The proteasome cap RPT5/Rpt5p subunit prevents aggregation of unfolded ricin A chain

Paola Pietroni,Lynne M Roberts,David M Roberts,Jonathan P Cook,Rasmus Hartmann-Petersen,Robert A Spooner,J Michael Lord,Nishi Vasisht

doi:10.1042/bj20130133

Paola Pietroni, Lynne M Roberts + Show 6 more

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https://doi.org/10.1042/bj20130133

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Abstract

The plant cytotoxin ricin enters mammalian cells by receptor-mediated endocytosis, undergoing retrograde transport to the ER (endoplasmic reticulum) where its catalytic A chain (RTA) is reductively separated from the holotoxin to enter the cytosol and inactivate ribosomes. The currently accepted model is that the bulk of ER-dislocated RTA is degraded by proteasomes. We show in the present study that the proteasome has a more complex role in ricin intoxication than previously recognized, that the previously reported increase in sensitivity of mammalian cells to ricin in the presence of proteasome inhibitors simply reflects toxicity of the inhibitors themselves, and that RTA is a very poor substrate for proteasomal degradation. Denatured RTA and casein compete for a binding site on the regulatory particle of the 26S proteasome, but their fates differ. Casein is degraded, but the mammalian 26S proteasome AAA (ATPase associated with various cellular activities)-ATPase subunit RPT5 acts as a chaperone that prevents aggregation of denatured RTA and stimulates recovery of catalytic RTA activity in vitro. Furthermore, in vivo, the ATPase activity of Rpt5p is required for maximal toxicity of RTA dislocated from the Saccharomyces cerevisiae ER. The results of the present study implicate RPT5/Rpt5p in the triage of substrates in which either activation (folding) or inactivation (degradation) pathways may be initiated.

Highlights

The eukaryotic 26S proteasome is a multi-component endoproteolytic complex that cleaves most cytosolic proteins, regulating protein turnover and maintaining cellular homoeostasis
In the present study, we examined the interactions of Abbreviations used: AAA, ATPase associated with various cellular activities; ALLN, N-acetyl-L-leucyl-L-leucylnorleucinal; BFA, brefeldin A; cLβ-l, clasto lactacystin β-lactone; CP, core particle; DSS, disuccinimidyl suberate; ER, endoplasmic reticulum; ERAD, ER-associated degradation; GdnHCl, guanidinium chloride; Pi1, proteasome inhibitor 1; RP, regulatory particle; RTA, ricin toxin A chain; nonnative forms of RTA (nnRTA), non-native forms of RTA
The peptide inhibitor ALLN protected cells from ricin at early time points, sensitizing only at later time points (Figure 1B, middle panel). These proteasome inhibitors inhibit the major endocytic pathway cathepsins, so we tested the cathepsin inhibitors leupeptin and pepstatin in combination and this time observed little or no effect on ricin toxicity under all assay conditions tested (Figure 1B, bottom panel). These data suggest that the changing protection/sensitization profiles seen with Pi1 and ALLN were a result of proteasome inhibition

Summary

Introduction

The eukaryotic 26S proteasome is a multi-component endoproteolytic complex that cleaves most cytosolic proteins, regulating protein turnover and maintaining cellular homoeostasis. Denatured RTA and casein compete for the 19S RP In the presence of a 2-fold excess (Figure 4A) or equimolar GdnHCl-denatured RTA (Figure 4B), casein (white arrowhead) was degraded more slowly by intact proteasomes, but in a c The Authors Journal compilation c 2013 Biochemical Society

Results

Conclusion