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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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Stability of different PMA1-STE3 fusion proteins.  Turnover analysis performed on cells of the MATα strain SY1574  transformed either by a CEN/ARS plasmid carrying an HA  epitope-tagged version of Pma1p under the control of the GAL1  promoter (pND542) is shown in the top panel. The five panels  below show the same analysis applied to cells transformed by five  versions of the pND542 plasmid, having the two COOH-terminal  Pma1p codons replaced by the indicated Ste3p sequence interval.  For this analysis, galactose (2%) was added for 1 h, followed by  the addition of glucose to 3%, and at the indicated times thereafter, culture aliquots were removed and protein extracts prepared.  Extracts were subjected to SDS-PAGE, followed by immunoblot  analysis using anti-HA mAb.
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Figure 5: Stability of different PMA1-STE3 fusion proteins. Turnover analysis performed on cells of the MATα strain SY1574 transformed either by a CEN/ARS plasmid carrying an HA epitope-tagged version of Pma1p under the control of the GAL1 promoter (pND542) is shown in the top panel. The five panels below show the same analysis applied to cells transformed by five versions of the pND542 plasmid, having the two COOH-terminal Pma1p codons replaced by the indicated Ste3p sequence interval. For this analysis, galactose (2%) was added for 1 h, followed by the addition of glucose to 3%, and at the indicated times thereafter, culture aliquots were removed and protein extracts prepared. Extracts were subjected to SDS-PAGE, followed by immunoblot analysis using anti-HA mAb.

Mentions: Five different PMA1-STE3 fusion proteins were constructed, each with a different segment of the STE3 CTD fused at the COOH terminus of PMA1. The parental HA-tagged Pma1p showed no evidence of turnover during the 2-h course of this experiment (Fig. 5). Attachment of the 347–398 STE3 sequence interval was without effect; this PMA1-STE3 fusion protein shows a stability equivalent to that of the starting PMA1 construct (Fig. 5). In contrast, attachment of the 400–470 interval, encoding the COOH-terminal 71 amino acids of Ste3p, does destabilize the resulting PMA1-STE3 fusion protein (Fig. 5). This fusion protein is degraded with a t1/2 of 40 min (Table II). Likewise, the fusion carrying the 400–449 STE3 sequence interval also turns over (Fig. 5); a t1/2 of 50 min is calculated for this protein (Table II). Turnover slows dramatically when the critical 414–449 interval is violated as it is for PMA1-STE3(400–440) (Fig. 5); a t1/2 of 90 min is calculated (Table II). Finally, removal of the entire 414–449 interval, as for PMA1-STE3(400–412), effectively abolishes turnover (Fig. 5; Table II).


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)

Stability of different PMA1-STE3 fusion proteins.  Turnover analysis performed on cells of the MATα strain SY1574  transformed either by a CEN/ARS plasmid carrying an HA  epitope-tagged version of Pma1p under the control of the GAL1  promoter (pND542) is shown in the top panel. The five panels  below show the same analysis applied to cells transformed by five  versions of the pND542 plasmid, having the two COOH-terminal  Pma1p codons replaced by the indicated Ste3p sequence interval.  For this analysis, galactose (2%) was added for 1 h, followed by  the addition of glucose to 3%, and at the indicated times thereafter, culture aliquots were removed and protein extracts prepared.  Extracts were subjected to SDS-PAGE, followed by immunoblot  analysis using anti-HA mAb.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132879&req=5

Figure 5: Stability of different PMA1-STE3 fusion proteins. Turnover analysis performed on cells of the MATα strain SY1574 transformed either by a CEN/ARS plasmid carrying an HA epitope-tagged version of Pma1p under the control of the GAL1 promoter (pND542) is shown in the top panel. The five panels below show the same analysis applied to cells transformed by five versions of the pND542 plasmid, having the two COOH-terminal Pma1p codons replaced by the indicated Ste3p sequence interval. For this analysis, galactose (2%) was added for 1 h, followed by the addition of glucose to 3%, and at the indicated times thereafter, culture aliquots were removed and protein extracts prepared. Extracts were subjected to SDS-PAGE, followed by immunoblot analysis using anti-HA mAb.
Mentions: Five different PMA1-STE3 fusion proteins were constructed, each with a different segment of the STE3 CTD fused at the COOH terminus of PMA1. The parental HA-tagged Pma1p showed no evidence of turnover during the 2-h course of this experiment (Fig. 5). Attachment of the 347–398 STE3 sequence interval was without effect; this PMA1-STE3 fusion protein shows a stability equivalent to that of the starting PMA1 construct (Fig. 5). In contrast, attachment of the 400–470 interval, encoding the COOH-terminal 71 amino acids of Ste3p, does destabilize the resulting PMA1-STE3 fusion protein (Fig. 5). This fusion protein is degraded with a t1/2 of 40 min (Table II). Likewise, the fusion carrying the 400–449 STE3 sequence interval also turns over (Fig. 5); a t1/2 of 50 min is calculated for this protein (Table II). Turnover slows dramatically when the critical 414–449 interval is violated as it is for PMA1-STE3(400–440) (Fig. 5); a t1/2 of 90 min is calculated (Table II). Finally, removal of the entire 414–449 interval, as for PMA1-STE3(400–412), effectively abolishes turnover (Fig. 5; Table II).

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