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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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Turnover rate of different COOH-terminally truncated  a-factor receptors. The wild-type MATα strain NDY334 and  three isogenic strains with different transplaced STE3 alleles:  STE3Δ450-468, STE3Δ441-468, or STE3Δ413-468 were subjected  to pulse-chase analysis and otherwise treated as described for Fig.  1 A. (A) Effect of COOH-terminal deletions on receptor turnover. Autoradiography of the SDS-PAGE of the immunoprecipitated receptor mutants. (B) Determination of the turnover t1/2 for  the receptor mutants. Phosphorimager analysis was applied to  the polyacrylamide gel of A (Materials and Methods). For each  strain, the radioactivity associated with receptor bands at the initial 1-min chase time-point was normalized to 100%. A semi-log  plot of the radioactivity remaining associated with the receptor  bands at each of the subsequent chase time-points is shown. For  each strain, a turnover t1/2 was calculated (indicated for each as  t1/2) from the slope of the line drawn through the linear portion of  each data set.
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Figure 2: Turnover rate of different COOH-terminally truncated a-factor receptors. The wild-type MATα strain NDY334 and three isogenic strains with different transplaced STE3 alleles: STE3Δ450-468, STE3Δ441-468, or STE3Δ413-468 were subjected to pulse-chase analysis and otherwise treated as described for Fig. 1 A. (A) Effect of COOH-terminal deletions on receptor turnover. Autoradiography of the SDS-PAGE of the immunoprecipitated receptor mutants. (B) Determination of the turnover t1/2 for the receptor mutants. Phosphorimager analysis was applied to the polyacrylamide gel of A (Materials and Methods). For each strain, the radioactivity associated with receptor bands at the initial 1-min chase time-point was normalized to 100%. A semi-log plot of the radioactivity remaining associated with the receptor bands at each of the subsequent chase time-points is shown. For each strain, a turnover t1/2 was calculated (indicated for each as t1/2) from the slope of the line drawn through the linear portion of each data set.

Mentions: To identify the sequence element(s) that direct the constitutive endocytosis of Ste3p we have constructed a finer series of deletions within the 413–468 interval. First we tested the effects of three deletions extending progressively in from the receptor COOH terminus. The in vivo rate of turnover was assessed for wild-type Ste3p and for each mutant receptor via a pulse-chase regimen similar to that described for Fig. 1 A. Immunoprecipitated receptors were subjected to SDS-PAGE (Fig. 2 A) and then to PhosphorImaging analysis to quantitate their rate of loss to degradation (Fig. 2 B). As seen previously (Fig. 1 A), turnover is rapid for wild-type Ste3p and wholly blocked for Ste3Δ413–468p. The two intermediate deletion mutants, Δ450–468 and Δ441–450 show intermediate rates of turnover. Quantitation of the receptor-associated radioactivity at each time-point is shown in Fig. 2 B. Most of the data points for each receptor conform to a linear plot, allowing the calculation of turnover half-life for each mutant (Fig. 2 B). For wild-type Ste3p, a t1/2 of 16 min is calculated. For Ste3Δ450–468p, turnover remains rapid, though somewhat less so than wild-type: a t1/2 of 21 min is calculated. With the progressive extension of these deletions, a progressive impairment is evident. A t1/2 of 65 min is calculated for the Δ441–468 receptor and the turnover block appears to be complete for the Δ413–468 receptor.


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)

Turnover rate of different COOH-terminally truncated  a-factor receptors. The wild-type MATα strain NDY334 and  three isogenic strains with different transplaced STE3 alleles:  STE3Δ450-468, STE3Δ441-468, or STE3Δ413-468 were subjected  to pulse-chase analysis and otherwise treated as described for Fig.  1 A. (A) Effect of COOH-terminal deletions on receptor turnover. Autoradiography of the SDS-PAGE of the immunoprecipitated receptor mutants. (B) Determination of the turnover t1/2 for  the receptor mutants. Phosphorimager analysis was applied to  the polyacrylamide gel of A (Materials and Methods). For each  strain, the radioactivity associated with receptor bands at the initial 1-min chase time-point was normalized to 100%. A semi-log  plot of the radioactivity remaining associated with the receptor  bands at each of the subsequent chase time-points is shown. For  each strain, a turnover t1/2 was calculated (indicated for each as  t1/2) from the slope of the line drawn through the linear portion of  each data set.
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Figure 2: Turnover rate of different COOH-terminally truncated a-factor receptors. The wild-type MATα strain NDY334 and three isogenic strains with different transplaced STE3 alleles: STE3Δ450-468, STE3Δ441-468, or STE3Δ413-468 were subjected to pulse-chase analysis and otherwise treated as described for Fig. 1 A. (A) Effect of COOH-terminal deletions on receptor turnover. Autoradiography of the SDS-PAGE of the immunoprecipitated receptor mutants. (B) Determination of the turnover t1/2 for the receptor mutants. Phosphorimager analysis was applied to the polyacrylamide gel of A (Materials and Methods). For each strain, the radioactivity associated with receptor bands at the initial 1-min chase time-point was normalized to 100%. A semi-log plot of the radioactivity remaining associated with the receptor bands at each of the subsequent chase time-points is shown. For each strain, a turnover t1/2 was calculated (indicated for each as t1/2) from the slope of the line drawn through the linear portion of each data set.
Mentions: To identify the sequence element(s) that direct the constitutive endocytosis of Ste3p we have constructed a finer series of deletions within the 413–468 interval. First we tested the effects of three deletions extending progressively in from the receptor COOH terminus. The in vivo rate of turnover was assessed for wild-type Ste3p and for each mutant receptor via a pulse-chase regimen similar to that described for Fig. 1 A. Immunoprecipitated receptors were subjected to SDS-PAGE (Fig. 2 A) and then to PhosphorImaging analysis to quantitate their rate of loss to degradation (Fig. 2 B). As seen previously (Fig. 1 A), turnover is rapid for wild-type Ste3p and wholly blocked for Ste3Δ413–468p. The two intermediate deletion mutants, Δ450–468 and Δ441–450 show intermediate rates of turnover. Quantitation of the receptor-associated radioactivity at each time-point is shown in Fig. 2 B. Most of the data points for each receptor conform to a linear plot, allowing the calculation of turnover half-life for each mutant (Fig. 2 B). For wild-type Ste3p, a t1/2 of 16 min is calculated. For Ste3Δ450–468p, turnover remains rapid, though somewhat less so than wild-type: a t1/2 of 21 min is calculated. With the progressive extension of these deletions, a progressive impairment is evident. A t1/2 of 65 min is calculated for the Δ441–468 receptor and the turnover block appears to be complete for the Δ413–468 receptor.

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