Txl1 and Txc1 are co-factors of the 26S proteasome in fission yeast.

The 26S proteasome is a large proteolytic particle present in the cytosol and nucleus of eukaryotic cells. Most intracellular proteins, including those affected by oxidative damage, are degraded by the proteasome. The human thioredoxin, Txnl1, is known to associate with the 26S proteasome and thereby equips proteasomes with redox capabilities. Here, we characterize the fission yeast orthologue of Txnl1, called Txl1. Txl1 associates with the 26S proteasome via its C-terminal domain. This domain is also found in the uncharacterized protein, Txc1, which was also found to interact with 26S proteasomes. A txl1 null mutant, but not a txc1 null, displayed a synthetic growth defect with cut8, encoding a protein that tethers the proteasome to the nuclear membrane. Txc1 is present throughout the cytoplasm and nucleus, whereas Txl1 co-localizes with 26S proteasomes in both wild-type cells and in cut8 mutants, indicating that Txl1 is tightly associated with 26S proteasomes, while Txc1 might be only transiently bound to the complex. Finally, we show that Txl1 is an active thioredoxin. Accordingly, Txl1 was able to reduce and mediate the degradation of an oxidized model proteasome substrate in vitro. Thus, Txl1 and Txc1 are proteasome co-factors connected with oxidative stress.

The 20S proteasome as an assembly platform for the 19S regulatory complex.

26S proteasomes consist of cylindrical 20S proteasomes with 19S regulatory complexes attached to the ends. Treatment with high concentrations of salt causes the regulatory complexes to separate into two sub-complexes, the base, which is in contact with the 20S proteasome, and the lid, which is the distal part of the 19S complex. Here, we describe two assembly intermediates of the human regulatory complex. One is a dimer of the two ATPase subunits, Rpt3 and Rpt6. The other is a complex of nascent Rpn2, Rpn10, Rpn11, Rpn13, and Txnl1, attached to preexisting 20S proteasomes. This early assembly complex does not yet contain Rpn1 or any of the ATPase subunits of the base. Thus, assembly of 19S regulatory complexes takes place on preexisting 20S proteasomes, and part of the lid is assembled before the base.

Mammalian 26S proteasomes remain intact during protein degradation.

It has been suggested that degradation of polyubiquitylated proteins is coupled to dissociation of 26S proteasomes. In contrast, using several independent types of experiments, we find that mammalian proteasomes can degrade polyubiquitylated proteins without disassembling. Thus, immobilized, (35)S-labeled 26S proteasomes degraded polyubiquitylated Sic1 and c-IAP1 without releasing any subunits. In addition, it is predicted that if 26S proteasomes dissociate into 20S proteasomes and regulatory complexes during a degradation cycle, the reassembly rate would be limiting at low proteasome concentrations. However, the rate with which each proteasome degraded polyubiquitylated Sic1 was independent of the proteasome concentration. Likewise, substrate-dependent dissociation of 26S proteasomes could not be detected by nondenaturing electrophoresis. Lastly, epoxomicin-inhibited 20S proteasomes can trap released regulatory complexes, forming inactive 26S proteasomes, but addition of epoxomicin-inhibited 20S proteasomes had no effect on the degradation of either polyubiquitylated Sic1 or UbcH10 by 26S proteasomes or of endogenous substrates in cell extracts.