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26 S proteasomes function as stable entities
Klavs B Hendil1, Rasmus Hartmann-Petersen, Keiji Tanaka
1August Krogh Institute, Copenhagen O, DK-2100, Denmark. KBHendil@aki.ku.dk
Abstract:
Most proteins in eukaryotic cells are degraded by 26-S proteasomes, usually after being conjugated to ubiquitin. In the absence of ATP, 26-S proteasomes fall apart into their two sub-complexes, 20-S proteasomes and PA700, which reassemble upon addition of ATP. Conceivably, 26-S proteasomes dissociate and reassemble during initiation of protein degradation in a ternary complex with the substrate, as in the dissociation-reassembly cycles found for ribosomes and the chaperonin GroEL/GroES. Here we followed disassembly and assembly of 26-S proteasomes in cell extracts as the exchange of PA700 subunits between mouse and human 26-S proteasomes. Compared to the rate of proteolysis in the same extract, the disassembly-reassembly cycle was much too slow to present an obligatory step in a degradation cycle. It has been suggested that subunit S5a (Mcb1, Rpn10), which binds poly-ubiquitin substrates, shuttles between a free state and the 26-S proteasome, bringing substrate to the complex. However, S5a was not found in the free state in HeLa cells. Besides, all subunits in PA700, including S5a, exchanged at similar low rates. It therefore seems that 26-S proteasomes function as stable entities during degradation of proteins.
Insights
The 26-S proteasome, crucial for protein degradation in eukaryotic cells, appears to function as a stable complex. Its disassembly-reassembly cycle is too slow to be essential for protein degradation initiation.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The 26-S proteasome is the primary machinery for regulated protein degradation in eukaryotic cells.
- Ubiquitin-conjugated proteins are targeted for degradation by the 26-S proteasome.
- The 26-S proteasome can reversibly dissociate into 20-S proteasomes and PA700 sub-complexes in an ATP-dependent manner.
Purpose of the Study:
- To investigate the dynamics of 26-S proteasome disassembly and reassembly during protein degradation.
- To determine if 26-S proteasome dissociation-reassembly cycles are obligatory steps in protein degradation.
- To examine the proposed role of subunit S5a in substrate shuttling.
Main Methods:
- Monitoring the exchange of PA700 subunits between mouse and human 26-S proteasomes in cell extracts.
- Comparing the rates of proteolysis with the rates of 26-S proteasome disassembly-reassembly.
- Assessing the free state of S5a in HeLa cell extracts.
Main Results:
- The disassembly-reassembly cycle of 26-S proteasomes was significantly slower than the rate of proteolysis.
- Subunit S5a was not detected in a free state in HeLa cells.
- All PA700 subunits, including S5a, exhibited similarly low exchange rates.
Conclusions:
- The disassembly-reassembly cycle of the 26-S proteasome is unlikely to be an obligatory step in protein degradation.
- 26-S proteasomes likely function as stable, intact complexes during the process of protein degradation.
- The proposed substrate-shuttling role of S5a is not supported by these findings.