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A large PEST-like sequence directs the ubiquitination, endocytosis, and vacuolar degradation of the yeast a-factor receptor.

Roth AF, Sullivan DM, Davis NG - J. Cell Biol. (1998)

Bottom Line: Both modes are associated with receptor ubiquitination (Roth, A.F., and N.G.Mutants deleted for this sequence show undetectable levels of ubiquitination, and mutants having intermediate endocytosis defects show a correlated reduced level of ubiquitination.Alanine scanning mutagenesis across the 36-residue-long interval highlights its overall complexity-no singular sequence motif or signal is found, instead required sequence elements distribute throughout the entire interval.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.

ABSTRACT
The yeast a-factor receptor (encoded by STE3) is subject to two modes of endocytosis, a ligand-dependent endocytosis as well as a constitutive, ligand-independent mode. Both modes are associated with receptor ubiquitination (Roth, A.F., and N.G. Davis. 1996. J. Cell Biol. 134:661-674) and both depend on sequence elements within the receptor's regulatory, cytoplasmically disposed, COOH-terminal domain (CTD). Here, we concentrate on the Ste3p sequences required for constitutive endocytosis. Constitutive endocytosis is rapid. Receptor is synthesized, delivered to the cell surface, endocytosed, and then delivered to the vacuole where it is degraded, all with a t1/2 of 15 min. Deletion analysis has defined a 36-residue-long sequence mapping near the COOH-terminal end of the Ste3p CTD that is the minimal sequence required for this rapid turnover. Deletions intruding into this interval block or severely slow the rate of endocytic turnover. Moreover, the same 36-residue sequence directs receptor ubiquitination. Mutants deleted for this sequence show undetectable levels of ubiquitination, and mutants having intermediate endocytosis defects show a correlated reduced level of ubiquitination. Not only necessary for ubiquitination and endocytosis, this sequence also is sufficient. When transplanted to a stable cell surface protein, the plasma membrane ATPase Pma1p, the 36-residue STE3 signal directs both ubiquitination of the PMA1-STE3 fusion protein as well as its endocytosis and consequent vacuolar degradation. Alanine scanning mutagenesis across the 36-residue-long interval highlights its overall complexity-no singular sequence motif or signal is found, instead required sequence elements distribute throughout the entire interval. The high proportion of acidic and hydroxylated amino acid residues in this interval suggests a similarity to PEST sequences-a broad class of sequences which have been shown to direct the ubiquitination and subsequent proteosomal degradation of short-lived nuclear and cytoplasmic proteins. A likely possibility, therefore, is that this sequence, responsible for both endocytosis and ubiquitination, may be first and foremost a ubiquitination signal. Finally, we present evidence suggesting that the true signal in the wild-type receptor extends beyond the 36-residue-long sequence defined as a minimal signal to include contiguous PEST-like sequences which extend another 21 residues to the COOH terminus of Ste3p. Together with sequences identified in two other yeast plasma membrane proteins, the STE3 sequence defines a new class of ubiquitination/endocytosis signal.

