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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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Analysis of the CTD sequences that contribute to the rapid, constitutive endocytosis of  Ste3p. Yeast strains were subjected to [35S]methionine pulse-chase analysis as described in the Materials and Methods. (A) Contribution of CTD sequences to Ste3p constitutive turnover. Cells from  four isogenic strains were tested: NDY334, a wild-type MATα strain, as well as three strains differing  only in their STE3 allele, these being either  STE3Δ413-468, STE3Δ365-414, or STE3Δ320-363.  Receptor immunoprecipitated from labeled extracts prepared at the indicated chase times was subjected to SDS-PAGE, and then visualized by autoradiography. (B) Surface-localization assessment for the turnover defective deletion mutant receptor Ste3Δ413-468p. The  STE3Δ413-468 strain of part A was subjected to pulse-chase analysis as above. At the two indicated chase times, 45 min and 90 min after  addition of the excess non-radioactive methionine and cysteine, culture aliquots were removed and whole, energy-poisoned cells were  treated with proteases (+), or mock-treated (−), as described in the Materials and Methods. Protein extracts prepared from each sample  were subjected to immunoprecipitation with either Ste3p-specific (top panel) or Pgk1p-specific (bottom panel) antibodies, followed by  SDS-PAGE and autoradiography. The arrow at right indicates the position of the protected, CTD digestion product for Ste3Δ413-468p.
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Figure 1: Analysis of the CTD sequences that contribute to the rapid, constitutive endocytosis of Ste3p. Yeast strains were subjected to [35S]methionine pulse-chase analysis as described in the Materials and Methods. (A) Contribution of CTD sequences to Ste3p constitutive turnover. Cells from four isogenic strains were tested: NDY334, a wild-type MATα strain, as well as three strains differing only in their STE3 allele, these being either STE3Δ413-468, STE3Δ365-414, or STE3Δ320-363. Receptor immunoprecipitated from labeled extracts prepared at the indicated chase times was subjected to SDS-PAGE, and then visualized by autoradiography. (B) Surface-localization assessment for the turnover defective deletion mutant receptor Ste3Δ413-468p. The STE3Δ413-468 strain of part A was subjected to pulse-chase analysis as above. At the two indicated chase times, 45 min and 90 min after addition of the excess non-radioactive methionine and cysteine, culture aliquots were removed and whole, energy-poisoned cells were treated with proteases (+), or mock-treated (−), as described in the Materials and Methods. Protein extracts prepared from each sample were subjected to immunoprecipitation with either Ste3p-specific (top panel) or Pgk1p-specific (bottom panel) antibodies, followed by SDS-PAGE and autoradiography. The arrow at right indicates the position of the protected, CTD digestion product for Ste3Δ413-468p.

Mentions: Our previous work showed that the rapid constitutive endocytosis of Ste3p requires sequence elements within the COOH-terminal 72–amino acid residues of the receptor (Davis et al., 1993). To better delineate this requirement we have constructed a series of in-frame deletion mutants within the receptor's dispensable CTD. First we have first investigated if CTD sequences in addition to the 72-residue COOH-terminal segment are required for rapid constitutive endocytosis. The 187-residue-long Ste3p CTD extends from Asp284 (the first charged residue after the seventh transmembrane segment) to a COOH-terminal Pro470. Three large deletion mutants, deleting residues 320– 363 (STE3Δ320–363), residues 365–414 (STE3Δ365–414), and the COOH-terminal residues 413–468 (STE3Δ413–468) were used. As a first assessment of endocytosis, the rate of degradative turnover of each of the different receptor mutants was determined (Fig. 1 A). While wild-type Ste3p turns over rapidly with a t1/2 estimated to be ∼15 min, the Δ413–468 mutant receptor is completely stable, indicating that the COOH-terminal 58 residues are required for this ligand-independent receptor turnover. Like wild-type, both the Δ365–414 and Δ320–363 receptors show rapid turnover consistent with endocytosis that is largely unimpaired. Thus, of the STE3 CTD sequences, the COOH-terminal 58 residues play the primary role in specifying rapid turnover. We have therefore concentrated our analyses on this interval.


