The cloning and characterisation of a mammalian proteasome subunit.

The proteasome is a high molecular weight (~700 kDa), multisubunit complex involved in non-lysosomal intracellular proteolysis. The eukaryotic complex is composed of fourteen different subunits and has at least five distinct catalytic activities. cDNA cloning of one subunit of the rat proteasome, RN3, was achieved through the use of a PCR based strategy. The cDNA sequence obtained contained an open reading frame of 699 nucleotides encoding a protein of 232 amino acids with a predicted molecular mass of 25.5 kDa. Sequence analysis showed it to have no homology to other known protein sequences, except for other proteasome subunits. Sequence alignments revealed that it belongs to the ?-type family of proteasome subunits. The yeast homologue of RN3, Pre4 (40% identity), has been implicated in the peptidylglutamyl-peptide hydrolase activity of the yeast proteasome. It is suggested that the RN3 subunit has a similar function in the rat enzyme. Immunoblot analysis, using anti-RN3 polyclonal antibodies, of purified rat liver proteasomes have revealed the subunit to be present as a ~24 kDa form with a pi value of ~5. The RN3 subunit is also present within the 26S proteasome complex, further supporting the suggestion that the proteasome forms the proteolytic core of this larger complex. Pulse/chase experiments have revealed that the N3 subunit is synthesised as a 28.5 kDa precursor protein which is post-translationaly processed to a 24 kDa form upon or before incorporation into the proteasome. The half-life of this processing event has been determined to be 31.5 min, suggesting it to be a very early event in the life-span of the subunit. The size of the precursor protein suggested that the original clone lacked some 5' sequence. The remaining sequence was obtained using 5' RACE and extended the open reading frame to 789 nucleotides encoding a protein of 263 amino acids.