I am a bit short on time, but I have seen that the Glickman paper on the proteasome base structure has finally appeared in print – this event should not go unnoticed. Before I begin to discuss the paper, I must admit that I haven’t really read it – I have heard Michael talk about this model at least three times, and had lengthy discussions with him and others during the Lake Garda meeting. The new model, now published in Nature Structural and Molecular Biology, departs from the old dogma how subunits of the 19S proteasome regulator complex are arranged. Not surprisingly, reactions from the proteasome field are mixed. It is no coincidence that it took more than a year to get this story published.
Before describing the new model, let me briefly recount the conventional wisdom on proteasome architecture. In the center, there is the 20S core particle, whose structure is very well understood. It consists of four stacked rings of seven different subunits each. The two middle rings contain the beta subunits – three of them with catalytic activity, the other ones are closely related but inactive. The two outer rings contain the alpha subunits, which are distantly related to the beta subunits; however, none of the alphas is catalytically active.
The outer faces of the core particle, formed by the alpha subunits, can associate with at least two different regulatory particles. The more familiar (and probably also more important) one is the ’19S regulatory particle’. The 19S particle itself consists of a ‘base’ structure proximal to the 20S core proteasome, and a ‘lid’ sitting on top of the base. By the way, it was also Michael Glickman (while working with Dan Finley), who showed that base and lid are separate particles held together by the Rpn10/S5a subunit. The lid will probably be a topic for another post; here, I will only talk about the ‘base’ and how it attaches to the 20S core.
The ‘base complex’ consists of at least 10 proteins: 6 homologous AAA-ATPases forming a hexameric ring like most other AAA ATPases, 2 large proteins called Rpn1 and Rpn2 in yeast, and finally Rpn10/S5a, which is the classical ubiquitin receptor of the proteasome and is also required for holding the lid in place. A very recent addition is Rpn13, which is probably also a component of the base.
Archea have a proteasome very much like the eukaryotic one, the same is true for a group of eubacteria, the actinomycetes (which include the notorious Mycobacterium tuberculosis). These prokaryotic proteasomes are somewhat simpler: they contain only the 20S core (made from homomeric rings of only one alpha and beta subunit each) and a hexameric ring of ATPases very similar to the eukaryotic base. In prokaryotes, there is no doubt that the ATPase ring is stacked on top of the alpha ring of the 20S core, and the same architecture was assumed for the eukaryotic base. The big Rpn1 and Rpn2 proteins are usually depicted as blobs, sitting somewhere on top of the ATPase ring, together with Rpn10 forming a ‘hinge structure’ for attaching the lid.
This architecture has now been challenged by the new paper from Michael Glickman’s group in Haifa. He describes a number of results from very different approaches, which all converge on an alternative model in which Rpn1 and Rpn2 are more proximal to the 20S core than the AAA ATPases are. Apparently, the whole thing got started by a bioinformatical paper by Andrey Kajava, coincidentally a former office-mate of mine during my Lausanne years. Andrey had predicted that the repeat motifs of Rpn1 and Rpn2 would give the proteins a toroid structure. When this first paper came out, it was greeted by some skepticism, in particular from the cryo-EM people who did not see a convincing toroid electron density on top of the ATPase ring.
Rina Rosenzweig in Michael Glickman group took this idea one step further; they collaborated with Pawel Osmulski and Maria Gaczynska, who use atomic force microscopy (AFM) for studying macromolecular assemblies. According to the published AFM data, both Rpn1 and Rpn2 form individual rings. When analyzed together, a ring with the same diameter was observed, which was twice as high, suggesting that Rpn1 and Rpn2 stack on top of each other. Crosslinking experiments showed that Rpn2, but not Rpn1, appeared to be a direct neighbor of the 20S core particle: according to MS analysis, Rpn2 was crosslinked to five different alpha subunits.
Finally, a series of atomic force micrographs of 20S core particles reconstituted with various factors, had been obtained. I think the figure to the right looks quite convincing. The rightmost panel shows the 20S core with a relatively flat surface. In the 2nd panel from the right, Rpn2 appears to form a circular structure on top of the 20S core. In the 3rd panel from the right, Rpn2 and Rpn1 combined result in a circular structure of higher profile (bottom row), while the addition of the full base particle, including the ATPases, does broaden but not further heighten the profile.
The obvious model for explaining these results looks like the one shown on the left. The two rings of Rpn2 (yellow) and Rpn1(red) form a funnel-like structure on top of the ‘entrance’ of the 20S core particle (green). The six AAA ATPases (orange) form a ring encircling the funnel.
The two most important questions are i) is this model correct? and ii) will it have a strong impact on the future of proteasome research?
I mentioned above that the reactions to the new model and the underlying data have been mixed. During the last year, I have discussed it with several people from the field and heard comments ranging from ‘certainly correct, is in perfect agreement with my new data’ to ‘complete rubbish, my new data prove that the model is wrong’. I for myself find that the data look convincing, but I am not much of an expert in atomic force microscopy. I am aware of the specificity problems with photochemical crosslinking, but as far as I can tell this part of the paper doesn’t look too bad, either. So, judging by the data, I don’t think that the new model can be easily dismissed. What I don’t like about the model are two things. First, I find it hard to believe that the arrangement of core particle and ATPases should be different in eukaryotes and prokaryotes (which lack Rpn1/2). Second, I see a problem with Rpn2 being completely buried inside the proteasome structure. After all, Rpn13 has been found to attach to the complex via binding to Rpn2, which is not really compatible with its purely internal position. Michael (sort of) addressed this problem by admitting that some part of Rpn2 might stick out somewhere. I don’t know what to think, probably time will tell.
With regard to the relevance of the new model, it is interesting to note the complete absence of both associated ‘news & views’ material and press releases claiming new avenues to treat cancer. I find this somewhat surprising, as this new finding is much closer to a ‘gatekeeping’ role than Rpn13 is. It should be kept in mind that the entrance pore of isolated 20S proteasome core partciles is closed. For opening this pore, an interaction with the base complex is required. According to the new model, Rpn2 would be in the best position to do this job. I am convinced that – as soon as more data supporting the new model becomes available – a lot of the accepted truths about ubiquitin recognition by the proteasome and shuttling substrates into the 20S catalytic chamber will have to be reconsidered.
Addition: After discussing this post with Michael Glickman, I should point out that even in his new model there are contacts between the ATPases and the 20S core. Thus, the new model is not that much different from the archaeal proteasome. The major difference is that in the revised eukaryotic model, the opening in the ATPase ring is large enough to accomodate the toroidal Rpn2/Rpn1 subcomplex.