Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE: The Case for a Combined Approach

Summary

This protocol uses both subunit coexpression and postlysis subunit mixing for a more thorough examination of recombinant proteasome assembly.

Abstract

Proteasomes are found in all domains of life. They provide the major route of intracellular protein degradation in eukaryotes, though their assembly is not completely understood. All proteasomes contain a structurally conserved core particle (CP), or 20S proteasome, containing two heptameric β subunit rings sandwiched between two heptameric α subunit rings. Archaeal 20S proteasomes are compositionally simpler compared to their eukaryotic counterparts, yet they both share a common assembly mechanism. Consequently, archaeal 20S proteasomes continue to be important models for eukaryotic proteasome assembly. Specifically, recombinant expression of archaeal 20S proteasomes coupled with nondenaturing polyacrylamide gel electrophoresis (PAGE) has yielded many important insights into proteasome biogenesis. Here, we discuss a means to improve upon the usual strategy of coexpression of archaeal proteasome α and β subunits prior to nondenaturing PAGE. We demonstrate that although rapid and efficient, a coexpression approach alone can miss key assembly intermediates. In the case of the proteasome, coexpression may not allow detection of the half-proteasome, an intermediate containing one complete α-ring and one complete β-ring. However, this intermediate is readily detected via lysate mixing. We suggest that combining coexpression with lysate mixing yields an approach that is more thorough in analyzing assembly, yet remains labor nonintensive. This approach may be useful for the study of other recombinant multiprotein complexes.

Introduction

Multiprotein complexes carry out numerous critical cellular activities¹. For many of these complexes, much more is known about their structure and function than about their assembly^2,3. The proteasome is one such complex and is found in all domains of life. In eukaryotes, this molecular machine is at the core of the Ubiquitin/Proteasome System (UPS) and provides the major route of intracellular protein degradation⁴. The eukaryotic proteasome (referred to as the 26S proteasome) is comprised of two major sub assemblies: a 20S proteasome, or Core Particle (CP)⁵, that can be capped on one or both ends by a 19S Regulatory Particl....

Protocol

1. Bacterial Expression.

Note: Expression plasmids used in this study are described in Table 1. Solutions, media, and buffers used in this study are described in Table 2. The cloning of archaeal proteasome subunit genes and the generation of expression plasmids are described elsewhere^18,20. In brief, plasmids for recombinant coexpression of subunits employ a bicistronic operon strategy which helps in obtaining comparable expression levels of individual subunits^18,20. The expression parameters listed below were empirically determined to be optimal for the proteasome subun....

Results

Proteasome assembly (Figure 1) begins when α subunits combine to form rings⁹. This can be illustrated when α subunits from the archaeon Methanococcus maripaludis S2 are expressed in E. coli as C-terminally hexahistidine tagged (his-tagged) derivatives (Table 1). When the recombinant α-his protein was purified by ICAR and analyzed by nondenaturing PAGE, two bands were observed (Figure 2A, lane 1). We have previously demonstrated that these cor.......

Discussion

We demonstrate the benefit of a combined approach to analyzing proteasome assembly by nondenaturing PAGE using recombinant archaeal proteasomes. The usual method^9,11 of bacterial coexpression of proteasome subunits allows for rapid analysis but may not reveal key assembly intermediates. We suggest combining coexpression with lysate mixing to develop a broader picture of assembly events.

The advantage of this combined approach is that despite requiring separate expression of the ^.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acrylamide (40%) solution	Biorad	1610104	Unpolymerized acrylamide is a neurotoxin. Wear proper protective gear
Amicon ultra 0.5 mL centrifugal filters	EMDMillipore	UFC501024
Ammonium Persulfate	Sigma	A3678
ATP	Sigma	A7699
BCA assay kit	Pierce	23225
Bisacrylamide (2%) solution	Biorad	1610142
Bromophenol blue	Sigma	B8026
DNaseI	Sigma	DN25
Dithiothreitol (DTT)	Thermo Fisher	BP172
E. coli BL21 competent cells	EMD Millipore	69450
GelCode Blue	Thermo Fisher	24592	Colloidal coomassie stain reagent for gels
Gel doc EZ system	Biorad	1708270	Gel documentation system
Gel releasers	Biorad	1653320	Wedge shaped plastic used to separate gel plates; useful for spreading liquid.
Glass rod	Thermo Fisher	11-380B
Glycerol	Sigma	49767
Glycine	Thermo Fisher	BP3865
Hamilton syringe	Thermo Fisher	14-813-38	Glass syringe for loading gels
HEPES	US Biologicals	H2010
HMW Native calibration kit	GE Healthcare	170445-01	High molecular weight protein standards
Hoefer SG30	Thermo Fisher	03-500-277	Gradient maker
Imidazole	US Biologicals	280671
IPTG	US Biologicals	I8500	For induction of protein expression
Isopropanol	Thermo Fisher	BP26181
Kanamycin sulfate	US Biologicals	K0010
Lysozyme	Sigma	L6876
MgCl2	Fluka analytical	630680
Mini Protean Tetra Cell	Biorad	1658002EDU	Gel electrophoresis apparatus
NaCl	Thermo Fisher	S640-3
NaOH	Thermo Fisher	S318-1
Pefabloc SC	Roche	11429876001	Protease inhibitor
pET42	EMD Millipore	70562	Expression plasmid
Precision plus all blue standard	Biorad	1610373	Molecular protein standard for SDS-PAGE
Quickchange mutagenesis kit	Agilent technologies	200521
Sodium Dodecyl Sulfate (SDS)	Thermo Fisher	BP166
Suc-LLVY-AMC	Enzo lifesciences	BML P802-0005	Fluorogenic substrate
Talon Metal Affinity Resin	Clontech	635502	Immobilized Cobalt Affinity Resin
TEMED	Sigma	T7024
Tris	US Biologicals	T8600
Triton-X100	Sigma	93426
Tryptone	Bacto BD	211699
UV sample tray	Biorad	1708271	For UV imaging of gels
Yeast extract	Bacto BD	212720