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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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Assessment of receptor ubiquitination levels for STE3  deletion mutants. (A) Phosphatase-treated STE3 deletion mutant  receptors expressed from the GAL1 promoter in a pep4Δ cells. 2 h  of growth in galactose medium was used to induce receptor expression in MATα ste3Δ pep4Δ cells (strain SY2602) transformed  by a CEN/ARS plasmid with wild-type (wt) STE3 under the control of the GAL1 promoter (pSL552), or by the equivalent plasmid having one of three different STE3 mutant alleles, either  STE3Δ450-468, STE3Δ413-468, or STE3Δ413-451. Extracts were  prepared and subjected to phosphatase digestion, SDS-PAGE,  and finally anti-Ste3p immunoblot analysis. Positions of the putative mono- and di-ubiquitinated forms of wild-type Ste3p (lane 1)  are indicated by the arrows at left. The arrow at right indicates  the position of a band that cross-reacts with the Ste3p-specific antibodies in these immunoblot analyses. (B) The effect of the  end4-1 mutation on the ubiquitination levels of the STE3 deletion mutants. Cells of the MATα ste3Δ end4-1 strain (NDY344)  transformed by the GAL1-STE3 plasmid pSL552 (wt), or by the  equivalent plasmid with one of three STE3 mutant alleles, either  STE3Δ450-468, STE3Δ441-468, or STE3Δ413-468 were grown for  2 h in galactose medium, to induce receptor expression. Further  receptor synthesis was then repressed with 3% glucose and  growth was continued for an additional 2 h. Extracts were prepared, treated with phosphatase, and otherwise analyzed as described above for A. Though the end4 strain is temperature sensitive, the Ste3p is wholly defective for endocytosis even at  permissive temperatures (Roth and Davis, 1996). Thus, in this  and other experiments using the end4-1 mutation all growth of  cells is at 30°C.
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Figure 4: Assessment of receptor ubiquitination levels for STE3 deletion mutants. (A) Phosphatase-treated STE3 deletion mutant receptors expressed from the GAL1 promoter in a pep4Δ cells. 2 h of growth in galactose medium was used to induce receptor expression in MATα ste3Δ pep4Δ cells (strain SY2602) transformed by a CEN/ARS plasmid with wild-type (wt) STE3 under the control of the GAL1 promoter (pSL552), or by the equivalent plasmid having one of three different STE3 mutant alleles, either STE3Δ450-468, STE3Δ413-468, or STE3Δ413-451. Extracts were prepared and subjected to phosphatase digestion, SDS-PAGE, and finally anti-Ste3p immunoblot analysis. Positions of the putative mono- and di-ubiquitinated forms of wild-type Ste3p (lane 1) are indicated by the arrows at left. The arrow at right indicates the position of a band that cross-reacts with the Ste3p-specific antibodies in these immunoblot analyses. (B) The effect of the end4-1 mutation on the ubiquitination levels of the STE3 deletion mutants. Cells of the MATα ste3Δ end4-1 strain (NDY344) transformed by the GAL1-STE3 plasmid pSL552 (wt), or by the equivalent plasmid with one of three STE3 mutant alleles, either STE3Δ450-468, STE3Δ441-468, or STE3Δ413-468 were grown for 2 h in galactose medium, to induce receptor expression. Further receptor synthesis was then repressed with 3% glucose and growth was continued for an additional 2 h. Extracts were prepared, treated with phosphatase, and otherwise analyzed as described above for A. Though the end4 strain is temperature sensitive, the Ste3p is wholly defective for endocytosis even at permissive temperatures (Roth and Davis, 1996). Thus, in this and other experiments using the end4-1 mutation all growth of cells is at 30°C.

Mentions: In Fig. 4 A, the ubiquitination of three receptor mutants is compared with wild-type. For wild-type, the majority species (∼80%) has no attached ubiquitin (Roth and Davis, 1996). The remaining 20% distributes almost equally between two, more slowly-migrating, ubiquitinated forms (indicated in Fig. 4 A, arrows at left), the mono- and di-ubiquitinated receptor species (Roth and Davis, 1996). Mutants that block endocytosis, namely Δ413–468 and Δ413–451 (Fig. 3), also blocked receptor ubiquitination (Fig. 4 A). Furthermore, the Δ450–468 mutant receptor that showed diminished turnover (Fig. 2) also shows diminished ubiquitination (Fig. 4 A). Quantitation of these results along with similar analyses applied to the other mutant receptors is reported in Fig. 3. Comparison of the turnover half-life with ubiquitination levels shows a consistent correlation between the two—further compelling evidence for a functional connection linking ubiquitination and endocytosis.


