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Title: Molecular Dissection of the Arabidopsis 26S Proteasome

Abstract

The 26S proteasome is a 2.5-MDa, ATP-dependent protease complex responsible for degrading many important cell regulators in plants and animals, especially those targeted by ubiquitylation. Its 64 or more subunits generate two subparticles: a 20S core protease (CP) that houses the proteolytic active sites and a 19S regulatory particle (RP) that binds to both ends of the CP and recruits appropriate substrates for breakdown. Despite its importance, we had only a rudimentary appreciation of the structure and functions of the core 26S proteasome from plants, and hints that the previously defined complex actually represents the nucleus of an even more elaborate and dynamic particle harboring multiple routes for substrate recognition and sophisticated mechanisms to control its assembly/location. Genomic analyses also suggested that plants in particular exploit this heterogeneity to generate a wide array of proteasome types, each with distinct compositions, locations, and/or functions/specificities. The goal of this DOE BES-funded research was to better define the 26S proteasome and its diversity in plants by a thorough analysis of the particle from Arabidopsis thaliana. During this completed work, we developed stringent affinity methods to rapidly isolate 26S proteasomes intact from Arabidopsis seedlings, and identified by mass spectrometry all the main CP andmore » RP subunits along with a suite of interacting proteins. We also identified a number of post-translational modifications, and detected an alternative proteasome containing the CP capped by the PA200 protein. We then amassed a large collection of mutants eliminating most CP and RP subunits, and notable accessory proteins such as PA200. Phenotypic analyses of a few RP mutants support the notion that the Arabidopsis 26S proteasome is functionally heterogeneous. Through both genetic and biochemical analyses, we characterized a collection of shuttle proteins that help deliver ubiquitylated substrates, and discovered a subset of accessory proteins that likely act as chaperones that facilitate CP and RP assembly. These chaperone activities were supported by examining 26S proteasome assembly in plants missing these factors, which showed that the mutants accumulated high levels of partially assembled subparticles. In an effort to understand how the numerous genes encoding the various proteasome subunits are coordinately regulated to assemble the 64-subunit core complex and ultimately maintain adequate proteolytic capacity, we discovered by RNA-seq that most Arabidopsis genes required to synthesize the 26S proteasome are coordinately regulated transcriptionally. Through analysis of this ‘proteasome-stress’ regulon by protein-DNA binding, and reverse genetic methods, we identified the cis and trans-acting elements involved, that center around a pair of NAC transcription factors (NAC53 and NAC78) binding to a consensus proteasome-related cis element (PRCE) upstream of most proteasome-associated genes. And finally, we discovered that proteasomes are rapidly degraded by autophagy in response to nutrient stress or inactivation via a process we called proteaphagy. Whereas the former turnover is triggered by the ATG1-kinase complex working downstream of stress sensors such as the TOR kinase, the latter turnover is induced by substantial ubiquitylation of the complex followed by recognition of the appended ubiquitin moieties by the ubiquitin-binding autophagy receptor RPN10. Surprisingly, studies with RPN10 revealed that it represents the founding member of a new class of autophagy receptors that exploit a novel way to tether cargo to autophagic membranes. Instead of using the canonical AIM motif to bind ATG8 lining the autophagic membranes, it and relatives exploit a UIM motif to bind ATG8 at an alternative site. Studying the same inhibitor-induced proteaphagy in yeast revealed that an unrelated autophagy receptor Cue5 is required to recognize the bound ubiquitins. Strikingly, we found that carbon starvation also induces the accumulation novel cytoplasmic particles called proteasome storage granules that appear to protect proteasomes from autophagy. Their accumulation in yeast requires the CP-binding protein PA200 along with the RP-binding proteins Spg5. Taken together, this research revealed the composition of plant 26S proteasome, how it is assembled, and how is abundance is regulated both transcriptionally and by autophagy. Collectively, this research enhanced our appreciation of the plant 26S proteasome and its roles in ubiquitin-mediated protein turnover, and identified points of opportunity where its abundance or activity can be manipulated to improve food and biofuel crops.« less

