Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of the triple alanine mutations on the turnover  rate of Ste3Δ450-468p. The amino acid sequence of the 45  COOH-terminal residues of Ste3Δ450-468p is shown. Numbers  above correspond to residue numbering of wild-type Ste3p and  the minimal signal domain residues 414–449 is highlighted with  bold face type. The Δ450–468 deletion is indicated with the leucine-aspartate-arginine (LDR) tripeptide in place of the removed  19 STE3 residues. 12 mutants alleles were constructed each having three consecutive residues (underlined) replaced by three alanines. Strains isogenic with NDY334 except for the mutant triple  alanine STE3 allele replacement at the STE3 locus were subjected to pulse-chase analysis identical to that of Fig. 2. The turnover t1/2 calculated for each is indicated below.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132879&req=5

Figure 8: Effect of the triple alanine mutations on the turnover rate of Ste3Δ450-468p. The amino acid sequence of the 45 COOH-terminal residues of Ste3Δ450-468p is shown. Numbers above correspond to residue numbering of wild-type Ste3p and the minimal signal domain residues 414–449 is highlighted with bold face type. The Δ450–468 deletion is indicated with the leucine-aspartate-arginine (LDR) tripeptide in place of the removed 19 STE3 residues. 12 mutants alleles were constructed each having three consecutive residues (underlined) replaced by three alanines. Strains isogenic with NDY334 except for the mutant triple alanine STE3 allele replacement at the STE3 locus were subjected to pulse-chase analysis identical to that of Fig. 2. The turnover t1/2 calculated for each is indicated below.

Mentions: The 12 triple alanine mutations created within the 414–449 interval of STE3 (see Fig. 8) also were constructed via oligonucleotide-directed mutagenesis (Kunkel et al., 1987). For each, three adjacent STE3 codons are replaced by the sequence GCTGCAGCC, encoding three consecutive alanines. This substitution also introduces a PstI site, allowing identification of the mutant clone by restriction analysis. The ssDNA template for mutagenesis was from pND210, the Δ450–468 mutant version of pSL1839. Fidelity of mutagenesis was confirmed for each via DNA sequencing in the vicinity of mutation site.


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)

Effect of the triple alanine mutations on the turnover  rate of Ste3Δ450-468p. The amino acid sequence of the 45  COOH-terminal residues of Ste3Δ450-468p is shown. Numbers  above correspond to residue numbering of wild-type Ste3p and  the minimal signal domain residues 414–449 is highlighted with  bold face type. The Δ450–468 deletion is indicated with the leucine-aspartate-arginine (LDR) tripeptide in place of the removed  19 STE3 residues. 12 mutants alleles were constructed each having three consecutive residues (underlined) replaced by three alanines. Strains isogenic with NDY334 except for the mutant triple  alanine STE3 allele replacement at the STE3 locus were subjected to pulse-chase analysis identical to that of Fig. 2. The turnover t1/2 calculated for each is indicated below.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Effect of the triple alanine mutations on the turnover rate of Ste3Δ450-468p. The amino acid sequence of the 45 COOH-terminal residues of Ste3Δ450-468p is shown. Numbers above correspond to residue numbering of wild-type Ste3p and the minimal signal domain residues 414–449 is highlighted with bold face type. The Δ450–468 deletion is indicated with the leucine-aspartate-arginine (LDR) tripeptide in place of the removed 19 STE3 residues. 12 mutants alleles were constructed each having three consecutive residues (underlined) replaced by three alanines. Strains isogenic with NDY334 except for the mutant triple alanine STE3 allele replacement at the STE3 locus were subjected to pulse-chase analysis identical to that of Fig. 2. The turnover t1/2 calculated for each is indicated below.
Mentions: The 12 triple alanine mutations created within the 414–449 interval of STE3 (see Fig. 8) also were constructed via oligonucleotide-directed mutagenesis (Kunkel et al., 1987). For each, three adjacent STE3 codons are replaced by the sequence GCTGCAGCC, encoding three consecutive alanines. This substitution also introduces a PstI site, allowing identification of the mutant clone by restriction analysis. The ssDNA template for mutagenesis was from pND210, the Δ450–468 mutant version of pSL1839. Fidelity of mutagenesis was confirmed for each via DNA sequencing in the vicinity of mutation site.

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