This work was supported in part by a Research Support Funds Grant (RSFG) from Indiana University-Purdue University, Indianapolis, and in part by an award from the American Heart Association 14GRNT20390154, to A.R.K.

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 elsewhere18,20. In brief, plasmids for recombinant coexpression of subunits employ a bicistronic operon strategy which helps in obtaining comparable expression le...

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We demonstrate the benefit of a combined approach to analyzing proteasome assembly by nondenaturing PAGE using recombinant archaeal proteasomes. The usual method9,11 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 &#94...

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 assembly2,3. 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 Particle (RP)6.
The 20S proteasome&#160;is a large compartmentalized protease. Its quaternary structure is absolutely conserved across all domains of life and consists of a stack of four seven-membered rings containing two types of structurally related subunits, &#945; and &#946;5,7,8. In eukaryotes, the two outer rings are each comprised of seven distinct &#945; subunits and the two inner rings are each comprised of seven distinct &#946; subunits; proteolytic activity resides within three of the &#946; subunits. By contrast, the CP rings of archaea and bacteria are usually comprised of only one type of &#945; and one type of &#946; subunit. Archaeal proteasomes have provided an important model system to study proteasome assembly due to both their compositional simplicity and their sharing a common assembly mechanism with their eukaryotic counterparts9-13. In brief, &#945; subunits assemble into &#945; rings first, which serve as a scaffold onto which &#946; subunits assemble. The resulting half-proteasomes (&#945;7&#946;7) dimerize, giving rise to fully assembled CP (&#945;7&#946;7&#946;7&#945;7). During dimerization, the propeptides present on &#946; subunits are autocatalytically removed, exposing the catalytic N-terminal threonines. The use of archaeal proteasomes to model assembly frequently takes advantage of the production of recombinant archaeal proteasome proteins in Escherichia coli. This is a worthwhile approach because it enables the subunits to be produced in various combinations, as both WT and mutant versions, in a host organism that does not produce its own proteasomes.
Monitoring the assembly of multi-protein complexes biochemically requires some kind of fractionation method that separates fully assembled complexes from assembly intermediates and precursors. Due to its superior resolving capacity, nondenaturing Polyacrylamide Gel Electrophoresis (PAGE) has proven to be especially useful in the fractionation of various large multiprotein complexes14-17. The combination of recombinant archaeal proteasome production and nondenaturing PAGE has become a powerful approach in dissecting proteasome assembly9,11,12,18. However, the usual method by which this approach is applied (i.e. via the recombinant coexpression of &#945; and &#946; subunits) has an important drawback. Assembly reactions are cooperative and strongly concentration-dependent3. Given that the protein concentration inside cells is very high19, due to excluded volume effects, assembly reactions proceed rapidly in vivo. Hence it is possible to miss important assembly intermediates when &#945; and &#946; subunits are coexpressed.
Here, we argue for a combined approach in the study of proteasome assembly using recombinant archaeal proteasome subunits. In this approach, both coexpression and lysate mixing methods are employed. The former allows for rapid analysis of assembly because coexpression is less labor intensive. The latter depends on separate expression of &#945; and &#946; subunits, followed by mixing. Though this requires a bit more effort than coexpression, it is more than compensated for by the ability to detect intermediates that are missed during coexpression. Together, these two methods can provide a more complete picture of proteasome assembly.

