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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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Ubp2, Rsp5, and Rup1 interact physically.(A) Silver-stained SDS polyacrylamide gel showing affinity purified Ubp2-TAP and Rup1-TAP protein complexes. Arrows indicate polypeptides identified by gel band excision followed by MALDI-ToF mass spectrometry. The asterisk indicates degradation products of Ubp2 and Rup1. (B) Ubp2 recovery from Ubp2-TAP, Ubp2-TAP rup1Δ, and Rup1-TAP strains. Arrow indicates gel band identified as Ubp2 by MALDI-ToF mass spectrometry. (C) Co-immunoprecipitation of a fraction of cellular Rsp5 with Ubp2 and Rup1. Cell extracts were depleted of endogenously TAP-tagged Ubp2 or Rup1 with IgG (IP lanes), and the remaining soluble Rsp5 (post-IP lanes) and bait proteins probed by Western blot with anti-Rsp5 antibodies which also recognized the protein A portion of the TAP tag. The ‘input’ lane shows the Rsp5 level in the extract prior to IP. (D) Rup1 tethers Ubp2 to Rsp5. IgG-based immunoprecipitation of Rup1-TAP and Ubp2-TAP from WT, ubp2Δ or rup1Δ strains. The co-purification of Rsp5 with the baits was determined by Western blotting using anti-Rsp5 antibodies. (E) Plasmids expressing FLAG(F/H)-tagged wildtype Rup1 or the Y135F point mutant were transformed into cells lacking endogenous Rup1. After immunoprecipitation with anti-FLAG antibodies, Rup1 and Rsp5 were detected by Western blotting. The bottom panel show the expression levels of Rsp5 in whole cell extracts by Western blotting against Rsp5.
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pone-0004259-g001: Ubp2, Rsp5, and Rup1 interact physically.(A) Silver-stained SDS polyacrylamide gel showing affinity purified Ubp2-TAP and Rup1-TAP protein complexes. Arrows indicate polypeptides identified by gel band excision followed by MALDI-ToF mass spectrometry. The asterisk indicates degradation products of Ubp2 and Rup1. (B) Ubp2 recovery from Ubp2-TAP, Ubp2-TAP rup1Δ, and Rup1-TAP strains. Arrow indicates gel band identified as Ubp2 by MALDI-ToF mass spectrometry. (C) Co-immunoprecipitation of a fraction of cellular Rsp5 with Ubp2 and Rup1. Cell extracts were depleted of endogenously TAP-tagged Ubp2 or Rup1 with IgG (IP lanes), and the remaining soluble Rsp5 (post-IP lanes) and bait proteins probed by Western blot with anti-Rsp5 antibodies which also recognized the protein A portion of the TAP tag. The ‘input’ lane shows the Rsp5 level in the extract prior to IP. (D) Rup1 tethers Ubp2 to Rsp5. IgG-based immunoprecipitation of Rup1-TAP and Ubp2-TAP from WT, ubp2Δ or rup1Δ strains. The co-purification of Rsp5 with the baits was determined by Western blotting using anti-Rsp5 antibodies. (E) Plasmids expressing FLAG(F/H)-tagged wildtype Rup1 or the Y135F point mutant were transformed into cells lacking endogenous Rup1. After immunoprecipitation with anti-FLAG antibodies, Rup1 and Rsp5 were detected by Western blotting. The bottom panel show the expression levels of Rsp5 in whole cell extracts by Western blotting against Rsp5.

Mentions: Neither Ubp2 or Rup1 are essential for normal cell growth and deletion of either gene does not result in any obvious growth defects or overt morphological phenotypes [15], [25]. Hence, to investigate the possible role(s) of Ubp2 in yeast, we performed large-scale tandem affinity purifications (TAP) to isolate endogenous Ubp2 from S. cerevisiae strains bearing a C-terminal TAP tag [26]. Stably interacting partner proteins were resolved by SDS-PAGE, stained with silver, and identified by peptide mass fingerprinting using MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) mass spectrometry. Two polypeptides, encoding the E3 ubiquitin ligase Rsp5, and a protein of previously unknown function, YOR138c, which we have named Rup1 (Rsp5-Ubp2 interacting Protein 1), were found to reproducibly and specifically co-purify with Ubp2 (Figure 1A). Neither of these binding partners were detected in parallel purifications from an untagged negative control strain or from several hundred other TAP-tagged bait proteins [27]. These data were independently confirmed by highly-sensitive gel-free tandem mass spectrometry-based peptide shotgun sequencing (LC-MS/MS) of an aliquot of each protein mixture after in solution digestion with trypsin (http://tap.med.utoronto.ca/). Conversely, no known substrate or other previously reported Rsp5-interacting proteins [28]–[31] co-purified reproducibly with either Ubp2-TAP or Rup1-TAP (data not shown).