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The STE3 signal directs ubiquitination of PMA1-STE3 fusion proteins. (A) Immune precipitation of PMA1-STE3 fusion proteins from end4 cells overexpressing a myc  epitope–tagged ubiquitin. Two plasmids were  used to doubly transform cells of the MATa  end4-1 strain RH268-1C: (1) a LEU2/CEN/ ARS GAL1-HA-PMA1-STE3 plasmid derived  from pND542, with the STE3 contribution being either the 400–412 Ste3p interval, the 400– 449 interval, or the 400–470 interval, and with  (2) a URA3/2μ plasmid for CUP1-driven overexpression of wild-type or myc-tagged ubiquitin, either pND186 (u, wild-type ubiquitin), pND747 (m, myc-ubiquitin), pND187 (−, no ubiquitin). As an immunoprecipitation control, one cell was transformed both with the myc-ubiquitin plasmid pND747 and with the vector plasmid pRS315 (Sikorski and Hieter,  1989) in place of a PMA1-STE3–expression plasmid (no fusion). Transformants were subjected to a culture protocol that included growth  at 30°C in media containing 100 μM CuSO4 for 1 h (to induce ubiquitin expression), and then 2 h in the presence of 2% galactose (to induce PMA1-STE3 fusion protein expression), and finally 1 h in the presence of 3% glucose (to repress PMA1-STE3 expression). Extracts  prepared from these cells were immunoprecipitated using the HA.11 mAb conjugated to Sepharose beads, and then subjected to SDS-PAGE followed by Western analysis. Duplicate nitrocellulose transfer membranes were developed with either the HA.11 mAb (top  panel) or the myc mAb (bottom panel). (B) The STE3 sequence requirement for ubiquitination. Cells of the MATα end4-1 strain  NDY335 were transformed by various GAL1-HA-PMA1-STE3 plasmids derived from pND542. The plasmids differ in terms of the STE3  sequences fused at the COOH terminus. For each, the contributed STE3 sequence extends from Ste3p residue 400 to the residue indicated above each lane. The six different transformants were subjected to a culture protocol at 30°C, which included a 1 h exposure to 2%  galactose, followed by 1 h in 3% glucose. Protein extracts were prepared and analyzed as described for Fig. 5. The brackets to the right indicate the position of the more slowly migrating modified forms of the PMA1-STE3(400–449) and PMA1-STE3(400–470) fusion proteins.
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Figure 7: The STE3 signal directs ubiquitination of PMA1-STE3 fusion proteins. (A) Immune precipitation of PMA1-STE3 fusion proteins from end4 cells overexpressing a myc epitope–tagged ubiquitin. Two plasmids were used to doubly transform cells of the MATa end4-1 strain RH268-1C: (1) a LEU2/CEN/ ARS GAL1-HA-PMA1-STE3 plasmid derived from pND542, with the STE3 contribution being either the 400–412 Ste3p interval, the 400– 449 interval, or the 400–470 interval, and with (2) a URA3/2μ plasmid for CUP1-driven overexpression of wild-type or myc-tagged ubiquitin, either pND186 (u, wild-type ubiquitin), pND747 (m, myc-ubiquitin), pND187 (−, no ubiquitin). As an immunoprecipitation control, one cell was transformed both with the myc-ubiquitin plasmid pND747 and with the vector plasmid pRS315 (Sikorski and Hieter, 1989) in place of a PMA1-STE3–expression plasmid (no fusion). Transformants were subjected to a culture protocol that included growth at 30°C in media containing 100 μM CuSO4 for 1 h (to induce ubiquitin expression), and then 2 h in the presence of 2% galactose (to induce PMA1-STE3 fusion protein expression), and finally 1 h in the presence of 3% glucose (to repress PMA1-STE3 expression). Extracts prepared from these cells were immunoprecipitated using the HA.11 mAb conjugated to Sepharose beads, and then subjected to SDS-PAGE followed by Western analysis. Duplicate nitrocellulose transfer membranes were developed with either the HA.11 mAb (top panel) or the myc mAb (bottom panel). (B) The STE3 sequence requirement for ubiquitination. Cells of the MATα end4-1 strain NDY335 were transformed by various GAL1-HA-PMA1-STE3 plasmids derived from pND542. The plasmids differ in terms of the STE3 sequences fused at the COOH terminus. For each, the contributed STE3 sequence extends from Ste3p residue 400 to the residue indicated above each lane. The six different transformants were subjected to a culture protocol at 30°C, which included a 1 h exposure to 2% galactose, followed by 1 h in 3% glucose. Protein extracts were prepared and analyzed as described for Fig. 5. The brackets to the right indicate the position of the more slowly migrating modified forms of the PMA1-STE3(400–449) and PMA1-STE3(400–470) fusion proteins.

Mentions: We next tested if the STE3 mutants defective for constitutive endocytosis are also defective for the associated constitutive ubiquitination. Receptor ubiquitination levels of the various STE3 mutants were assessed via Western analysis: ubiquitinated receptor species manifest an 8-kD shift with each added ubiquitin moiety. Detection of the ubiquitinated forms is improved with the use of several key experimental conditions, including receptor overproduction (∼10-fold) from the GAL1 promoter. This enhances detection of the ubiquitinated forms, yet neither alters receptor turnover rate nor the proportion of the receptor subject to ubiquitination (data not shown). Secondly, to avoid loss of the ubiquitinated forms to vacuolar degradation, pep4Δ mutants deficient in vacuolar protease activity were used. Finally, treatment of protein extracts with phosphatase before electrophoresis eliminates the substantial heterogeneity in gel mobility because of the heterogenous phosphorylation of Ste3p (Fig. 7; also Roth and Davis, 1996).


