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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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Summary of turnover rates and the proportion of receptor proteins that are ubiquitinated for a collection of STE3  deletion mutants constructed within the COOH-terminal 73  amino acid residues of Ste3p. Shown in the upper left is the hypothesized transmembrane structure for Ste3p. The region corresponding to the COOH-terminal 57 residues of the Ste3p CTD is  highlighted, the 414–451 interval indicated with a striped box and  the 451–470 interval by a stippled box. Shown below are sequences deleted (dotted line) for each of the different in-frame  STE3 deletion mutants tested. To the right, the exact amino acids  residues removed for each mutant are indicated. The two columns at the right summarize the results of the two different experiments that were performed for each receptor, these being: (1)  measurement of the turnover t1/2, and (2) measurement of the  proportion of the receptors found to be ubiquitinated. Measurement of turnover rate used a collection of strains isogenic to  NDY334, except for the indicated STE3 mutant alleles. As described in Fig. 2, each strain was subjected to pulse-chase labeling, immunoprecipitation, SDS-PAGE, phosphorimaging, and  turnover t1/2 calculation. In terms of experimental variability,  half-lives calculated from different experiments were within 20%  of the reported value for each mutant. Different conditions were  required to assess receptor ubiquitination levels. Cells of the  MATα ste3Δ pep4Δ strain SY2602 transformed by either pSL552  (GAL1-STE3) or by the identical plasmid with the indicated  STE3 deletion allele in place of the wild-type STE3 were grown  and processed as described for Fig. 4 A. Quantitation of the proportion of receptor antigen localizing to the putative mono- and  di-ubiquitinated receptor species was as described in the Materials and Methods. The values reported in the far right-hand column represent the combined sum of the receptor antigen present  in the mono- and di-ubiquitinated forms. For the Δ398-468 receptor mutant the proportion of receptor ubiquitinated was not determined (nd).
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Figure 3: Summary of turnover rates and the proportion of receptor proteins that are ubiquitinated for a collection of STE3 deletion mutants constructed within the COOH-terminal 73 amino acid residues of Ste3p. Shown in the upper left is the hypothesized transmembrane structure for Ste3p. The region corresponding to the COOH-terminal 57 residues of the Ste3p CTD is highlighted, the 414–451 interval indicated with a striped box and the 451–470 interval by a stippled box. Shown below are sequences deleted (dotted line) for each of the different in-frame STE3 deletion mutants tested. To the right, the exact amino acids residues removed for each mutant are indicated. The two columns at the right summarize the results of the two different experiments that were performed for each receptor, these being: (1) measurement of the turnover t1/2, and (2) measurement of the proportion of the receptors found to be ubiquitinated. Measurement of turnover rate used a collection of strains isogenic to NDY334, except for the indicated STE3 mutant alleles. As described in Fig. 2, each strain was subjected to pulse-chase labeling, immunoprecipitation, SDS-PAGE, phosphorimaging, and turnover t1/2 calculation. In terms of experimental variability, half-lives calculated from different experiments were within 20% of the reported value for each mutant. Different conditions were required to assess receptor ubiquitination levels. Cells of the MATα ste3Δ pep4Δ strain SY2602 transformed by either pSL552 (GAL1-STE3) or by the identical plasmid with the indicated STE3 deletion allele in place of the wild-type STE3 were grown and processed as described for Fig. 4 A. Quantitation of the proportion of receptor antigen localizing to the putative mono- and di-ubiquitinated receptor species was as described in the Materials and Methods. The values reported in the far right-hand column represent the combined sum of the receptor antigen present in the mono- and di-ubiquitinated forms. For the Δ398-468 receptor mutant the proportion of receptor ubiquitinated was not determined (nd).

Mentions: Quantitating the proportion of receptor proteins with attached ubiquitin was by Western analysis of phosphatase-treated protein extracts for the various STE3 mutants. Quantitative AMBIS densitometry of films (AMBIS Systems, Inc., San Diego, CA) resulting from ECL development of the Western blot (Amersham Corp., Arlington Heights, IL) was used to estimate the relative amount of the receptor protein present in the two ubiquitin-modified electrophoretic positions (the presumptive mono- and di-ubiquitinated forms; Roth and Davis, 1996), and at the unmodified position. Many aspects of this analysis rely on techniques wherein the measured output is produced non-linearly. To circumvent this potential source of error, we have compared signals only of similar intensity levels. This was achieved by applying the Western analysis to a dilution series for each sample. For instance, with phosphatase-treated extracts derived from wild-type Ste3p, the intensity level of the signal associated with the mono-ubiquitinated band was found to be intermediate between the level of the unmodified receptor band present from the 1:5 and 1:25 dilutions of the same extract. Quantitative densitometry of the band signals from the same compared samples led to the determination that 10% of the wild-type receptor was present in the mono-ubiquitinated species band. As 10% was also present in the di-ubiquitinated band, the summed estimate of 20% was reported in Fig. 3 as proportion of receptor ubiquitinated.


