Remodeling Membrane Binding by Mono-Ubiquitylation

Biomolecules
Neta TannerGali Prag

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 finding...Continue Reading

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Citations

Sep 23, 2020·The Journal of Clinical Investigation·Kai SongHong Chen

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Methods Mentioned

BETA
DNA shearing
size-exclusion chromatography
reverse-phase chromatography
acetylation
Pull-Down
PCR
PIP-Array

Software Mentioned

Trans Proteomic Pipeline ( TPP )
PyMol
Protein Prophet
Refmac5
! Tandem
Protein Homology /
Peptide Prophet
analogY Recognition Engine
Coot

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