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, Rsp5, and Rup1 interact physically.(A) Silver-stained SDS polyacrylamide gel showing affinity purified Ubp2-TAP and Rup1-TAP protein complexes. Arrows indicate polypeptides identified by gel band excision followed by MALDI-ToF mass spectrometry. The asterisk indicates degradation products of Ubp2 and Rup1. (B) Ubp2 recovery from Ubp2-TAP, Ubp2-TAP rup1Δ, and Rup1-TAP strains. Arrow indicates gel band identified as Ubp2 by MALDI-ToF mass spectrometry. (C) Co-immunoprecipitation of a fraction of cellular Rsp5 with Ubp2 and Rup1. Cell extracts were depleted of endogenously TAP-tagged Ubp2 or Rup1 with IgG (IP lanes), and the remaining soluble Rsp5 (post-IP lanes) and bait proteins probed by Western blot with anti-Rsp5 antibodies which also recognized the protein A portion of the TAP tag. The ‘input’ lane shows the Rsp5 level in the extract prior to IP. (D) Rup1 tethers Ubp2 to Rsp5. IgG-based immunoprecipitation of Rup1-TAP and Ubp2-TAP from WT, ubp2Δ or rup1Δ strains. The co-purification of Rsp5 with the baits was determined by Western blotting using anti-Rsp5 antibodies. (E) Plasmids expressing FLAG(F/H)-tagged wildtype Rup1 or the Y135F point mutant were transformed into cells lacking endogenous Rup1. After immunoprecipitation with anti-FLAG antibodies, Rup1 and Rsp5 were detected by Western blotting. The bottom panel show the expression levels of Rsp5 in whole cell extracts by Western blotting against Rsp5.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004259-g001: Ubp2, Rsp5, and Rup1 interact physically.(A) Silver-stained SDS polyacrylamide gel showing affinity purified Ubp2-TAP and Rup1-TAP protein complexes. Arrows indicate polypeptides identified by gel band excision followed by MALDI-ToF mass spectrometry. The asterisk indicates degradation products of Ubp2 and Rup1. (B) Ubp2 recovery from Ubp2-TAP, Ubp2-TAP rup1Δ, and Rup1-TAP strains. Arrow indicates gel band identified as Ubp2 by MALDI-ToF mass spectrometry. (C) Co-immunoprecipitation of a fraction of cellular Rsp5 with Ubp2 and Rup1. Cell extracts were depleted of endogenously TAP-tagged Ubp2 or Rup1 with IgG (IP lanes), and the remaining soluble Rsp5 (post-IP lanes) and bait proteins probed by Western blot with anti-Rsp5 antibodies which also recognized the protein A portion of the TAP tag. The ‘input’ lane shows the Rsp5 level in the extract prior to IP. (D) Rup1 tethers Ubp2 to Rsp5. IgG-based immunoprecipitation of Rup1-TAP and Ubp2-TAP from WT, ubp2Δ or rup1Δ strains. The co-purification of Rsp5 with the baits was determined by Western blotting using anti-Rsp5 antibodies. (E) Plasmids expressing FLAG(F/H)-tagged wildtype Rup1 or the Y135F point mutant were transformed into cells lacking endogenous Rup1. After immunoprecipitation with anti-FLAG antibodies, Rup1 and Rsp5 were detected by Western blotting. The bottom panel show the expression levels of Rsp5 in whole cell extracts by Western blotting against Rsp5.
Mentions: Neither Ubp2 or Rup1 are essential for normal cell growth and deletion of either gene does not result in any obvious growth defects or overt morphological phenotypes [15], [25]. Hence, to investigate the possible role(s) of Ubp2 in yeast, we performed large-scale tandem affinity purifications (TAP) to isolate endogenous Ubp2 from S. cerevisiae strains bearing a C-terminal TAP tag [26]. Stably interacting partner proteins were resolved by SDS-PAGE, stained with silver, and identified by peptide mass fingerprinting using MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) mass spectrometry. Two polypeptides, encoding the E3 ubiquitin ligase Rsp5, and a protein of previously unknown function, YOR138c, which we have named Rup1 (Rsp5-Ubp2 interacting Protein 1), were found to reproducibly and specifically co-purify with Ubp2 (Figure 1A). Neither of these binding partners were detected in parallel purifications from an untagged negative control strain or from several hundred other TAP-tagged bait proteins [27]. These data were independently confirmed by highly-sensitive gel-free tandem mass spectrometry-based peptide shotgun sequencing (LC-MS/MS) of an aliquot of each protein mixture after in solution digestion with trypsin (http://tap.med.utoronto.ca/). Conversely, no known substrate or other previously reported Rsp5-interacting proteins [28]–[31] co-purified reproducibly with either Ubp2-TAP or Rup1-TAP (data not shown).

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