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Distinct Elements in the Proteasomal ?5 Subunit Propeptide Required for Autocatalytic Processing and Proteasome Assembly.

ABSTRACT: Eukaryotic 20S proteasome assembly remains poorly understood. The subunits stack into four heteroheptameric rings; three inner-ring subunits (?1, ?2, and ?5) bear the protease catalytic residues and are synthesized with N-terminal propeptides. These propeptides are removed autocatalytically late in assembly. In Saccharomyces cerevisiae, ?5 (Doa3/Pre2) has a 75-residue propeptide, ?5pro, that is essential for proteasome assembly and can work in trans. We show that deletion of the poorly conserved N-terminal half of the ?5 propeptide nonetheless causes substantial defects in proteasome maturation. Sequences closer to the cleavage site have critical but redundant roles in both assembly and self-cleavage. A conserved histidine two residues upstream of the autocleavage site strongly promotes processing. Surprisingly, although ?5pro is functionally linked to the Ump1 assembly factor, trans-expressed ?5pro associates only weakly with Ump1-containing precursors. Several genes were identified as dosage suppressors of trans-expressed ?5pro mutants; the strongest encoded the ?7 proteasome subunit. Previous data suggested that ?7 and ?5pro have overlapping roles in bringing together two half-proteasomes, but the timing of ?7 addition relative to half-mer joining was unclear. Here we report conditions where dimerization lags behind ?7 incorporation into the half-mer. Our results suggest that ?7 insertion precedes half-mer dimerization, and the ?7 tail and ?5 propeptide have unequal roles in half-mer joining.

SUBMITTER: Li X

PROVIDER: S-EPMC4722473 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Distinct Elements in the Proteasomal β5 Subunit Propeptide Required for Autocatalytic Processing and Proteasome Assembly.

Li Xia X Li Yanjie Y Arendt Cassandra S CS Hochstrasser Mark M

The Journal of biological chemistry 20151201 4

Eukaryotic 20S proteasome assembly remains poorly understood. The subunits stack into four heteroheptameric rings; three inner-ring subunits (β1, β2, and β5) bear the protease catalytic residues and are synthesized with N-terminal propeptides. These propeptides are removed autocatalytically late in assembly. In Saccharomyces cerevisiae, β5 (Doa3/Pre2) has a 75-residue propeptide, β5pro, that is essential for proteasome assembly and can work in trans. We show that deletion of the poorly conserved ...[more]

PMID: 26627836

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Project description:Proteasome subunits are encoded by members of the same gene family and can be divided into two groups based on their similarity to the alpha and beta subunits of the simpler proteasome isolated from Thermoplasma acidophilum. RN3 is the beta-type subunit, N3, of rat proteasomes which has been implicated in the peptidylglutamyl-peptide hydrolase activity of the proteinase complex. We have expressed recombinant RN3 protein in Escherichia coli in order to raise subunit-specific polyclonal antibodies. Identification of the position of RN3 on two-dimensional PAGE gels of purified rat liver proteasomes showed a single protein spot of molecular mass 24 kDa and of pI value of about 5. This protein has a free N-terminus, having undergone post-translational processing. After immunoprecipitation from [35S]methionine-labelled human embryo lung L-132 cells using anti-RN3 antibodies, two radiolabelled spots were observed on two-dimensional PAGE gels, one corresponding to the mature N3, the other of molecular mass 28.5 kDa and pI value around 5, which was probably the unprocessed form of N3. However, the latter protein had a higher molecular mass (31 kDa) than was predicted from the sequence of previously cloned cDNA. Therefore rapid amplification of cDNA ends ("RACE') was carried out to determine the full sequence. The lack of detectable RN3 precursor in purified rat liver proteasomes suggests that the processing probably accompanies assembly of the complex. The half-life of the processing was determined to be 31 min in growing L-132 cells. The unprocessed form of N3 was not observed after immunoprecipitation of 35S-labelled complexes with anti-proteasome antibodies. There was no evidence to suggest that unprocessed N3 is found in precursor complexes which have been implicated in the assembly of some other unprocessed beta-type subunits. Interestingly also, the site of cleavage of N3 (ITR decreases TQN) differs significantly from those of other processed animal beta-type proteasome subunits [(H/T)G decreases TT(T/L)], many of which resemble more closely the cleavage site of the Thermoplasma acidophilum beta subunit.

Dataset Information

Distinct Elements in the Proteasomal ?5 Subunit Propeptide Required for Autocatalytic Processing and Proteasome Assembly.

Publications

Distinct Elements in the Proteasomal β5 Subunit Propeptide Required for Autocatalytic Processing and Proteasome Assembly.

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