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)

Assessment of receptor ubiquitination levels for STE3  deletion mutants. (A) Phosphatase-treated STE3 deletion mutant  receptors expressed from the GAL1 promoter in a pep4Δ cells. 2 h  of growth in galactose medium was used to induce receptor expression in MATα ste3Δ pep4Δ cells (strain SY2602) transformed  by a CEN/ARS plasmid with wild-type (wt) STE3 under the control of the GAL1 promoter (pSL552), or by the equivalent plasmid having one of three different STE3 mutant alleles, either  STE3Δ450-468, STE3Δ413-468, or STE3Δ413-451. Extracts were  prepared and subjected to phosphatase digestion, SDS-PAGE,  and finally anti-Ste3p immunoblot analysis. Positions of the putative mono- and di-ubiquitinated forms of wild-type Ste3p (lane 1)  are indicated by the arrows at left. The arrow at right indicates  the position of a band that cross-reacts with the Ste3p-specific antibodies in these immunoblot analyses. (B) The effect of the  end4-1 mutation on the ubiquitination levels of the STE3 deletion mutants. Cells of the MATα ste3Δ end4-1 strain (NDY344)  transformed by the GAL1-STE3 plasmid pSL552 (wt), or by the  equivalent plasmid with one of three STE3 mutant alleles, either  STE3Δ450-468, STE3Δ441-468, or STE3Δ413-468 were grown for  2 h in galactose medium, to induce receptor expression. Further  receptor synthesis was then repressed with 3% glucose and  growth was continued for an additional 2 h. Extracts were prepared, treated with phosphatase, and otherwise analyzed as described above for A. Though the end4 strain is temperature sensitive, the Ste3p is wholly defective for endocytosis even at  permissive temperatures (Roth and Davis, 1996). Thus, in this  and other experiments using the end4-1 mutation all growth of  cells is at 30°C.
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Figure 4: Assessment of receptor ubiquitination levels for STE3 deletion mutants. (A) Phosphatase-treated STE3 deletion mutant receptors expressed from the GAL1 promoter in a pep4Δ cells. 2 h of growth in galactose medium was used to induce receptor expression in MATα ste3Δ pep4Δ cells (strain SY2602) transformed by a CEN/ARS plasmid with wild-type (wt) STE3 under the control of the GAL1 promoter (pSL552), or by the equivalent plasmid having one of three different STE3 mutant alleles, either STE3Δ450-468, STE3Δ413-468, or STE3Δ413-451. Extracts were prepared and subjected to phosphatase digestion, SDS-PAGE, and finally anti-Ste3p immunoblot analysis. Positions of the putative mono- and di-ubiquitinated forms of wild-type Ste3p (lane 1) are indicated by the arrows at left. The arrow at right indicates the position of a band that cross-reacts with the Ste3p-specific antibodies in these immunoblot analyses. (B) The effect of the end4-1 mutation on the ubiquitination levels of the STE3 deletion mutants. Cells of the MATα ste3Δ end4-1 strain (NDY344) transformed by the GAL1-STE3 plasmid pSL552 (wt), or by the equivalent plasmid with one of three STE3 mutant alleles, either STE3Δ450-468, STE3Δ441-468, or STE3Δ413-468 were grown for 2 h in galactose medium, to induce receptor expression. Further receptor synthesis was then repressed with 3% glucose and growth was continued for an additional 2 h. Extracts were prepared, treated with phosphatase, and otherwise analyzed as described above for A. Though the end4 strain is temperature sensitive, the Ste3p is wholly defective for endocytosis even at permissive temperatures (Roth and Davis, 1996). Thus, in this and other experiments using the end4-1 mutation all growth of cells is at 30°C.
Mentions: In Fig. 4 A, the ubiquitination of three receptor mutants is compared with wild-type. For wild-type, the majority species (∼80%) has no attached ubiquitin (Roth and Davis, 1996). The remaining 20% distributes almost equally between two, more slowly-migrating, ubiquitinated forms (indicated in Fig. 4 A, arrows at left), the mono- and di-ubiquitinated receptor species (Roth and Davis, 1996). Mutants that block endocytosis, namely Δ413–468 and Δ413–451 (Fig. 3), also blocked receptor ubiquitination (Fig. 4 A). Furthermore, the Δ450–468 mutant receptor that showed diminished turnover (Fig. 2) also shows diminished ubiquitination (Fig. 4 A). Quantitation of these results along with similar analyses applied to the other mutant receptors is reported in Fig. 3. Comparison of the turnover half-life with ubiquitination levels shows a consistent correlation between the two—further compelling evidence for a functional connection linking ubiquitination and endocytosis.

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