Katrine Andersen:
Identification and Characterisation of a Proteasome-Associated Thioredoxin

Date: 15-02-2010    Supervisor: Rasmus Hartmann-Petersen

The 26S proteasome is a giant and complex protease that is present in the cytoplasm and nucleus of all eukaryotic cells. It is composed of two large subcomplexes, the 20S core particle and the 19S regulatory particle, which in total contain more than 30 different subunits. The 26S proteasome is responsible for the degradation of most intracellular proteins. To sustain its function, the proteasome is supported by a still increasing number of interacting proteins or co-factors. In the work presented here, two new proteasome interacting proteins are identified and characterised in humans and in the fission yeast S. pombe. One of the proteins is the thioredoxin-like protein 1 (Txnl1 in humans/Txl1 in S. pombe), which is an active thioredoxin and a near stoichiometric component of the 26S proteasome. Besides having a thioredoxin (TRX) domain, Txnl1/Txl1 also contains a proteasome-interacting thioredoxin (PITH) domain, which is necessary and sufficient for binding to the proteasome. Txnl1/Txl1 is found to interact with Rpn11, a subunit of the 19S regulatory particle. The other identified protein, Txc1, is also a PITH domain containing protein, and accordingly interacts with the proteasome via this domain. A txl1 yeast knockout mutant displays a synthetic growth defect with a cut8 knockout, whereas the txc1 knockout does not. In fission yeast, Cut8 is a nuclear protein that tethers the 26S proteasome to the nuclear membrane. In both wild type cells and in a cut8 null mutant Txl1 co-localises with the proteasome at the nuclear rim, whereas Txc1 is distributed throughout the cytoplasm and nucleus. This indicates that Txl1 is closely associated with the 26S proteasome while Txc1 is perhaps only transiently bound to the complex. A slight stabilisation of ubiquitin conjugates was observed upon knock-down of Txnl1 in HeLa cells, whereas no stabilisation was detected in txl1Δ cells in S. pombe. In human cells, an active site Txnl1 mutant formed a mixed disulfide intermediate with eEF1A1, a reported substrate-recruiting factor of the 26S proteasome. Fission yeast Txl1 can reduce an oxidised proteasomal model substrate in vitro and thereby mediate its degradation. In conclusion, although the fields of intracellular protein degradation and oxidative stress have been connected for years, Txnl1/Txl1 is the first protein to provide a direct link between proteolysis and protein reduction.