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 alpha- and beta-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 alpha-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 alpha-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 alpha-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 beta-type subunits, HsBPROS26 and HsDelta. Both these beta-type subunits, either in the proprotein or in the mature form, exist in monomers up to tetramers. In contrast to the alpha- and beta-subunit of T. acidophilum, coexpression of the human beta-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.
Highlights
The 20 S proteasome is a multicatalytic protease complex which functions as the catalytic core of the larger 26 S proteasome
The N-terminal threonine results from proteolytic processing of the N terminus, which is an essential step in the maturation of the proteasome
It was shown that the proteolytic processing of these -subunits is autocatalytic and that their folding and assembly is chaperoned by the ␣-subunits [24]
Summary
Mutation and PCR1 primers were purchased from Eurogentec. The NcoI and NdeI sites in the primer sequences are underlined and the. For coexpression of HsC8 with -type proteasomal subunits, the XbaI-BamHI fragment of pET16bHsC8 was subcloned into the XbaI-BamHI sites of pET24d expression vector (Novagen, pET24dHsC8). Both vectors encode the complete HsC8 protein with only a Ser to Gly mutation at position 2. To construct the vector for expression of the mature protein (HsBPROS26mat) in bacteria, an NdeI site comprising a new ATG start codon 39 base pairs downstream of the original start position the HsBPROS26 cDNA was generated by sitedirected mutagenesis using the oligonucleotide-directed in vitro mutagenesis system (Amersham Corp.) with the oligonucleotide 5Ј-CAGAGGTCCAATCCATATGACCCAGAACCCC-3Ј as the mutagenic primer. For electron microscopy (see below) HsC8 protein was further purified on a Superose 6 HR 10/30 prepacked size exclusion column (PharmaciaLKB) in 10 mM Tris/HCl, pH 7.5, followed by concentration of the HsC8 peak fractions in a microsep device (Filtron) with a cutoff of 100 kDa
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