Ste4 is the β subunit of a heterotrimeric G protein that mediates mating responses in Saccharomyces cerevisiae. Here we show that Ste4 undergoes ubiquitination in response to pheromone stimulation. Ubiquitination of Ste4 is dependent on the E3 ligase Rsp5. Disrupting the activity of Rsp5 abolishes ubiquitination of Ste4 in vivo, and recombinant Rsp5 is capable of ubiquitinating Ste4 in vitro. We find also that Lys-340 is a major ubiquitination site on Ste4, as pheromone-induced ubiquitination of the protein is prevented when this residue is mutated to an arginine. Functionally, ubiquitination does not appear to regulate the stability of Ste4, as blocking ubiquitination has no apparent effect on either the abundance or the half-life of the protein. However, when presented with a concentration gradient of pheromone, Ste4(K340R) mutant cells polarize significantly faster than wild-type cells, indicating that ubiquitination limits pheromone-directed polarized growth. Together, these findings reveal a novel stimulus-dependent posttranslational modification of a Gβ subunit, establish Ste4 as a new substrate of the E3 ligase Rsp5, and demonstrate a role for G protein ubiquitination in cell polarization.
Increase the total number of rows showing on this page using the pull-down located below the table, or use the page scroll at the table's top right to browse through the table's pages; use the arrows to the right of a column header to sort by that column; filter the table using the "Filter" box at the top of the table.
Increase the total number of rows showing on this page using the pull-down located below the table, or use the page scroll at the table's top right to browse through the table's pages; use the arrows to the right of a column header to sort by that column; filter the table using the "Filter" box at the top of the table; click on the small "i" buttons located within a cell for an annotation to view further details.
| Gene | Phenotype | Experiment Type | Mutant Information | Strain Background | Chemical | Details |
|---|---|---|---|---|---|---|
| RSP5 | protein/peptide modification: absent Reporter: Ste4p | classical genetics | repressible | S288C | Treatment: alpha-factor Details: monoubiquitination of Ste4p is completely blocked | |
| RSP5 | protein/peptide modification: absent Reporter: Ste4p | classical genetics | conditional Allele: rsp5-1 L733S (nucleotide T2198C); HECT domain mutated | S288C | Temperature: elevated temperature, 37 °C Treatment: alpha-factor Details: monoubiquitination of Ste4p is completely blocked | |
| STE4 | shmoo formation: increased rate | classical genetics | unspecified Allele: ste4-K340R pheromone-induced monoubiquitination site | S288C | Treatment: alpha-factor Details: enhanced rate of alpha-factor induced initiation of polarized growth |
Increase the total number of rows displayed on this page using the pull-down located below the table, or use the page scroll at the table's top right to browse through the table's pages; use the arrows to the right of a column header to sort by that column; to filter the table by a specific experiment type, type a keyword into the Filter box (for example, “microarray”); download this table as a .txt file using the Download button or click Analyze to further view and analyze the list of target genes using GO Term Finder, GO Slim Mapper, or SPELL.
Increase the total number of rows showing on this page by using the pull-down located below the table, or use the page scroll at the table's top right to browse through its pages; use the arrows to the right of a column header to sort by that column; filter the table using the "Filter" box at the top of the table.
Increase the total number of rows showing on this page by using the pull-down located below the table, or use the page scroll at the table's top right to browse through the table's pages; use the arrows to the right of a column header to sort by that column; filter the table using the "Filter" box at the top of the table; click on the small "i" buttons located within a cell for an annotation to view further details about experiment type and any other genes involved in the interaction.
| Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
|---|
Increase the total number of rows showing on this page by using the pull-down located below the table, or use the page scroll at the table's top right to browse through the table's pages; use the arrows to the right of a column header to sort by that column; filter the table using the "Filter" box at the top of the table; click on the small "i" buttons located within a cell for an annotation to view further details about experiment type and any other genes involved in the interaction.
| Interactor | Interactor | Assay | Annotation | Action | Modification | |
|---|---|---|---|---|---|---|
| RSP5 | STE4 | Affinity Capture-Western | manually curated | Bait-Hit | No Modification | |
| RSP5 | STE4 | Biochemical Activity | manually curated | Bait-Hit | ubiquitinylated lysine | |
absence or presence of E1 activating enzyme (Uba1). All reactions contained Ubc5b, ATP, and ubiquitin. | RSP5 | UBC5 | Reconstituted Complex | manually curated | Bait-Hit | No Modification |