SGTA binding to Rpn13 selectively modulates protein quality control

Abstract

Rpn13 is an intrinsic ubiquitin receptor of the 26S proteasome regulatory subunit that facilitates substrate capture prior to degradation. Here we show that the C-terminal region of Rpn13 binds to the tetratricopeptide repeat (TPR) domain of SGTA, a cytosolic factor implicated in the quality control of mislocalised membrane proteins (MLPs). The overexpression of SGTA results in a substantial increase in steady-state MLP levels, consistent with an effect on proteasomal degradation. However, this effect is strongly dependent upon the interaction of SGTA with the proteasomal component Rpn13. Hence, overexpression of the SGTA-binding region of Rpn13 or point mutations within the SGTA TPR domain both inhibit SGTA binding to the proteasome and substantially reduce MLP levels. These findings suggest that SGTA can regulate the access of MLPs to the proteolytic core of the proteasome, implying that a protein quality control cycle that involves SGTA and the BAG6 complex can operate at the 19S regulatory particle. We speculate that the binding of SGTA to Rpn13 enables specific polypeptides to escape proteasomal degradation and/or selectively modulates substrate degradation.

SGTA interacts with the proteasomal ubiquitin receptor Rpn13 via a carboxylate clamp mechanism

“…In order to understand molecular details underlying the interaction between SGTA and Rpn13, solution NMR studies were carried out on excised domains of SGTA and Rpn13 guided by earlier mapping studies24. Reciprocal chemical shift perturbation (CSP) experiments were performed by titrating unlabelled Rpn13 C-terminal domain (residues 260–407; hereafter described as Rpn13 260-407 ) into 15 N-labelled TPR domain of SGTA (residues 84–211; hereafter referred to as SGTA_TPR) and vice versa.…”

“…Hence, SGTA overexpression typically increases the steady-state levels of model MLPs, including OP91, an N-terminal fragment of the polytopic integral membrane protein opsin that is inefficiently targeted to the endoplasmic reticulum4524. However, the ability of exogenous SGTA to enhance steady state MLP levels is offset when key residues of the carboxylate clamp, located in its TPR domain, are mutated24. We recapitulated this experiment and found that the co-expression of exogenous SGTA-V5 led to a three-fold increase in OP91 levels when compared to the expression of either the K160E/R164E SGTA_TPR mutant or a PEX19 control (Fig.…”

“…Rpn10 and Rpn13 are two intrinsic proteasomal ubiquitin receptors, present at the 19S regulatory particle, that selectively recognise ubiquitinated substrates2122. Furthermore, whilst the N-terminal region of BAG6 associates with Rpn101223, the central TPR domain of SGTA was recently shown to bind to the C-terminal domain of Rpn1324. These observations led to the proposal that the BAG6/SGTA quality control cycle may be operational at the 19S regulatory particle of the proteasome, thereby regulating the access of MLPs to the proteolytic core24.…”

“…Furthermore, whilst the N-terminal region of BAG6 associates with Rpn101223, the central TPR domain of SGTA was recently shown to bind to the C-terminal domain of Rpn1324. These observations led to the proposal that the BAG6/SGTA quality control cycle may be operational at the 19S regulatory particle of the proteasome, thereby regulating the access of MLPs to the proteolytic core24. Rpn13 consists of an N-terminal pleckstrin-like receptor for ubiquitin (PRU) domain that binds to both ubiquitin and the proteasome, a conserved C-terminal DEUBiquitinase ADaptor (DEUBAD) domain that binds to the deubiquitinating enzyme UCH37 (UCH-L5) and, lastly, a flexible C-terminal extension25.…”

“…Upon activation, the UCH37 deubiquitinase can bind to the Rpn13 DEUBAD domain in a manner that allows UCH37 access to its ubiquitinated substrates2627. Therefore in the context of the BAG6/SGTA cycle, it has been speculated that the interaction between SGTA and Rpn13 provides SGTA-bound substrates with access to the UCH37 deubiquitinase, and could thereby facilitate a rescue option for prematurely ubiquitinated substrates24252627.…”