A large PEST-like sequence directs the ubiquitination, endocytosis, and vacuolar degradation of the yeast a-factor receptor.

Roth AF, Sullivan DM, Davis NG - J. Cell Biol. (1998)

The STE3 signal directs ubiquitination of PMA1-STE3 fusion proteins. (A) Immune precipitation of PMA1-STE3 fusion proteins from end4 cells overexpressing a myc  epitope–tagged ubiquitin. Two plasmids were  used to doubly transform cells of the MATa  end4-1 strain RH268-1C: (1) a LEU2/CEN/ ARS GAL1-HA-PMA1-STE3 plasmid derived  from pND542, with the STE3 contribution being either the 400–412 Ste3p interval, the 400– 449 interval, or the 400–470 interval, and with  (2) a URA3/2μ plasmid for CUP1-driven overexpression of wild-type or myc-tagged ubiquitin, either pND186 (u, wild-type ubiquitin), pND747 (m, myc-ubiquitin), pND187 (−, no ubiquitin). As an immunoprecipitation control, one cell was transformed both with the myc-ubiquitin plasmid pND747 and with the vector plasmid pRS315 (Sikorski and Hieter,  1989) in place of a PMA1-STE3–expression plasmid (no fusion). Transformants were subjected to a culture protocol that included growth  at 30°C in media containing 100 μM CuSO4 for 1 h (to induce ubiquitin expression), and then 2 h in the presence of 2% galactose (to induce PMA1-STE3 fusion protein expression), and finally 1 h in the presence of 3% glucose (to repress PMA1-STE3 expression). Extracts  prepared from these cells were immunoprecipitated using the HA.11 mAb conjugated to Sepharose beads, and then subjected to SDS-PAGE followed by Western analysis. Duplicate nitrocellulose transfer membranes were developed with either the HA.11 mAb (top  panel) or the myc mAb (bottom panel). (B) The STE3 sequence requirement for ubiquitination. Cells of the MATα end4-1 strain  NDY335 were transformed by various GAL1-HA-PMA1-STE3 plasmids derived from pND542. The plasmids differ in terms of the STE3  sequences fused at the COOH terminus. For each, the contributed STE3 sequence extends from Ste3p residue 400 to the residue indicated above each lane. The six different transformants were subjected to a culture protocol at 30°C, which included a 1 h exposure to 2%  galactose, followed by 1 h in 3% glucose. Protein extracts were prepared and analyzed as described for Fig. 5. The brackets to the right indicate the position of the more slowly migrating modified forms of the PMA1-STE3(400–449) and PMA1-STE3(400–470) fusion proteins.
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Related In: Results  -  Collection

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Figure 7: The STE3 signal directs ubiquitination of PMA1-STE3 fusion proteins. (A) Immune precipitation of PMA1-STE3 fusion proteins from end4 cells overexpressing a myc epitope–tagged ubiquitin. Two plasmids were used to doubly transform cells of the MATa end4-1 strain RH268-1C: (1) a LEU2/CEN/ ARS GAL1-HA-PMA1-STE3 plasmid derived from pND542, with the STE3 contribution being either the 400–412 Ste3p interval, the 400– 449 interval, or the 400–470 interval, and with (2) a URA3/2μ plasmid for CUP1-driven overexpression of wild-type or myc-tagged ubiquitin, either pND186 (u, wild-type ubiquitin), pND747 (m, myc-ubiquitin), pND187 (−, no ubiquitin). As an immunoprecipitation control, one cell was transformed both with the myc-ubiquitin plasmid pND747 and with the vector plasmid pRS315 (Sikorski and Hieter, 1989) in place of a PMA1-STE3–expression plasmid (no fusion). Transformants were subjected to a culture protocol that included growth at 30°C in media containing 100 μM CuSO4 for 1 h (to induce ubiquitin expression), and then 2 h in the presence of 2% galactose (to induce PMA1-STE3 fusion protein expression), and finally 1 h in the presence of 3% glucose (to repress PMA1-STE3 expression). Extracts prepared from these cells were immunoprecipitated using the HA.11 mAb conjugated to Sepharose beads, and then subjected to SDS-PAGE followed by Western analysis. Duplicate nitrocellulose transfer membranes were developed with either the HA.11 mAb (top panel) or the myc mAb (bottom panel). (B) The STE3 sequence requirement for ubiquitination. Cells of the MATα end4-1 strain NDY335 were transformed by various GAL1-HA-PMA1-STE3 plasmids derived from pND542. The plasmids differ in terms of the STE3 sequences fused at the COOH terminus. For each, the contributed STE3 sequence extends from Ste3p residue 400 to the residue indicated above each lane. The six different transformants were subjected to a culture protocol at 30°C, which included a 1 h exposure to 2% galactose, followed by 1 h in 3% glucose. Protein extracts were prepared and analyzed as described for Fig. 5. The brackets to the right indicate the position of the more slowly migrating modified forms of the PMA1-STE3(400–449) and PMA1-STE3(400–470) fusion proteins.
Mentions: We next tested if the STE3 mutants defective for constitutive endocytosis are also defective for the associated constitutive ubiquitination. Receptor ubiquitination levels of the various STE3 mutants were assessed via Western analysis: ubiquitinated receptor species manifest an 8-kD shift with each added ubiquitin moiety. Detection of the ubiquitinated forms is improved with the use of several key experimental conditions, including receptor overproduction (∼10-fold) from the GAL1 promoter. This enhances detection of the ubiquitinated forms, yet neither alters receptor turnover rate nor the proportion of the receptor subject to ubiquitination (data not shown). Secondly, to avoid loss of the ubiquitinated forms to vacuolar degradation, pep4Δ mutants deficient in vacuolar protease activity were used. Finally, treatment of protein extracts with phosphatase before electrophoresis eliminates the substantial heterogeneity in gel mobility because of the heterogenous phosphorylation of Ste3p (Fig. 7; also Roth and Davis, 1996).

