Remodeling Membrane Binding by Mono-Ubiquitylation.

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.

E. coli-Based Selection and Expression Systems for Discovery, Characterization, and Purification of Ubiquitylated Proteins.

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.

A bacterial genetic selection system for ubiquitylation cascade discovery.

About one-third of the eukaryotic proteome undergoes ubiquitylation, but the enzymatic cascades leading to substrate modification are largely unknown. We present a genetic selection tool that utilizes Escherichia coli, which lack deubiquitylases, to identify interactions along ubiquitylation cascades. Coexpression of split antibiotic resistance protein tethered to ubiquitin and ubiquitylation target together with a functional ubiquitylation apparatus results in a covalent assembly of the resistance protein, giving rise to bacterial growth on selective media.

Purification and crystallization of yeast Ent1 ENTH domain.

Members of the Epsin protein family regulate the ubiquitin/clathrin-dependent trafficking of transmembrane proteins. The yeast Epsin-1 (ent1) gene was cloned and expressed in Escherichia coli. The protein product of a construct containing the ENTH-UIM modules was purified to homogeneity and subjected to crystallization screening using the sitting-drop vapour-diffusion method.

Synthetic biology approach to reconstituting the ubiquitylation cascade in bacteria.

Covalent modification of proteins with ubiquitin (Ub) is widely implicated in the control of protein function and fate. Over 100 deubiquitylating enzymes rapidly reverse this modification, posing challenges to the biochemical and biophysical characterization of ubiquitylated proteins. We circumvented this limitation with a synthetic biology approach of reconstructing the entire eukaryotic Ub cascade in bacteria.