Characterisation of the newly identified human Ump1 homologue POMP and analysis of LMP7(beta 5i) incorporation into 20 S proteasomes.
Biogenesis of mammalian 20 S proteasomes occurs via precursor complexes containing alpha and unprocessed beta subunits. A human homologue of the yeast proteasome maturation factor Ump1 was identified in 2D gels of 16 S precursor preparations and designated as POMP (proteasome maturation protein). We show that POMP is detected only in precursor fractions and not in fractions containing mature 20 S proteasome. Northern blot experiments revealed that expression of POMP is induced after treatment with interferon gamma. To analyse the role of the beta 5 propeptide for proper maturation and incorporation of the beta 5 subunit into the complex, human T2 cells, which highly express derivatives of the beta 5i subunit (LMP7), were studied. In contrast to yeast, the presence of the beta 5 propeptide is not essential for incorporation of LMP7 into the proteasome complex. Mutated LMP7 subunits either carrying the prosequence of beta 2i (LMP2) or containing a mutation in the active threonine site are incorporated like wild-type LMP7, while a LMP7 derivative lacking the prosequence completely is incorporated to a lesser extent. Although the absence of the prosequence does not affect incorporation of LMP7, its deletion leads to delayed proteasome maturation and thereby to an accumulation of precursor complexes. As a result of the precursor accumulation, an increased amount of the POMP protein can be detected in these cells.
Witt
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E
Institut für Biochemie, Charité-Humboldt University Medical School, Monbijoustr.2, Berlin, 10117, Germany.
Zantopf
D
D
Schmidt
M
M
Kraft
R
R
Kloetzel
P M
PM
Krüger
E
E
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D046988:Proteasome Endopeptidase Complex; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015854:Up-Regulation / Q000187:drug effects
D018832:Molecular Chaperones; D009097:Multienzyme Complexes; D011506:Proteins; D012333:RNA, Messenger; C110856:proteasome maturation protein; D007371:Interferon-gamma; D003546:Cysteine Endopeptidases; C077436:LMP7 protein; D046988:Proteasome Endopeptidase Complex