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

Effects of pep4 and end4 mutations on the turnover of  the PMA1-STE3(400–449) fusion protein. For the experiment in  the left panel, the GAL1-HA-PMA1-STE3(400–449) plasmid described for Fig. 5 was used to transform either the wild-type  MATα strain SY1574 (PEP4+) or the isogenic pep4Δ version  SY2601. For the experiment shown in the right-hand panel, the  same plasmid was used to transform either the wild-type MATα  strain NDY334 (END4+) or the isogenic end4-1 version  NDY335. For both experiments, after a 2-h exposure to galactose  (2%), glucose was added to 3%, and at the indicated times thereafter, culture aliquots were removed and protein extracts were  prepared. Extracts were subjected to SDS-PAGE, followed by  immunoblot analysis using anti-HA mAb. The arrowhead to the  right of the right-hand panel indicates the position of a modified  form of the PMA1-STE3(400–449) fusion protein that is accentuated in extracts from the end4 cells.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132879&req=5

Figure 6: Effects of pep4 and end4 mutations on the turnover of the PMA1-STE3(400–449) fusion protein. For the experiment in the left panel, the GAL1-HA-PMA1-STE3(400–449) plasmid described for Fig. 5 was used to transform either the wild-type MATα strain SY1574 (PEP4+) or the isogenic pep4Δ version SY2601. For the experiment shown in the right-hand panel, the same plasmid was used to transform either the wild-type MATα strain NDY334 (END4+) or the isogenic end4-1 version NDY335. For both experiments, after a 2-h exposure to galactose (2%), glucose was added to 3%, and at the indicated times thereafter, culture aliquots were removed and protein extracts were prepared. Extracts were subjected to SDS-PAGE, followed by immunoblot analysis using anti-HA mAb. The arrowhead to the right of the right-hand panel indicates the position of a modified form of the PMA1-STE3(400–449) fusion protein that is accentuated in extracts from the end4 cells.

Mentions: Degradation of Ste3p depends on transport from the cell surface to the vacuole (Davis et al., 1993; Roth and Davis, 1996). Is the turnover of the PMA1-STE3 constructs instigated by a similar route of transport? To test involvement of vacuolar proteases in PMA1-STE3 fusion turnover, constructs with the STE3 400–449 peptide sequence interval were introduced into isogenic PEP4+ and pep4Δ strains and turnover assessed. While turnover proceeds in the PEP4+ background, in the pep4Δ context, turnover is completely blocked (Fig. 6, left panel). This is also true for the longer PMA1-STE3(400–470) fusion protein (data not shown). Thus, like wild-type Ste3p, turnover of these PMA1-STE3 fusions also depends on vacuolar proteases and therefore also likely depends on transport to the vacuole.


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)

Effects of pep4 and end4 mutations on the turnover of  the PMA1-STE3(400–449) fusion protein. For the experiment in  the left panel, the GAL1-HA-PMA1-STE3(400–449) plasmid described for Fig. 5 was used to transform either the wild-type  MATα strain SY1574 (PEP4+) or the isogenic pep4Δ version  SY2601. For the experiment shown in the right-hand panel, the  same plasmid was used to transform either the wild-type MATα  strain NDY334 (END4+) or the isogenic end4-1 version  NDY335. For both experiments, after a 2-h exposure to galactose  (2%), glucose was added to 3%, and at the indicated times thereafter, culture aliquots were removed and protein extracts were  prepared. Extracts were subjected to SDS-PAGE, followed by  immunoblot analysis using anti-HA mAb. The arrowhead to the  right of the right-hand panel indicates the position of a modified  form of the PMA1-STE3(400–449) fusion protein that is accentuated in extracts from the end4 cells.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Effects of pep4 and end4 mutations on the turnover of the PMA1-STE3(400–449) fusion protein. For the experiment in the left panel, the GAL1-HA-PMA1-STE3(400–449) plasmid described for Fig. 5 was used to transform either the wild-type MATα strain SY1574 (PEP4+) or the isogenic pep4Δ version SY2601. For the experiment shown in the right-hand panel, the same plasmid was used to transform either the wild-type MATα strain NDY334 (END4+) or the isogenic end4-1 version NDY335. For both experiments, after a 2-h exposure to galactose (2%), glucose was added to 3%, and at the indicated times thereafter, culture aliquots were removed and protein extracts were prepared. Extracts were subjected to SDS-PAGE, followed by immunoblot analysis using anti-HA mAb. The arrowhead to the right of the right-hand panel indicates the position of a modified form of the PMA1-STE3(400–449) fusion protein that is accentuated in extracts from the end4 cells.
Mentions: Degradation of Ste3p depends on transport from the cell surface to the vacuole (Davis et al., 1993; Roth and Davis, 1996). Is the turnover of the PMA1-STE3 constructs instigated by a similar route of transport? To test involvement of vacuolar proteases in PMA1-STE3 fusion turnover, constructs with the STE3 400–449 peptide sequence interval were introduced into isogenic PEP4+ and pep4Δ strains and turnover assessed. While turnover proceeds in the PEP4+ background, in the pep4Δ context, turnover is completely blocked (Fig. 6, left panel). This is also true for the longer PMA1-STE3(400–470) fusion protein (data not shown). Thus, like wild-type Ste3p, turnover of these PMA1-STE3 fusions also depends on vacuolar proteases and therefore also likely depends on transport to the vacuole.

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