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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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Comparison of effects on turnover rate of mutants constructed in the Ste3Δ450-468p receptor versus the wild-type Ste3p  receptor context. Cells of strains isogenic to NDY334, except for  the transplacement of the wild-type STE3 by the indicated STE3  mutant allele, were subjected to a pulse-chase analysis equivalent  to that of Fig. 1. (A) Comparison of effects on turnover of two triple alanine mutations constructed either in the Ste3Δ450-468p  context (top panel) or in the wild-type Ste3p context (bottom  panel). The two triple alanine substitutions used for this experiment replace either the 426–428 threonine-leucine-aspartate tripeptide or the 417–419 isoleucine-serine-leucine tripeptide. (B)  Comparison of the effects of the Δ441–450 deletion constructed  either in the Ste3Δ450-468p receptor context or in the wild-type  Ste3p context.
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Figure 9: Comparison of effects on turnover rate of mutants constructed in the Ste3Δ450-468p receptor versus the wild-type Ste3p receptor context. Cells of strains isogenic to NDY334, except for the transplacement of the wild-type STE3 by the indicated STE3 mutant allele, were subjected to a pulse-chase analysis equivalent to that of Fig. 1. (A) Comparison of effects on turnover of two triple alanine mutations constructed either in the Ste3Δ450-468p context (top panel) or in the wild-type Ste3p context (bottom panel). The two triple alanine substitutions used for this experiment replace either the 426–428 threonine-leucine-aspartate tripeptide or the 417–419 isoleucine-serine-leucine tripeptide. (B) Comparison of the effects of the Δ441–450 deletion constructed either in the Ste3Δ450-468p receptor context or in the wild-type Ste3p context.

Mentions: Two of the triple alanine mutations were also constructed within the context of wild-type (i.e., full-length) STE3. Reconstruction of STE3 with either the 417–419 or 426–428 triple alanine mutations (see Fig. 9) used the HindIII site at codons 433, 434, allowing the wild-type COOH-terminal STE3 sequences to be restored to the STE3Δ450–468 versions of these mutations.


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)

Comparison of effects on turnover rate of mutants constructed in the Ste3Δ450-468p receptor versus the wild-type Ste3p  receptor context. Cells of strains isogenic to NDY334, except for  the transplacement of the wild-type STE3 by the indicated STE3  mutant allele, were subjected to a pulse-chase analysis equivalent  to that of Fig. 1. (A) Comparison of effects on turnover of two triple alanine mutations constructed either in the Ste3Δ450-468p  context (top panel) or in the wild-type Ste3p context (bottom  panel). The two triple alanine substitutions used for this experiment replace either the 426–428 threonine-leucine-aspartate tripeptide or the 417–419 isoleucine-serine-leucine tripeptide. (B)  Comparison of the effects of the Δ441–450 deletion constructed  either in the Ste3Δ450-468p receptor context or in the wild-type  Ste3p context.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 9: Comparison of effects on turnover rate of mutants constructed in the Ste3Δ450-468p receptor versus the wild-type Ste3p receptor context. Cells of strains isogenic to NDY334, except for the transplacement of the wild-type STE3 by the indicated STE3 mutant allele, were subjected to a pulse-chase analysis equivalent to that of Fig. 1. (A) Comparison of effects on turnover of two triple alanine mutations constructed either in the Ste3Δ450-468p context (top panel) or in the wild-type Ste3p context (bottom panel). The two triple alanine substitutions used for this experiment replace either the 426–428 threonine-leucine-aspartate tripeptide or the 417–419 isoleucine-serine-leucine tripeptide. (B) Comparison of the effects of the Δ441–450 deletion constructed either in the Ste3Δ450-468p receptor context or in the wild-type Ste3p context.
Mentions: Two of the triple alanine mutations were also constructed within the context of wild-type (i.e., full-length) STE3. Reconstruction of STE3 with either the 417–419 or 426–428 triple alanine mutations (see Fig. 9) used the HindIII site at codons 433, 434, allowing the wild-type COOH-terminal STE3 sequences to be restored to the STE3Δ450–468 versions of these mutations.

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