PROCESSING OF N3, A MAMMALIAN PROTEASOME BETA-TYPE SUBUNIT

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 Thermoplasm a acidophilum. RN3 is the beta-type subunit, N3, of rat proteasomes wh ich has been implicated in the peptidylglutamyl-peptide hydrolase acti vity of the proteinase complex. We have expressed recombinant RN3 prot ein in Escherichia coli in order to raise subunit-specific polyclonal antibodies. Identification of the position of RN3 on two-dimensional P AGE gels of purified rat liver proteasomes showed a single protein spo t of molecular mass 24 kDa and of pI value of about 5. This protein ha s a free N-terminus, having undergone post-translational processing. A fter immunoprecipitation from [S-35]methionine-labelled human embryo l ung L-132 cells using anti-RN3 antibodies, two radiolabelled spots wer e observed on two-dimensional PAGE gels, one corresponding to the matu re N3, the other of molecular mass 28.5 kDa and pI value around 5, whi ch was probably the unprocessed form of N3. However, the latter protei n had a higher molecular mass (31 kDa) than was predicted from the seq uence of previously cloned cDNA. Therefore rapid amplification of cDNA ends ('RACE') was carried out to determine the full sequence. The lac k of detectable RN3 precursor in purified rat liver proteasomes sugges ts that the processing probably accompanies assembly of the complex. T he 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 immunop recipitation of S-35-labelled complexes with anti-proteasome antibodie s. There was no evidence to suggest that unprocessed N3 is found in pr ecursor complexes which have been implicated in the assembly of some o ther unprocessed beta-type subunits. Interestingly also, the site of c leavage of N3 (ITR down arrow TQN) differs significantly from those of other processed animal beta-type proteasome subunits [(H/T)G down arr ow TT(T/L)], many of which resemble more closely the cleavage site of the Thermoplasma acidophilum beta subunit.