Abstract
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.
Highlights
Ubiquitylation regulates most cellular pathways in eukaryotes [1]
To reassess whether Ent1 undergoes mono-ubiquitylation in vivo, we transformed yeast cells with with a plasmid that over-expresses RGS (Arg-Gly-Ser) –His8–UbK0 (in which all seven lysine residues a plasmid that over-expresses RGS (Arg-Gly-Ser) –His8 –UbK0 and extracted the ubiquitylated proteins from whole-cell lysate using substituted for arginine) and extracted the ubiquitylated proteins from whole-cell lysate using rapid rapid TCA (TriChloroacetic Acid) lysis [16]
Coli-based expression system for the ubiquitylation cascade allowed us to purify milligram quantities of ubiquitylated proteins, which played a pivotal role in this work [7]
Summary
Ubiquitylation regulates most cellular pathways in eukaryotes [1]. Ubiquitin (Ub) signals are written and edited by a multiplexed network of E1 (Ub-activating enzyme), E2 (Ub-conjugating enzyme), E3 (Ub-ligase), and deubiquitylating enzymes in a tightly regulated manner. The same Ub molecules code many different cellular outcomes [2]. How do cells correctly interpret these Ub codes? It has become evident that hundreds of Ub receptors read and respond to Ub signals by tethering Ub binding domains (UBDs) to a “response element”. Ub receptors possess modules that sense the cellular context (hereafter “context domain”), which allows them to function in an appropriate spatiotemporal manner. Ub receptors are themselves regulated by ubiquitylation [3,4,5].
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