Authors:
 [1]
  1. Washington Univ., St. Louis, MO (United States); Univ. of Wisconsin, Madison, WI (United States)
Publication Date:
Research Org.:
Washington Univ., St. Louis, MO (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1483794
Report Number(s):
DOE-WASHU-0016589
DOE Contract Number:  
SC0016589
Resource Type:
Technical Report
Resource Relation:
Related Information: Hua, Z., and R.D. Vierstra (2016) Spotlight: Ubiquitin goes green. Trends Cell Biol. 26: 3-5.Marshall, R.S., F. Li, D.C. Gemperline, A.J. Book, and R.D. Vierstra (2015) Autophagic degradation of the 26S proteasome is mediated by the dual ATG8/ubiquitin receptor RPN10 in Arabidopsis. Mol. Cell 58: 1053-1066Marshall, R.S., and R.D. Vierstra (2015) Eat or be eaten: the autophagic plight of inactive 26S proteasomes (Commentary). Autophagy 11: 1927-1928. doi:10.1080/15548627.2015.1078961.Gemperline, D.C., M. Scalf, L.M. Smith, and R.D. Vierstra (2016) Morpheus Spectral Counter: a computational tool for quantitative mass spectrometry using the Morpheus search engine. Proteomics 16: 920-924 doi:10.1002/pmic.201500420.Gladman, N.P., R.S. Marshall, K.-H. Lee, and R.D. Vierstra (2016) The proteasome stress regulon is controlled by a pair of NAC transcription factors in Arabidopsis. Plant Cell 28: 1279-1296.Marshall, R.S., F. McLoughlin, and R.D. Vierstra (2016) Autophagic turnover of inactive 26S proteasomes in yeast is directed by the ubiquitin receptor Cue5 and the Hsp42 chaperone. Cell Rep. 16: 1717-1732.Marshall, R.S., D.C. Gemperline, and R.D. Vierstra (2017) Purification of 26S proteasomes and their subcomplexes from plants (Chapter 24). In: The Isolation of Plant Organelles and Structures: Methods and Protocols (Methods in Molecular Biology series). (N. Taylor and H. Miller eds) Springer Science, Berlin. 1511: 301-334.Marshall, R.S., and R.D. Vierstra (2018) Autophagy; the master of selective recycling. Annu.Rev. Plant Biol. 69: 173-208. doi:10.1146/annurev-arplant-042817-040606.Ustun, S, A. Hafren, Q. Liu, R.S. Marshall, E.A. Minina, P.V. Boshkov, R.D. Vierstra, and D. Hofius. (2018) Bacteria exploit autophagy for proteasome degradation and enhanced virulence in plants. Plant Cell 30: 668-685. doi:10.1105/tpc.17.00815.Marshall, R.S., and R.D. Vierstra (2018) Proteasome storage granules protect 26S proteasomes from autophagic clearance during carbon starvation. eLife2018;7:e34532. doi:10.7554/eLife.34532.Marshall, R.S., and R.D. Vierstra (2018) To save or degrade: balancing proteasome homeostasis to maximize cell survival. Autophagy doi.org/10.1080/15548627.2018. 1515531.
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES

