Regulation of the RSP5 Ubiquitin Ligase by an Intrinsic Ubiquitin-binding Site

Michael E French,Benjamin R Kretzmann,Linda Hicke

doi:10.1074/jbc.m901106200

Michael E French, Benjamin R Kretzmann + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m901106200

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Abstract

Rsp5 is a homologous to E6AP C terminus (HECT) ubiquitin ligase (E3) that controls many different cellular processes in budding yeast. Although Rsp5 targets a number of different substrates for ubiquitination, the mechanisms that regulate Rsp5 activity remain poorly understood. Here we demonstrate that Rsp5 carries a noncovalent ubiquitin-binding site in its catalytic HECT domain. The N-terminal lobe of the HECT domain mediates binding to ubiquitin, and point mutations that disrupt interactions with ubiquitin alter the ability of the Rsp5 HECT domain to assemble polyubiquitin chains in vitro. Point mutations that disrupt ubiquitin binding also result in temperature-sensitive growth defects in yeast, indicating that the Rsp5 ubiquitin-binding site is important for Rsp5 function in vivo. The Nedd4 HECT domain N-lobe also contains ubiquitin-binding activity, suggesting that interactions between the N-lobe and ubiquitin are conserved within the Nedd4 family of ubiquitin ligases. We propose that a subset of HECT E3s are regulated by a conserved ubiquitin-binding site that functions to restrict the length of polyubiquitin chains synthesized by the HECT domain.

Highlights

Ubiquitin is conjugated to proteins by a series of enzymes that act in a well defined, sequential manner [7]
The Rsp5 homologous to E6AP C terminus (HECT) Domain Binds to Ubiquitin—To identify cellular proteins that bind to ubiquitin, a yeast genomic two-hybrid library was screened using ubiquitin as the bait
These observations are consistent with a model in which the N-terminal lobe (N-lobe) binds to ubiquitin in an orientation that favors polyubiquitin chain linkage through Lys-63

Summary

EXPERIMENTAL PROCEDURES

Plasmid Construction and Mutagenesis—Plasmids encoding fragments of Rps fused to glutathione S-transferase (GST) were constructed in pGEX vectors (GE Healthcare). Ubiquitin and E2-binding Assays—Binding assays carried out with ubiquitin-agarose beads (Boston Biochem, Cambridge, MA) and lysates from yeast cells expressing HA-tagged Rsp were performed as previously described [34], except that 7.5 mg of lysate protein was incubated with the beads for 1 h at 4 °C. UbcH5a binding assays were carried out in the same manner, except that the beads carrying GST-HECT domain mutants were incubated with 25 nM to 0.1 ␮M purified UbcH5a (Boston Biochem) and bound proteins were analyzed by antiUbcH5a immunoblotting (Boston Biochem). All GST-HECT proteins were expressed in E. coli (BL21-CodonPlus cells), purified on glutathione resin according to the manufacturer’s instructions, and eluted from the resin in 10 mM Tris-HCl, pH 8.0, buffer containing 10 mM glutathione. Reaction aliquots were removed at the indicated times, added to an equal volume of 2ϫ Laemmli sample buffer, and analyzed by SDS-PAGE and anti-GST immunoblotting

RESULTS

To determine whether the ability

DISCUSSION

HECT domain is required for presumed interaction with ubiquitin