A novel proteasome assembly intermediate bypasses the need to form α-rings first.

Proteasomes provide the main route of intracellular protein degradation. They consist of a central protease, termed the 20S proteasome, or core particle (CP), that partners with one or more regulatory complexes. The quaternary structure of the CP is conserved across all domains of life and is comprised of four coaxially stacked heptameric rings formed by structurally related α and ß subunits. In eukaryotes, biogenesis of the CP is generally assumed to involve the obligate formation of α-rings. These serve as templates upon which ß subunits assemble to form half-proteasomes which dimerize to give rise to CP. Here, we demonstrate the in vivo existence of an assembly-competent intermediate containing an incomplete set of both α and ß subunits. The novel intermediate exhibits a precursor-product relationship with the well characterized CP assembly intermediate, the 13S. This is the first evidence that eukaryotic CP, like its archaeal and bacterial counterparts, can assemble in an α-ring independent manner.

Molecular chaperones of the Hsp70 family assist in the assembly of 20S proteasomes.

The eukaryotic 26S proteasome is a large protease comprised of two major sub assemblies, the 20S proteasome, or core particle (CP), and the 19S regulatory particle (RP). Assembly of the CP and RP is assisted by an expanding list of dedicated assembly factors. For the CP, this includes Ump1 and the heterodimeric Pba1-Pba2 and Pba3-Pba4 proteins. It is not known how many additional proteins that assist in proteasome biogenesis remain to be discovered. Here, we demonstrate that two members of the Hsp70 family in yeast, Ssa1 and Ssa2, play a direct role in CP assembly. Ssa1 and Ssa2 interact genetically and physically with proteasomal components. Specifically, they associate tightly with known CP assembly intermediates, but not with fully assembled CP, through an extensive purification protocol. And, in yeast lacking both Ssa1 and Ssa2, specific defects in CP assembly are observed.

Assembly of proteasome subunits into non-canonical complexes in vivo.

Proteasomes exist in all domains of life. In general, they are comprised of a compartmentalized protease whose activity is modulated by one or more regulatory complexes with which it interacts. The quaternary structure of this compartmentalized protease, called the 20S proteasome, is absolutely conserved and consists of four heptameric rings stacked coaxially. The rings are made of structurally related α and ß subunits. In eukaryotes, assembly factors chaperone the α and ß subunits during 20S biogenesis. Here we demonstrate that proteasome subunits can assemble into structures other than the canonical 20S proteasome in vivo. Specifically, the yeast α4 subunit forms high molecular weight complexes whose abundance increases when proteasome function is compromised. Results from a disulfide crosslinking approach are consistent with these complexes being ring-shaped. Though several eukaryotic α subunits can form rings when expressed recombinantly in bacteria, this is the first evidence that such non-canonical complexes exist in vivo.

Data on the identity of non-canonical complexes formed from proteasome subunits in vivo.

The dataset presented here represents analysis supplied by the local proteomics core facility on samples submitted to it in support of the article "Assembly of proteasome subunits into non-canonical complexes in vivo" Hammack and Kusmierczyk (2016) [1]. This article provides the detailed protein contents of gel slices, cut from non-denaturing polyacrylamide gels, containing distinct protein complexes visualized following gel staining. The identification of the protein contents of these complexes was carried out by liquid chromatography tandem mass-spectrometry (LC-MS/MS).