Citation Formats

Vierstra, Richard D. Molecular Dissection of the Arabidopsis 26S Proteasome. United States: N. p., 2016. Web. doi:10.2172/1483794.
Vierstra, Richard D. Molecular Dissection of the Arabidopsis 26S Proteasome. United States. doi:10.2172/1483794.
Vierstra, Richard D. Thu . "Molecular Dissection of the Arabidopsis 26S Proteasome". United States. doi:10.2172/1483794. https://www.osti.gov/servlets/purl/1483794.
@article{osti_1483794,
title = {Molecular Dissection of the Arabidopsis 26S Proteasome},
author = {Vierstra, Richard D.},
abstractNote = {The 26S proteasome is a 2.5-MDa, ATP-dependent protease complex responsible for degrading many important cell regulators in plants and animals, especially those targeted by ubiquitylation. Its 64 or more subunits generate two subparticles: a 20S core protease (CP) that houses the proteolytic active sites and a 19S regulatory particle (RP) that binds to both ends of the CP and recruits appropriate substrates for breakdown. Despite its importance, we had only a rudimentary appreciation of the structure and functions of the core 26S proteasome from plants, and hints that the previously defined complex actually represents the nucleus of an even more elaborate and dynamic particle harboring multiple routes for substrate recognition and sophisticated mechanisms to control its assembly/location. Genomic analyses also suggested that plants in particular exploit this heterogeneity to generate a wide array of proteasome types, each with distinct compositions, locations, and/or functions/specificities. The goal of this DOE BES-funded research was to better define the 26S proteasome and its diversity in plants by a thorough analysis of the particle from Arabidopsis thaliana. During this completed work, we developed stringent affinity methods to rapidly isolate 26S proteasomes intact from Arabidopsis seedlings, and identified by mass spectrometry all the main CP and RP subunits along with a suite of interacting proteins. We also identified a number of post-translational modifications, and detected an alternative proteasome containing the CP capped by the PA200 protein. We then amassed a large collection of mutants eliminating most CP and RP subunits, and notable accessory proteins such as PA200. Phenotypic analyses of a few RP mutants support the notion that the Arabidopsis 26S proteasome is functionally heterogeneous. Through both genetic and biochemical analyses, we characterized a collection of shuttle proteins that help deliver ubiquitylated substrates, and discovered a subset of accessory proteins that likely act as chaperones that facilitate CP and RP assembly. These chaperone activities were supported by examining 26S proteasome assembly in plants missing these factors, which showed that the mutants accumulated high levels of partially assembled subparticles. In an effort to understand how the numerous genes encoding the various proteasome subunits are coordinately regulated to assemble the 64-subunit core complex and ultimately maintain adequate proteolytic capacity, we discovered by RNA-seq that most Arabidopsis genes required to synthesize the 26S proteasome are coordinately regulated transcriptionally. Through analysis of this ‘proteasome-stress’ regulon by protein-DNA binding, and reverse genetic methods, we identified the cis and trans-acting elements involved, that center around a pair of NAC transcription factors (NAC53 and NAC78) binding to a consensus proteasome-related cis element (PRCE) upstream of most proteasome-associated genes. And finally, we discovered that proteasomes are rapidly degraded by autophagy in response to nutrient stress or inactivation via a process we called proteaphagy. Whereas the former turnover is triggered by the ATG1-kinase complex working downstream of stress sensors such as the TOR kinase, the latter turnover is induced by substantial ubiquitylation of the complex followed by recognition of the appended ubiquitin moieties by the ubiquitin-binding autophagy receptor RPN10. Surprisingly, studies with RPN10 revealed that it represents the founding member of a new class of autophagy receptors that exploit a novel way to tether cargo to autophagic membranes. Instead of using the canonical AIM motif to bind ATG8 lining the autophagic membranes, it and relatives exploit a UIM motif to bind ATG8 at an alternative site. Studying the same inhibitor-induced proteaphagy in yeast revealed that an unrelated autophagy receptor Cue5 is required to recognize the bound ubiquitins. Strikingly, we found that carbon starvation also induces the accumulation novel cytoplasmic particles called proteasome storage granules that appear to protect proteasomes from autophagy. Their accumulation in yeast requires the CP-binding protein PA200 along with the RP-binding proteins Spg5. Taken together, this research revealed the composition of plant 26S proteasome, how it is assembled, and how is abundance is regulated both transcriptionally and by autophagy. Collectively, this research enhanced our appreciation of the plant 26S proteasome and its roles in ubiquitin-mediated protein turnover, and identified points of opportunity where its abundance or activity can be manipulated to improve food and biofuel crops.},
doi = {10.2172/1483794},
journal = {},
number = ,
volume = ,
place = {United States},
year = {2016},
month = {9}
}