STUDIES ON RLMP7, A BETA-SUBUNIT OF THE MULTICATALYTIC PROTEINASE

Subunit rLMP7 of the multicatalytic proteinase (MCP), which has been a ssociated with chymotrypsin-like proteinase activity, was examined in rat liver and hepatocyte-derived cell. Lines. rLMP7 was detected in bo th nucleus and cytosol in liver by immunohistochemistry and immunoblot ting, using a peptide-specific anti-rLMP7 antibody. A M-r 30,000 precu rsor protein was present only in cytosol, as was a minor component of M-r 25,000. Mature rLMP7 (M-r 23,000) was present in MCP in both nucle us and cytosol, although it was not detectable in the nuclear scaffold . Two rLMP7 cDNAs (designated rLMP7.1 and rLMP7.s) were identified by rapid amplification of 5' ends using RT/PCR, a result which was confir med by Northern blot analysis and RNase protection assays. rLMP7.1 is 3-4x more abundant than rLMP7.s; it is 50 nt longer than the previousl y reported cDNA sequence and includes an upstream in-frame ATG within a consensus translation initiation sequence, which encodes the M-r 30, 000 rLMP7 precursor protein identified in vivo. rLMP7.s is 100 nt shor ter than rLMP7.1 and does not contain the most 5' ATG. Transient trans fection analyses with rLMP7.1 and rLMP7.s constructs coupled to green fluorescent protein showed that both transcripts were efficiently expr essed in vivo. In vitro expression of these two rLMP7 cDNAs showed tha t rLMP7.1 produces the M-r 30,000 precursor protein, whereas rLMP7.s p roduces two smaller peptides of M-r 25,000 and 23,000. Purified 205 MC P preparations were able to proteolytically process the M-r 30,000 pre cursor to the M-r 25,000 product but not to the mature rLMP7 form. How ever, incorporation of this processed M-r 25,000 product (or of either M-r form produced from rLMP7.s) did not occur in vitro. In vitro proc essing and pulse-chase experiments suggested that the mature M-r 23,00 0 subunit is derived, at least in part, from the M-r 30,000 precursor. The M-r 25,000 form may be a stable product produced directly from rL MP7.s. (C) 1997 Academic Press.