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)

Analysis of the CTD sequences that contribute to the rapid, constitutive endocytosis of  Ste3p. Yeast strains were subjected to [35S]methionine pulse-chase analysis as described in the Materials and Methods. (A) Contribution of CTD sequences to Ste3p constitutive turnover. Cells from  four isogenic strains were tested: NDY334, a wild-type MATα strain, as well as three strains differing  only in their STE3 allele, these being either  STE3Δ413-468, STE3Δ365-414, or STE3Δ320-363.  Receptor immunoprecipitated from labeled extracts prepared at the indicated chase times was subjected to SDS-PAGE, and then visualized by autoradiography. (B) Surface-localization assessment for the turnover defective deletion mutant receptor Ste3Δ413-468p. The  STE3Δ413-468 strain of part A was subjected to pulse-chase analysis as above. At the two indicated chase times, 45 min and 90 min after  addition of the excess non-radioactive methionine and cysteine, culture aliquots were removed and whole, energy-poisoned cells were  treated with proteases (+), or mock-treated (−), as described in the Materials and Methods. Protein extracts prepared from each sample  were subjected to immunoprecipitation with either Ste3p-specific (top panel) or Pgk1p-specific (bottom panel) antibodies, followed by  SDS-PAGE and autoradiography. The arrow at right indicates the position of the protected, CTD digestion product for Ste3Δ413-468p.
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Figure 1: Analysis of the CTD sequences that contribute to the rapid, constitutive endocytosis of Ste3p. Yeast strains were subjected to [35S]methionine pulse-chase analysis as described in the Materials and Methods. (A) Contribution of CTD sequences to Ste3p constitutive turnover. Cells from four isogenic strains were tested: NDY334, a wild-type MATα strain, as well as three strains differing only in their STE3 allele, these being either STE3Δ413-468, STE3Δ365-414, or STE3Δ320-363. Receptor immunoprecipitated from labeled extracts prepared at the indicated chase times was subjected to SDS-PAGE, and then visualized by autoradiography. (B) Surface-localization assessment for the turnover defective deletion mutant receptor Ste3Δ413-468p. The STE3Δ413-468 strain of part A was subjected to pulse-chase analysis as above. At the two indicated chase times, 45 min and 90 min after addition of the excess non-radioactive methionine and cysteine, culture aliquots were removed and whole, energy-poisoned cells were treated with proteases (+), or mock-treated (−), as described in the Materials and Methods. Protein extracts prepared from each sample were subjected to immunoprecipitation with either Ste3p-specific (top panel) or Pgk1p-specific (bottom panel) antibodies, followed by SDS-PAGE and autoradiography. The arrow at right indicates the position of the protected, CTD digestion product for Ste3Δ413-468p.
Mentions: Our previous work showed that the rapid constitutive endocytosis of Ste3p requires sequence elements within the COOH-terminal 72–amino acid residues of the receptor (Davis et al., 1993). To better delineate this requirement we have constructed a series of in-frame deletion mutants within the receptor's dispensable CTD. First we have first investigated if CTD sequences in addition to the 72-residue COOH-terminal segment are required for rapid constitutive endocytosis. The 187-residue-long Ste3p CTD extends from Asp284 (the first charged residue after the seventh transmembrane segment) to a COOH-terminal Pro470. Three large deletion mutants, deleting residues 320– 363 (STE3Δ320–363), residues 365–414 (STE3Δ365–414), and the COOH-terminal residues 413–468 (STE3Δ413–468) were used. As a first assessment of endocytosis, the rate of degradative turnover of each of the different receptor mutants was determined (Fig. 1 A). While wild-type Ste3p turns over rapidly with a t1/2 estimated to be ∼15 min, the Δ413–468 mutant receptor is completely stable, indicating that the COOH-terminal 58 residues are required for this ligand-independent receptor turnover. Like wild-type, both the Δ365–414 and Δ320–363 receptors show rapid turnover consistent with endocytosis that is largely unimpaired. Thus, of the STE3 CTD sequences, the COOH-terminal 58 residues play the primary role in specifying rapid turnover. We have therefore concentrated our analyses on this interval.

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