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)

Summary of turnover rates and the proportion of receptor proteins that are ubiquitinated for a collection of STE3  deletion mutants constructed within the COOH-terminal 73  amino acid residues of Ste3p. Shown in the upper left is the hypothesized transmembrane structure for Ste3p. The region corresponding to the COOH-terminal 57 residues of the Ste3p CTD is  highlighted, the 414–451 interval indicated with a striped box and  the 451–470 interval by a stippled box. Shown below are sequences deleted (dotted line) for each of the different in-frame  STE3 deletion mutants tested. To the right, the exact amino acids  residues removed for each mutant are indicated. The two columns at the right summarize the results of the two different experiments that were performed for each receptor, these being: (1)  measurement of the turnover t1/2, and (2) measurement of the  proportion of the receptors found to be ubiquitinated. Measurement of turnover rate used a collection of strains isogenic to  NDY334, except for the indicated STE3 mutant alleles. As described in Fig. 2, each strain was subjected to pulse-chase labeling, immunoprecipitation, SDS-PAGE, phosphorimaging, and  turnover t1/2 calculation. In terms of experimental variability,  half-lives calculated from different experiments were within 20%  of the reported value for each mutant. Different conditions were  required to assess receptor ubiquitination levels. Cells of the  MATα ste3Δ pep4Δ strain SY2602 transformed by either pSL552  (GAL1-STE3) or by the identical plasmid with the indicated  STE3 deletion allele in place of the wild-type STE3 were grown  and processed as described for Fig. 4 A. Quantitation of the proportion of receptor antigen localizing to the putative mono- and  di-ubiquitinated receptor species was as described in the Materials and Methods. The values reported in the far right-hand column represent the combined sum of the receptor antigen present  in the mono- and di-ubiquitinated forms. For the Δ398-468 receptor mutant the proportion of receptor ubiquitinated was not determined (nd).
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Related In: Results  -  Collection

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Figure 3: Summary of turnover rates and the proportion of receptor proteins that are ubiquitinated for a collection of STE3 deletion mutants constructed within the COOH-terminal 73 amino acid residues of Ste3p. Shown in the upper left is the hypothesized transmembrane structure for Ste3p. The region corresponding to the COOH-terminal 57 residues of the Ste3p CTD is highlighted, the 414–451 interval indicated with a striped box and the 451–470 interval by a stippled box. Shown below are sequences deleted (dotted line) for each of the different in-frame STE3 deletion mutants tested. To the right, the exact amino acids residues removed for each mutant are indicated. The two columns at the right summarize the results of the two different experiments that were performed for each receptor, these being: (1) measurement of the turnover t1/2, and (2) measurement of the proportion of the receptors found to be ubiquitinated. Measurement of turnover rate used a collection of strains isogenic to NDY334, except for the indicated STE3 mutant alleles. As described in Fig. 2, each strain was subjected to pulse-chase labeling, immunoprecipitation, SDS-PAGE, phosphorimaging, and turnover t1/2 calculation. In terms of experimental variability, half-lives calculated from different experiments were within 20% of the reported value for each mutant. Different conditions were required to assess receptor ubiquitination levels. Cells of the MATα ste3Δ pep4Δ strain SY2602 transformed by either pSL552 (GAL1-STE3) or by the identical plasmid with the indicated STE3 deletion allele in place of the wild-type STE3 were grown and processed as described for Fig. 4 A. Quantitation of the proportion of receptor antigen localizing to the putative mono- and di-ubiquitinated receptor species was as described in the Materials and Methods. The values reported in the far right-hand column represent the combined sum of the receptor antigen present in the mono- and di-ubiquitinated forms. For the Δ398-468 receptor mutant the proportion of receptor ubiquitinated was not determined (nd).
Mentions: Quantitating the proportion of receptor proteins with attached ubiquitin was by Western analysis of phosphatase-treated protein extracts for the various STE3 mutants. Quantitative AMBIS densitometry of films (AMBIS Systems, Inc., San Diego, CA) resulting from ECL development of the Western blot (Amersham Corp., Arlington Heights, IL) was used to estimate the relative amount of the receptor protein present in the two ubiquitin-modified electrophoretic positions (the presumptive mono- and di-ubiquitinated forms; Roth and Davis, 1996), and at the unmodified position. Many aspects of this analysis rely on techniques wherein the measured output is produced non-linearly. To circumvent this potential source of error, we have compared signals only of similar intensity levels. This was achieved by applying the Western analysis to a dilution series for each sample. For instance, with phosphatase-treated extracts derived from wild-type Ste3p, the intensity level of the signal associated with the mono-ubiquitinated band was found to be intermediate between the level of the unmodified receptor band present from the 1:5 and 1:25 dilutions of the same extract. Quantitative densitometry of the band signals from the same compared samples led to the determination that 10% of the wild-type receptor was present in the mono-ubiquitinated species band. As 10% was also present in the di-ubiquitinated band, the summed estimate of 20% was reported in Fig. 3 as proportion of receptor ubiquitinated.

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