The San1 Ubiquitin Ligase Avidly Recognizes Misfolded Proteins through Multiple Substrate Binding Sites.

Rebeca Ibarra,Richard G Gardner,Heather R Borror,Gary Kleiger,Bryce Hart

doi:10.3390/biom11111619

Rebeca Ibarra, Richard G Gardner + Show 3 more

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https://doi.org/10.3390/biom11111619

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Abstract

Cellular homeostasis depends on robust protein quality control (PQC) pathways that discern misfolded proteins from functional ones in the cell. One major branch of PQC involves the controlled degradation of misfolded proteins by the ubiquitin-proteasome system. Here ubiquitin ligases must recognize and bind to misfolded proteins with sufficient energy to form a complex and with an adequate half-life to achieve poly-ubiquitin chain formation, the signal for protein degradation, prior to its dissociation from the ligase. It is not well understood how PQC ubiquitin ligases accomplish these tasks. Employing a fully reconstituted enzyme and substrate system to perform quantitative biochemical experiments, we demonstrate that the yeast PQC ubiquitin ligase San1 contains multiple substrate binding sites along its polypeptide chain that appear to display specificity for unique misfolded proteins. The results are consistent with a model where these substrate binding sites enable San1 to bind to misfolded substrates avidly, resulting in high affinity ubiquitin ligase-substrate complexes.

Highlights

Uncovering how ubiquitin ligases recognize misfolded protein substrates is an important step towards understanding the molecular pathologies of human diseases that are linked to aberrant protein quality control (PQC) processes
We began our investigation by attempting to improve the reconstituted ubiquitylation system since full-length recombinant San1 protein is highly prone to proteolysis, resulting in degradation products occurring even after multiple rounds of purification and with cubated with the membrane for 1 h at room temperature
We began our investigation by attempting to improve the reconstituted ubiquitylation system since full-length recombinant San1 protein is highly prone to proteolysis, resulting in degradation products occurring even after multiple rounds of purification and with affinity-based tags at both the N- and C-terminus (Figure 1A,B)

Summary

Introduction

All living cells are burdened with the task of disposing of misfolded or damaged proteins [1]. Termed protein quality control (PQC) [2,3,4,5,6,7], these molecular pathways guard against the accumulation of misfolded proteins in cells, which if left unchecked, may result in the malfunction of normal cellular processes. Many mechanistic aspects regarding how PQC ubiquitin ligases function remain unresolved, such as how misfolded proteins are recognized, and how sufficient binding energy is achieved to stabilize ligase-substrate complexes that are held together presumably through weak, nonspecific hydrophobic interactions. Uncovering how ubiquitin ligases recognize misfolded protein substrates is an important step towards understanding the molecular pathologies of human diseases that are linked to aberrant PQC processes

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