|M.Sc Student||Harish Roni|
|Subject||Nuclear Targeting of Proteasome Particles: A Search|
for Molecular Players and Interactions
|Department||Department of Biology||Supervisor||Professor Amnon Harel|
|Full Thesis text|
Transport between the cytoplasm and nucleoplasm of eukaryotic cells is facilitated by nuclear pore complexes (NPCs) via free diffusion of small molecules or active transport of macromolecules mediated by shuttling transport receptors.
The 26S proteasome, a protein degradation machine, is comprised of a barrel-shaped catalytic core particle (20S) and one or two regulatory particles (19S).
We previously reported 20S as a novel proteasome particle in Xenopus egg extracts, consisting of the 20S barrel and two 19S complex proteins, namely Rpn1 and Rpn2. An additional co-purifying protein is the prevalent molecular chaperone heat-shock protein 90 (Hsp90). In nuclear targeting assays, 20S was imported into the nuclei, whereas 26S and 20S proteasomes docked on the cytoplasmic side of NPCs.
This study first aimed to elucidate the structure of 20S using biochemical methods. We followed partial models of the yeast proteasome that were available when we set to work. These models maintained that Rpn1 and Rpn2 were directly attached to each other, with Rpn2 connecting also to the 20S barrel. Surprisingly, our results did not match these models. More recent models published during the last year rule out direct Rpn1-Rpn2 and 20S-Rpn2 interactions. Although our current results comply more with these models, 20S structure is still a riddle, as neither of the models explain how 20S, Rpn1 and Rpn2 can assemble to form a stable particle.
Here we report a novel direct interaction between Hsp90 and Rpn1, hence we assume Hsp90 functions to stabilize 20S or facilitate its nuclear import. We observed reduced proteasome amount and activity in heat-treated egg cytosol. Although no recruitment of Hsp90 to the proteasome was observed, we cannot rule out a potential stress-induced “rescue activity” of Hsp90. While heat shock clearly inhibited the classical import pathway in nuclear targeting assays, 26S and 20S targeting was hardly affected and Hsp90 import was enhanced. These findings indicate that proteasome and Hsp90 nuclear targeting is carried out by non-classical pathways. Future work should test 20S import in this context.
The nature of the 20S particle is still unknown, but verifying the essence of its link to Hsp90 can contribute to our understanding of proteasomes nuclear targeting. As stressed cells are likely to favor pathways facilitating nuclear import of crucial determinants (e.g. proteasomes and Hsp90) rather than less essential cargoes, an intriguing hypothesis is that nuclear import of Hsp90 and 20S is conducted simultaneously using the same pathway.