Regulation of the RSP5 ubiquitin ligase by an intrinsic ubiquitin-binding site - PubMed (original) (raw)

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

Michael E French et al. J Biol Chem. 2009.

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.

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Figures

FIGURE 1.

FIGURE 1.

The Rsp5 HECT domain binds directly to ubiquitin. A, a lysate prepared from yeast cells expressing HA-tagged Rps5 (LHY856) was incubated with ubiquitin-agarose beads (Ub) or agarose beads alone (Beads). Bound proteins were eluted, and Rsp5 was detected on an anti-HA immunoblot. B, schematic representation of Rsp5 indicating the position of its functional domains. Fragments tested for ubiquitin binding in C are shown. C, bacterial lysates from cells expressing the indicated GST-tagged Rsp5 domains were incubated with ubiquitin-agarose beads or agarose beads alone. Total lysates and bound proteins were analyzed by anti-GST immunoblotting.

FIGURE 2.

FIGURE 2.

The Rsp5 and Nedd4 HECT domain N-lobes bind to ubiquitin. A, schematic representation of the Rsp5 HECT domain. Fragments tested for ubiquitin binding in B are shown. B, bacterial lysates from cells expressing the indicated GST-tagged HECT domain fragments were incubated with beads carrying immobilized His6-tagged ubiquitin (His6-Ub). Lysates and proteins eluted from the beads were analyzed by anti-GST immunoblotting (top panels) or Coomassie staining (bottom panel). C, GST-HECT domain and GST-HECT N-lobe fusions were immobilized on beads, and the beads were incubated with purified His6-tagged Lys-63- or Lys-48-linked polyubiquitin chains. Purified chains (10% Input) and proteins eluted from the beads were analyzed by anti-His immunoblotting (top panel) or Coomassie staining (bottom panel). D, the indicated N-lobes were purified from an E. coli lysate and incubated with equivalent amounts of immobilized His6-Ub or a control His6-tagged SH3 domain from Rvs167 (_His6_-SH3). Purified N-lobes (1% Input) and proteins eluted from Ub or SH3 beads were detected by Coomassie staining.

FIGURE 3.

FIGURE 3.

Ubiquitin binds to a region on the front surface of the Rsp5 HECT domain N-lobe. A, a representative experiment from the alanine-scanning mutagenesis of residues in the Rsp5 HECT domain N-lobe. Bacterial lysates from cells expressing the indicated GST-tagged N-lobe mutants were incubated with beads carrying immobilized His6-tagged ubiquitin. Lysates and proteins bound to ubiquitin were analyzed by anti-GST immunoblotting. Mutation of the acidic residues in the E600A/N601A/S602A and P628A/D629A mutants resulted in slightly altered electrophoretic mobility. B, bacterial lysates from cells expressing the indicated GST-tagged HECT domain mutants were incubated with immobilized His6-tagged ubiquitin. Lysates and proteins bound to ubiquitin were analyzed by anti-GST immunoblotting.C, surface representation of the Rsp5 HECT domain, created by modeling onto the WWP1 HECT domain crystal structure (Protein Data Bank accession code 1ND7). Results of the alanine mutagenesis are summarized as follows: red, mutation abolished binding; magenta, mutation reduced binding; blue, mutation enhanced binding; dark gray, mutation had no effect. D, the indicated GST-HECT domain mutants were immobilized on beads, and the beads were incubated with increasing concentrations of purified UbcH5a: 25 (_1_×), 50 (_2_×), and 100 n

m

(_4_×). Proteins eluted from the beads were analyzed by anti-UbcH5a immunoblotting (top panel) or Coomassie staining (bottom panel). A nonspecific band unrelated to UbcH5a is represented by an asterisk.

FIGURE 4.

FIGURE 4.

The Ile-44 hydrophobic patch of ubiquitin is required for binding. A, the indicated His6-tagged ubiquitin mutants were immobilized on beads and incubated with a bacterial lysate from cells expressing a wild-type GST-tagged N-lobe. Bound proteins and ubiquitin mutants eluted from the beads were detected by Coomassie staining. B, surface representation of ubiquitin based on its three-dimensional structure (Protein Data Bank accession code 1UBQ). Results from the alanine mutagenesis are summarized as follows: red, mutation abolished binding;magenta, mutation reduced binding; dark gray, mutation had no effect. The position of Lys-63 is shown for reference. No mutations were made on the back face of ubiquitin (not shown).

FIGURE 5.

FIGURE 5.

Phenotypic analysis of the rsp5Y516A and_rsp5F618A_ mutants. A, RSP5 (LHY5653), rsp5Y516A (LHY5655), and_rsp5F618A_ (LHY5657) cells were serially diluted, plated onto rich media and grown at 30 or 37 °C for 2 days. B, yeast strains described in A were grown to mid-log phase at 30 °C and then shifted to 37 °C for 1 h. Cells were harvested before and after the temperature shift. Cell lysates were prepared and analyzed for Rsp5 expression by immunoblotting with Rsp5 antiserum. C, RSP5 and_rsp5F618A_ yeast strains used in A were transformed with multicopy plasmids encoding either wild-type (WT) ubiquitin (pUb-WT) or 0K ubiquitin (pUb-0K). Serial dilutions of each strain were plated onto rich media and grown at 37 °C for 2 days. D, yeast strains tested in A were treated as described in B, except that cell lysates were analyzed for free ubiquitin levels by antiubiquitin immunoblotting.

FIGURE 6.

FIGURE 6.

The Rsp5 ubiquitin-binding site regulates the length of polyubiquitin chains assembled by the HECT domain. A, in vitro autoubiquitination assays were carried out with a wild-type GST-HECT fusion protein and wild-type ubiquitin (WT), lysine-less ubiquitin (0K), or one of the indicated single lysine ubiquitins (K63,K48, or K29). Reactions were quenched at the indicated times and ubiquitin conjugates were detected by anti-GST immunoblotting. The position of the unmodified HECT domain is indicated. B–E, reactions were carried out as described in A but in the presence of either a wild-type or mutant GST-HECT fusion protein (WT,F618Y, or Y516A) and the indicated ubiquitin: WT ubiquitin for B, 0K ubiquitin for C, K63-ubiquitin for D, and K48-ubiquitin for E.

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