Possible tetramerisation of the proteasome maturation factor POMP/proteassemblin/hUmp1 and its subcellular localisation.
The proteasome is a multisubunit complex with a central role in non-lysosomal proteolysis and the processing of proteins for presentation by the MHC class I pathway. The 16kDa proteasome maturation protein POMP (also named proteassemblin or hUmp1) acts as a chaperone and is essential for the maturation of the 20S proteasome proteolytic core complex. However, the exact mechanism, timing and localisation of mammalian proteasome assembly remains elusive. We sought to investigate the localisation of POMP within the cell and therefore purified the protein and produced a polyclonal antibody. For immunisation, POMP was overexpressed and purified from a bacterial GST-system. Interestingly, after removal of the GST-tag, POMP was hardly detectable by Coomassie blue- and Ponceau red-staining. However, with a reverse zinc-staining, the protein could easily be visualised. POMP was gel-filtrated and eluted from a calibrated chromatography column with an apparent molecular weight of approximately 64kDa, suggesting that it forms tetramers. Moreover, localisation studies by immunofluorescence stainings and confocal microscopy revealed that POMP is present in the cytoplasm as well as in the nucleus.
Hoefer
Melanie M
MM
Biotechnology Institute Thurgau, Taegerwilen, Switzerland. melanie.hoefer@bitg.ch
Boneberg
Eva-Maria
EM
Grotegut
Stefan
S
Kusch
Justine
J
Illges
Harald
H
D002138:Calibration; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D019281:Dimerization; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances / Q000737:chemistry; D018832:Molecular Chaperones / Q000737:chemistry*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D046911:Macromolecular Substances; D018832:Molecular Chaperones; C110856:proteasome maturation protein; C413862:proteassemblin; D005982:Glutathione Transferase; D046988:Proteasome Endopeptidase Complex