<table border="0" cellpadding="0" cellspacing="0" width="701">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:165px;">Acrylamide (40%) solution</td>
			<td style="width:123px;">Biorad</td>
			<td style="width:105px;">1610104</td>
			<td style="width:309px;">Unpolymerized acrylamide is a neurotoxin. Wear proper protective equipment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">Amicon ultra 0.5ml centrifugal filters</td>
			<td style="width:123px;">EMDMillipore</td>
			<td>UFC501024</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">Ammonium persulfate</td>
			<td>Sigma</td>
			<td>A3678</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">ATP</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:105px;">A7699</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">BCA assay kit</td>
			<td style="width:123px;">Pierce</td>
			<td style="width:105px;">23225</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">Bisacrylamide (2%) solution</td>
			<td style="width:123px;">Biorad</td>
			<td style="width:105px;">1610142</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Bromophenol blue</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:105px;">B8026</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">DNaseI</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:105px;">DN25</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Dithiothreitol (DTT)</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td style="width:105px;">BP172</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">E.coli BL21 competent cells</td>
			<td style="width:123px;">EMD Millipore</td>
			<td style="width:105px;">69450</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">GelCode Blue</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td style="width:105px;">24592</td>
			<td style="width:309px;">Colloidal coomassie stain reagent for gels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Gel doc EZ system</td>
			<td style="width:123px;">Biorad</td>
			<td>1708270</td>
			<td style="width:309px;">Gel documentation system</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:165px;">Gel releasers</td>
			<td style="width:123px;">Biorad</td>
			<td>1653320</td>
			<td style="width:309px;">Wedge shaped plastic used to separate gel plates; useful for spreading liquid.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Glass rod</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td>11-380B</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Glycerol</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:105px;">49767</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Glycine</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td style="width:105px;">BP3865</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Hamilton syringe</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td>14-813-38</td>
			<td style="width:309px;">Glass syringe for loading gels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">HEPES</td>
			<td style="width:123px;">US Biologicals</td>
			<td style="width:105px;">H2010</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">HMW Native calibration kit</td>
			<td style="width:123px;">GE Healthcare</td>
			<td style="width:105px;">170445-01</td>
			<td style="width:309px;">High molecular weight protein standards</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Hoefer SG30</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td style="width:105px;">03-500-277</td>
			<td style="width:309px;">Gradient maker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Imidazole</td>
			<td style="width:123px;">US Biologicals</td>
			<td style="width:105px;">280671</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">IPTG</td>
			<td style="width:123px;">US Biologicals</td>
			<td style="width:105px;">I8500</td>
			<td style="width:309px;">For induction of protein expression</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Isopropanol</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td style="width:105px;">BP26181</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Kanamycin sulfate</td>
			<td style="width:123px;">US Biologicals</td>
			<td style="width:105px;">K0010</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Lysozyme</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:105px;">L6876</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:165px;">MgCl2</td>
			<td style="width:123px;">Fluka analytical</td>
			<td style="width:105px;">630680</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Mini Protean Tetra Cell</td>
			<td style="width:123px;">Biorad</td>
			<td style="width:105px;">1658002EDU</td>
			<td style="width:309px;">Gel electrophoresis apparatus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">NaCl</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td style="width:105px;">S640-3</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">NaOH</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td style="width:105px;">S318-1</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Pefabloc SC</td>
			<td style="width:123px;">Roche</td>
			<td style="width:105px;">11429876001</td>
			<td style="width:309px;">Protease inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">pET42</td>
			<td style="width:123px;">EMD Millipore</td>
			<td style="width:105px;">70562</td>
			<td style="width:309px;">Expression plasmid</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">Precision plus all blue standard</td>
			<td style="width:123px;">Biorad</td>
			<td style="width:105px;">1610373</td>
			<td style="width:309px;">Molecular protein standard for SDS-PAGE</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">Quickchange mutagenesis kit</td>
			<td style="width:123px;">Agilent technologies</td>
			<td style="width:105px;">200521</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">Sodium dodecyl sulfate (SDS)</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td style="width:105px;">BP166</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">Suc-LLVY-AMC</td>
			<td style="width:123px;">Enzo lifesciences</td>
			<td style="width:105px;">BML P802-0005</td>
			<td style="width:309px;">Fluorogenic substrate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">Talon Metal Affinity Resin</td>
			<td style="width:123px;">Clontech</td>
			<td style="width:105px;">635502</td>
			<td style="width:309px;">Immobilized-cobalt affinity resin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">TEMED</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:105px;">T7024</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Tris</td>
			<td style="width:123px;">US Biologicals</td>
			<td style="width:105px;">T8600</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Triton-X100</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:105px;">93426</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Tryptone</td>
			<td style="width:123px;">Bacto BD</td>
			<td style="width:105px;">211699</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">UV sample tray</td>
			<td style="width:123px;">Biorad</td>
			<td>1708271</td>
			<td style="width:309px;">For UV imaging of gels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Yeast extract</td>
			<td style="width:123px;">Bacto BD</td>
			<td style="width:105px;">212720</td>
			<td style="width:309px;"></td>
		</tr>
	</tbody>
</table>

examining proteasome assembly with recombinant archaeal proteasomes and nondenaturing page:  the case for a combined approach

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&#160;&#946; subunit rings sandwiched between two heptameric&#160;&#945; 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 &#945; and &#946; 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 &#945;-ring and one complete &#946;-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.

Proteasome assembly (Figure 1) begins when &#945; subunits combine to form rings9. This can be illustrated when &#945; 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 &#945;-his protein was purified by ICAR&#160;and analyzed by nondenaturing PAGE, two bands were observed (Figure 2A, lane 1). We have previously demonst...

Watch this Scientific Journal Video about Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE:  The Case for a Combined Approach at JoVE.com

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

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

Proteasomes are found in all domains of life. They provide the major route of intracellular protein degradation in eukaryotes, though their assembly is ...

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Biochemistry

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