Ubiquitin (Ub) receptors respond to ubiquitylation signals. They bind ubiquitylated substrates and exert their activity in situ. Intriguingly, Ub receptors themselves undergo rapid ubiquitylation and deubiquitylation. Here we asked what is the function of ubiquitylation of Ub receptors? We focused on yeast epsin, a Ub receptor that decodes the ubiquitylation signal of plasma membrane proteins into an endocytosis response. Using mass spectrometry, we identified lysine-3 as the major ubiquitylation site in the epsin plasma membrane binding domain. By projecting this ubiquitylation site onto our crystal structure, we hypothesized that this modification would compete with phosphatidylinositol-4,5-bisphosphate (PIP2) binding and dissociate epsin from the membrane. Using an E. coli-based expression of an authentic ubiquitylation apparatus, we purified ubiquitylated epsin. We demonstrated in vitro that in contrast to apo epsin, the ubiquitylated epsin does not bind to either immobilized PIPs or PIP2-enriched liposomes. To test this hypothesis in vivo, we mimicked ubiquitylation by the fusion of Ub at the ubiquitylation site. Live cell imaging demonstrated that the mimicked ubiquitylated epsin dissociates from the membrane. Our findings suggest that ubiquitylation of the Ub receptors dissociates them from their products to allow binding to a new ubiquitylated substrates, consequently promoting cyclic activity of the Ub receptors.
- MeSH
- buněčná membrána metabolismus MeSH
- molekulární modely MeSH
- proteinové domény MeSH
- Saccharomyces cerevisiae - proteiny chemie metabolismus MeSH
- ubikvitinace * MeSH
- vazba proteinů MeSH
- vezikulární transportní proteiny chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Ubiquitylation is an eukaryotic signal that regulates most cellular pathways. However, four major hurdles pose challenges to study ubiquitylation: (1) high redundancy between ubiquitin (Ub) cascades, (2) ubiquitylation is tightly regulated in the cell, (3) the transient nature of the Ub signal, and (4) difficulties to purify functional ubiquitylation apparatus for in vitro assay. Here, we present systems that express functional Ub cascades in E. coli, which lacks deubiquitylases, Ub-dependent degradations, and control mechanisms for ubiquitylation. Therefore, expression of an ubiquitylation cascade results in the accumulation of stable ubiquitylated protein that can be genetically selected or purified, thus circumventing the above challenges. Co-expression of split antibiotic resistance protein fragments tethered to Ub and ubiquitylation targets along with ubiquitylation enzymes (E1, E2, and E3) gives rise to bacterial growth on selective media. We show that ubiquitylation rate is highly correlated with growth efficiency. Hence, genetic libraries and simple manipulations in the selection system facilitate the identification and characterization of components and interfaces along Ub cascades. The bacterial expression system also facilitates the detection of ubiquitylated proteins. Furthermore, the expression system allows affinity chromatography-based purification of milligram quantities of ubiquitylated proteins for downstream biochemical, biophysical, and structural studies.
- MeSH
- Escherichia coli genetika metabolismus MeSH
- exprese genu * MeSH
- genetické vektory genetika MeSH
- konformace proteinů MeSH
- molekulární modely MeSH
- pořadí genů MeSH
- proteiny chemie genetika izolace a purifikace metabolismus MeSH
- ubikvitin aktivující enzymy metabolismus MeSH
- ubikvitin konjugující enzymy metabolismus MeSH
- ubikvitin metabolismus MeSH
- ubikvitinace MeSH
- ubikvitinligasy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
