Studies on rLMP7, a beta-subunit of the multicatalytic proteinase.
Subunit rLMP7 of the multicatalytic proteinase (MCP), which has been associated with chymotrypsin-like proteinase activity, was examined in rat liver and hepatocyte-derived cell lines. rLMP7 was detected in both nucleus and cytosol in liver by immunohistochemistry and immunoblotting, using a peptide-specific anti-rLMP7 antibody. A M(r) 30,000 precursor 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 nucleus 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 confirmed 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 previously 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 shorter than rLMP7.1 and does not contain the most 5' ATG. Transient transfection analyses with rLMP7.1 and rLMP7.s constructs coupled to green fluorescent protein showed that both transcripts were efficiently expressed in vivo. In vitro expression of these two rLMP7 cDNAs showed that rLMP7.1 produces the M(r) 30,000 precursor protein, whereas rLMP7.s produces two smaller peptides of M(r) 25,000 and 23,000. Purified 20S MCP preparations were able to proteolytically process the M(r) 30,000 precursor to the M(r) 25,000 product but not to the mature rLMP7 form. However, 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 processing and pulse-chase experiments suggested that the mature M(r) 23,000 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 rLMP7.s.
Ren
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Department of Pathology, The Pennsylvania State University School of Medicine, Hershey 17033, USA.
Clawson
G A
GA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002461:Cell Line, Transformed / Q000737:chemistry / Q000378:metabolism; D002467:Cell Nucleus / Q000737:chemistry; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D049452:Green Fluorescent Proteins; D006801:Humans; D008099:Liver / Q000166:cytology; D008164:Luminescent Proteins / Q000235:genetics; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D011498:Protein Precursors / Q000032:analysis; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014162:Transfection
D018076:DNA, Complementary; D008164:Luminescent Proteins; D009097:Multienzyme Complexes; D011498:Protein Precursors; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D003546:Cysteine Endopeptidases