We report the discovery of a short-lived chaperone that is required for the correct maturation of the eukaryotic 20S proteasome and is destroyed at a specific stage of the assembly process. The S. cerevisiae Ump1p protein is a component of proteasome precursor complexes containing unprocessed beta subunits but is not detected in the mature 20S proteasome. Upon the association of two precursor complexes, Ump1p is encased and is rapidly degraded after the proteolytic sites in the interior of the nascent proteasome are activated. Cells lacking Ump1p exhibit a lack of coordination between the processing of beta subunits and proteasome assembly, resulting in functionally impaired proteasomes. We also show that the propeptide of the Pre2p/Doa3p beta subunit is required for Ump1p's function in proteasome maturation.
The assembly of individual mammalian proteasome subunits into catalytically active 20S proteasome is not well understood. Herein, we report the identification and characterization of human and mouse homologues of the yeast proteasome maturating factor Ump1p. We delineate the region of hUMP1 implicated in the specific interaction with proteasome precursors and show that hUMP1 protein is absent from the mature form of the 20S proteasome. We also show that the transcript level of mammalian UMP1 is increased after IFN-␥ treatment and that mammalian UMP1 is functionally related to but not interchangeable with its yeast homologue. The proteasome is one of the major nonlysosomal proteases present in the cytosol and nucleus of cells. Through its catabolic functions, it is implicated in many cellular processes including the progression of cell cycle, the removal of misfolded proteins, apoptosis, and the production of peptides for presentation by MHC class I molecules (1-3). The proteasome is composed of a 20S barrel-shaped core particle, capped on each side by a 19S protein complex. Whereas the 20S proteasome can degrade short peptides, its association with the 19S caps forms the 26S proteasome, which is able to degrade multiubiquitylated protein substrates (4).The basic three-dimensional architecture of the 20S proteasome has been maintained from archaebacteria to humans and is characterized by four heptameric rings (5). The two outer rings are identical and are composed of ␣-subunits. The two inner rings, within which the catalytic centers are located, are also identical and are composed of -subunits. Whereas the ␣-and -rings of the archaebacterial 20S proteasome are formed of 7 identical ␣-subunits and 7 identical -subunits, the more complex eukaryotic proteasomes contain at least 14 subunits, 7 distinct ␣-subunits, and 7 distinct -subunits (6). In mammals, the existence of three additional -subunits, which are expressed and incorporated into the 20S proteasome after IFN-␥ treatment, further increases the complexity of the proteasome composition. Contrary to the archaebacterial proteasome, in which all seven -subunits are catalytically active, only three of the seven different -subunits (1, 2, and 5) of the eukaryotic standard proteasome possess catalytic properties. In cells exposed to IFN-␥, these three catalytic subunits are replaced by three distinct ones (1i͞LMP2, 2i͞MECL-1, and 5i͞LMP7) resulting in the formation of a 20S proteasome termed immunoproteasome. The different cleavage specificity of the immunoproteasome compared with the standard proteasome has been shown to modulate the production of certain antigenic peptides presented by MHC class I molecules (2, 7).A host of sequential reactions is required to reach the complex quaternary structure of eukaryotic proteasomes. The active -subunits carry N-terminal propeptides, which have been shown to play an important role, not only in the assembly and maturation of the 20S proteasome (8), but also to protect the N-terminal catalytic Thr of the matur...
Phenotypes on-demand generated by controlling activation and accumulation of proteins of interest are invaluable tools to analyse and engineer biological processes. While temperature-sensitive alleles are frequently used as conditional mutants in microorganisms, they are usually difficult to identify in multicellular species. Here we present a versatile and transferable, genetically stable system based on a low-temperature-controlled N-terminal degradation signal (lt-degron) that allows reversible and switch-like tuning of protein levels under physiological conditions in vivo. Thereby, developmental effects can be triggered and phenotypes on demand generated. The lt-degron was established to produce conditional and cell-type-specific phenotypes and is generally applicable in a wide range of organisms, from eukaryotic microorganisms to plants and poikilothermic animals. We have successfully applied this system to control the abundance and function of transcription factors and different enzymes by tunable protein accumulation.
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