The human α-type proteasomal subunit HsC8 forms a double ringlike structure, but does not assemble into proteasome-like particles with the β- type subunits HsDelta or HsBPROS26

Abstract

The eukaryotic proteasome is a barrel-shaped protease complex made up of four seven-membered rings of which the outer and inner rings may contain up to seven different α- and β-type subunits, respectively. The assembly of the eukaryotic proteasome is not well understood. We cloned the cDNA for HsC8, which is one of the seven known human α-type subunits, and produced the protein in Escherichia coli. Recombinant HsC8 protein forms a complex of about 540 kDa consisting of double ringlike structures, each ring containing seven subunits. Such a structure has not earlier been reported for any eukaryotic proteasome subunit, but is similar to the complex formed by the recombinant α-subunit of the archaebacterium Thermoplasma acidophilum (Zwickl, P., Kleinz, J., and Baumeister, W. (1994) Nat. Struct. Biol. 1, 765- 770). The ability of HsC8 to form α-rings suggests that these complexes may play an important role in the initiation of proteasome assembly in eukaryotes. To test this, we used two human β-type subunits, HsBPROS26 and HsDelta. Both these β-type subunits, either in the proprotein or in the mature form, exist in monomers up to tetramers. In contrast to the α- and β-subunit of T. acidophilum, coexpression of the human β-type subunits with HsC8 does not result in the formation of proteasome-like particles, which would be in agreement with the notion that proteasome assembly in eukaryotes is much more complex than in archaebacteria.