Proteasome-associated RNAs are non-specific

The RNA isolated from RNase-treated proteasome preparations from human erythrocytes, HeLa cells, the archaeon Thermoplasma acidophilum and also from recombinant proteasomes of T. acidophilum expressed in Escherichia coli was characterized. The RNA associated with structurally similar protein particles, namely with the two molecular chaperones, groEL from E. coli and with the thermosome from T. acidophilum, served as controls. Electrophoretic analysis on polyacrylamide gels of the radioactively end-labelled RNA revealed a very similar size distribution pattern, irrespectively of the protein particles from which they had been isolated. The predominant RNA species were in the size ranges 80 nucleotides and 120 nucleotides, respectively. Partial sequencing of their terminal regions by mobility-shift analysis revealed that, of the proteasomes from human erythrocytes, the approximately 80-nucleotide-long RNA consists of a heterogenous population of mostly tRNA species because they carried the tRNA-specific 3'-terminal sequence motif 5'-CCA-3'. The RNA in the size range 120 nucleotides isolated from the proteasomes of human erythrocytes and of T. acidophilum was also heterogeneous and displayed, in the terminal regions, a remarkable sequence similarity to the corresponding regions of the 5S rRNA from the same and different organisms. The total content of RNA of all the protein particles was quantified and found to be consistently sub-stoichiometric. All these findings strongly suggest that RNA associated with the proteasomes and with the molecular chaperones originate from the abundant cellular pool of the tRNAs and 5S rRNAs which bind non-specifically to these large protein particles

Cloning, sequencing and expression of vat, a cdc48/p97 atpase homologue from the archaeon thermoplasma acidophilum

A member of the AAA family of Mg2+-ATPases from the archaeon Thermoplasma acidophilum has been cloned and expressed in Escherichia coli. The protein, VCP-like ATPase of Thermoplasma acidophilum (VAT), is a homologue of SAV from Sulfolobus acidocaldarius and CdcH of Halobacterium salinarium, and belongs to the CDC48/VCP/p97 subfamily. The deduced product of the vat gene is 745 residues long (M(r) 83 000), which has an optimal Mg2+-ATPase activity at 70 degrees C. Electron microscopy shows the purified protein to form single and double homo-hexameric rings, Although the symmetry is different, the appearance of the complexes formed of two rings resembles the 20S proteasome and Hsp60/GroEL. (C) 1997 Federation of European Biochemical Societies. [References: 42