Bottom Line: Both modes are associated with receptor ubiquitination (Roth, A.F., and N.G.Mutants deleted for this sequence show undetectable levels of ubiquitination, and mutants having intermediate endocytosis defects show a correlated reduced level of ubiquitination.Alanine scanning mutagenesis across the 36-residue-long interval highlights its overall complexity-no singular sequence motif or signal is found, instead required sequence elements distribute throughout the entire interval.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.

ABSTRACT
The yeast a-factor receptor (encoded by STE3) is subject to two modes of endocytosis, a ligand-dependent endocytosis as well as a constitutive, ligand-independent mode. Both modes are associated with receptor ubiquitination (Roth, A.F., and N.G. Davis. 1996. J. Cell Biol. 134:661-674) and both depend on sequence elements within the receptor's regulatory, cytoplasmically disposed, COOH-terminal domain (CTD). Here, we concentrate on the Ste3p sequences required for constitutive endocytosis. Constitutive endocytosis is rapid. Receptor is synthesized, delivered to the cell surface, endocytosed, and then delivered to the vacuole where it is degraded, all with a t1/2 of 15 min. Deletion analysis has defined a 36-residue-long sequence mapping near the COOH-terminal end of the Ste3p CTD that is the minimal sequence required for this rapid turnover. Deletions intruding into this interval block or severely slow the rate of endocytic turnover. Moreover, the same 36-residue sequence directs receptor ubiquitination. Mutants deleted for this sequence show undetectable levels of ubiquitination, and mutants having intermediate endocytosis defects show a correlated reduced level of ubiquitination. Not only necessary for ubiquitination and endocytosis, this sequence also is sufficient. When transplanted to a stable cell surface protein, the plasma membrane ATPase Pma1p, the 36-residue STE3 signal directs both ubiquitination of the PMA1-STE3 fusion protein as well as its endocytosis and consequent vacuolar degradation. Alanine scanning mutagenesis across the 36-residue-long interval highlights its overall complexity-no singular sequence motif or signal is found, instead required sequence elements distribute throughout the entire interval. The high proportion of acidic and hydroxylated amino acid residues in this interval suggests a similarity to PEST sequences-a broad class of sequences which have been shown to direct the ubiquitination and subsequent proteosomal degradation of short-lived nuclear and cytoplasmic proteins. A likely possibility, therefore, is that this sequence, responsible for both endocytosis and ubiquitination, may be first and foremost a ubiquitination signal. Finally, we present evidence suggesting that the true signal in the wild-type receptor extends beyond the 36-residue-long sequence defined as a minimal signal to include contiguous PEST-like sequences which extend another 21 residues to the COOH terminus of Ste3p. Together with sequences identified in two other yeast plasma membrane proteins, the STE3 sequence defines a new class of ubiquitination/endocytosis signal.

Show MeSH
Related in: MedlinePlus