phosphoglycerate kinase (PGK) was used as a loading control. The asterisk indicates an unknown background band.
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Related In: Results  -  Collection

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pone-0004259-g004: Uracil-induced Fur4 sorting is perturbed in ubp2Δ mutant cells.A FUR4-GFP fusion reporter was transformed into cells, which were then grown in raffinose overnight. Galactose was added (OD600 = 0.6) for 2 hours to induce synthesis, followed by glucose for 10 min to chase Fur4-GFP to the plasma membrane, and then uracil (40 µg/ml) to trigger transporter internalization. (A) Fur4-GFP signal viewed by fluorescence microscopy at the indicated time points (min) after uracil addition. T0 indicates cells immediately before addition. (B) Fur4-GFP protein levels as measured by Western blotting. Total protein extracts were generated from cells harvested at the indicated time points following uracil addition. Fur4-GFP was probed with anti-GFP antibodies. 3-phosphoglycerate kinase (PGK) was used as a loading control. The asterisk indicates an unknown background band.
Mentions: In order to define whether the 5-FU sensitivity of ubp2Δ cells indeed comes from defects in trafficking, or merely from impaired RNA metabolism [40], we followed the fate of a GFP-tagged version of Fur4 in ubp2Δ and rup1Δ cells, using as control rsp5 mutant cells (Figure 4, A and B). A galactose-inducible version of Fur4-GFP was similarly targeted to the plasma membrane after galactose induction in all the strains. Glucose was then added to stop Fur4-GFP synthesis and chase to the plasma membrane any Fur4-GFP still in the secretory pathway. Uracil was then added to trigger Fur4-GFP endocytosis. Cells were harvested at various time points after the addition of uracil, and subjected to whole-cell imaging (Figure 4A). Total protein extracts were also prepared in parallel for Western blotting against Fur4-GFP in the case of wildtype and ubp2Δ cells (Figure 4B). Uracil triggered a progressive loss of plasma membrane GFP fluorescence in wildtype cells, a transient apparition of punctuate intracellular fluorescent dots (likely endosomes), followed by apparition of luminal vacuolar fluorescence (corresponding to free GFP, not immediately degraded by vacuolar proteases). Fur4-GFP displayed almost the same fate in rup1Δ cells. In contrast, in rsp5-1 cells, even at a permissive temperature, plasma membrane fluorescence was still detectable after two hours of uracil treatment, together with very faint vacuolar fluorescence. In ubp2Δ cells, the situation was intermediate between that observed in wildtype and rsp5 cells. Plasma membrane staining was still observed after 90 min of uracil treatment, and after two hours, both plasma membrane and vacuolar fluorescence were evidenced. In agreement with this observation, Fur4-GFP degradation was delayed in ubp2Δ compared to wildtype cells (Figure 4B).

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