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The HECT domain of the ubiquitin ligase Rsp5 contributes to substrate recognition.

ABSTRACT: Ubiquitin modification of endosomal membrane proteins is a signal for active inclusion into the Multivesicular Body (MVB) pathway, resulting in lysosomal degradation. However, the endosome represents a dynamic site of protein sorting with a majority of proteins destined for recycling, rather than MVB targeting. Substrate recognition by ubiquitin ligases is therefore highly regulated. We have investigated substrate recognition by the Nedd4 ortholog Rsp5 as a model for understanding ligase-substrate interactions. Rsp5 interacts directly with its substrate Cps1 via a novel interaction mode. Perturbation of this mode of interaction revealed a compensatory role for the Rsp5 adaptor Bsd2. These results highlight the ability of Rsp5 to interact with substrates via multiple modalities, suggesting additional mechanisms of regulating this interaction and relevant outcomes.

SUBMITTER: Lee JR

PROVIDER: S-EPMC2797283 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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The HECT domain of the ubiquitin ligase Rsp5 contributes to substrate recognition.

Lee Jacqueline R E JR Oestreich Andrea J AJ Payne Johanna A JA Gunawan Mia S MS Norgan Andrew P AP Katzmann David J DJ

The Journal of biological chemistry 20090910 46

Ubiquitin modification of endosomal membrane proteins is a signal for active inclusion into the Multivesicular Body (MVB) pathway, resulting in lysosomal degradation. However, the endosome represents a dynamic site of protein sorting with a majority of proteins destined for recycling, rather than MVB targeting. Substrate recognition by ubiquitin ligases is therefore highly regulated. We have investigated substrate recognition by the Nedd4 ortholog Rsp5 as a model for understanding ligase-substra ...[more]

PMID: 19744925

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Redefining the catalytic HECT domain boundaries for the HECT E3 ubiquitin ligase family.

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The HECT domain ubiquitin ligase HUWE1 targets unassembled soluble proteins for degradation.

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Interactions between Yeast Proteins and Rsp5 Ubiquitin Ligase

2008-12-30 | GSE14218 | GEO

Interactions between Yeast Proteins and Rsp5 Ubiquitin Ligase

Project description:To identify substrates of the ubiquitinating E3 enzyme Rsp5 we applied purified Rsp5 to duplicate protein arrays. The Rsp proteins were expressed as fusion proteins to GST. We used as a control Ubr1, a RING domain containing E3 ligase We analyzed Rsp5 from S.cerevisiae on duplicate arrays, with four control chips, two without Rsp5 and two with Ubr1.

2008-12-30 | E-GEOD-14218 | biostudies-arrayexpress

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Project description:The HECT-type ubiquitin ligase Smurf1 (Smad ubiquitination regulatory factor-1) plays the prominent role in regulation of bone formation, embryonic development, and tumorigenesis by directing the ubiquitin-proteasomal degradation of specific targets. In contrast with RING-type E3s, the catalytic HECT domain of Smurf1 firstly binds to and then transfers ubiquitin (Ub) molecules onto the substrates. The Smurf1-Ub interaction is required for Smurf1 catalytic ligase activity to promote substrate degradation. However, so far specific regulators or compounds controlling Smurf1-Ub interaction and the ligase activity have not been identified. Here we report two small molecule compounds targeting Ub binding region of HECT domain interrupt Smurf1-Ub contact, inhibit Smurf1 ligase activity and stabilize BMP signal components Smad1/5 protein level. Furthermore, these compounds increase BMP signal responsiveness and enhance osteoblastic activity in cultured cells. These findings provide a novel strategy through targeting Smurf1 ligase activity to potentially treat bone disorders such as osteoporosis.

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Regulation of the RSP5 ubiquitin ligase by an intrinsic ubiquitin-binding site.

Project description: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.

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