The present studies provide a number of insights concerning the association of ubiquitinated substrates with the proteasome and the subsequent steps leading to their degradation. These findings were made possible by the development of a rapid, highly specific and quantitative method to assay the initial binding of ubiquitinated proteins to the 26S. A key difference from previous assays is that we use low temperatures (4°C) to prevent further proteasome-associated reactions (e.g. unfolding, deubiquitination). It is noteworthy that the purified 26S binds K48 and K63 Ub chains with similar high affinities (K1/2
~30nM), even though in vivo a K63 chain do not target proteins to the proteasome (Newton et al., 2008
). The present findings, however, are in accord with prior reports that attachment of K63 chains to a substrate can support its efficient degradation by pure proteasomes (Hofmann and Pickart, 2001
; Kim et al., 2007
). Therefore, in cells, some unknown factors must prevent this high affinity association of K63 chains with the 26S.
Our findings also extend the prior conclusion that the 19S particle contains two primary “receptor” proteins for Ub conjugates, Rpn10 and Rpn13 (Husnjak et al., 2008
). As shown here, Rpn10 and Rpn13 contribute equally to the high affinity binding site for the Ub chain (K1/2
was reduced 2-fold upon inactivation of either). Accordingly, recent NMR findings suggested that isolated Rpn10 and Rpn13 molecules can bind simultaneously to the same Ub chain (Zhang et al., 2009
). Our studies also demonstrated another conjugate binding site with 4-fold lower affinity in the proteasome. The existence of such an additional site had been proposed to explain why the deletion of rpn10, rpn13, rad23, dsk2
is not lethal in S. cerevisiae
(Husnjak et al., 2008
). Though dispensible, the high affinity site is probably important for the efficient binding of ubiquitinated substrates, which then may be translocated to the lower affinity site leading to degradation.
An important finding is that ATP binding to the 19S ATPases stimulates the association of ubiquitinated proteins with both high affinity and low affinity sites. With Rpn10, Rpn13 and the low affinity site, binding was maximal in the ATP bound conformation, as shown with ATPγS, and minimal with ADP bound. These effects of nucleotides closely resemble findings on the role of the ATPases in stimulating gate-opening in the 20S particle to allow substrate entry (Smith et al., 2005
). This step is also activated maximally with ATPγS and is not supported by ADP. Thus, ATP binding affects multiple aspects of the 19S structure and not just the conformations of the six ATPase subunits. Presumably, the ability of ATP to promote conjugate binding as well as gate-opening allows the coordination of these processes to promote efficient proteolysis.
Although Rpn10 and 13 lack nucleotide binding sequences, Rpn10 has been shown to directly interact with the ATPase subunit, Rpt3. Since both Rpn10 and 13 appear to form a single binding site, Rpn13 is most likely also located in close proximity to the ATPase ring (Davy et al., 2001
; Nickell et al., 2009
; Zhang et al., 2009
), and thus structural changes in the ATPase subunits upon ATP binding and hydrolysis probably alter the conformations of Rpn10 and 13. Interestingly, the magnitude of the stimulation of conjugate binding by ATP and ATPγS was consistently greater in cells lacking Rpn10 or Rpn13 (especially in the double mutant) than in the wt 26S particles. Perhaps the remaining low affinity binding site in Rpn10 mutants is the ATPase subunit, Rpt5, which has been reported to crosslink to Ub chains (Lam et al., 2002
), and nucleotide binding to Rpt5 directly affects its conformation.
Presently, it is only possible to measure conjugate binding in the two extreme states when either ADP or ATPγS is bound. However, both these states are transitory ones and during protein degradation, bound ATP is rapidly hydrolyzed to ADP, which then exchanges with new ATP molecules. Consequently, in vivo the 19S particle must be repeatedly cycling between ATP and ADP-bound states, which clearly differ in affinity for Ub conjugates. Possibly, these ATP-driven dynamic changes in affinity for Ub chains might facilitate its release and diffusion from the initial Rpn10/Rpn13 binding site to the lower affinity site and eventually to the DUBs. Furthermore, because ATP and ADP probably bind to the six different ATPase subunits in a asynchronous cyclic fashion, these ATP-driven changes in conjugate affinity may affect the different Ub binding sites asynchronously (e.g. if they reduce the affinity of the Rpn10/Rpn13 site while enhancing the affinity of the second site, it could provide a mechanism for efficient chain diffusion between sites).
A commitment step in which Ub conjugates become more tightly bound
These studies have uncovered a key step in the handling of ubiquitinated proteins by the proteasome. After the initial easily reversible binding, the association of the conjugate with the 26S becomes tighter (i.e. salt-resistant). This step appears to commit the substrate to degradation and probably reflects a quality control mechanism in which Ub conjugates that cannot be partially unfolded or translocated are released. The step to tighter binding requires the presence of a loose domain on the substrate. Such proteins as well as ones that are inherently structureless, partially misfolded or damaged postsynthetically should be efficiently hydrolyzed. By contrast, proteins containing only tightly folded globular domains that would resist unfolding and translocation into the 20S (Prakash et al., 2009
) are probably important to release from the 26S, because their continued association could prevent the degradation of other substrates.
These observations nicely account for the findings by Matouschek, Coffino and colleagues that efficient degradation by the 26S (e.g. Ub5
-DHFR or Ub4
-Barstar) requires an unstructured site in the protein (Prakash et al., 2009
; Takeuchi et al., 2007
). In vivo, the proteasome also does not degrade ubiquitinated DHFR that is stabilized through association with methotrexate (Johnston et al., 1995
). The ligand-stabilized Ub5
-DHFR initially associates with the 26S through its Ub chain as tightly as the ligand-free, degradable form, but it can't proceed to the more tightly bound state. Similarly Ub4
-Barstar without an unfolded domain could not be tightly bound or degraded by the 26S. Thus, the ability to undergo this transition correlates with susceptibility to proteolysis.
Although a loosely folded domain on a substrate is critical for the tighter association and rapid degradation, not every protein substrate contains such a domain. In fact, the presence of such features on regulatory proteins may have evolved to allow their rapid clearance, while their absence may be a feature of long-lived polypeptides. Recent studies indicate that the p97/VCP ATPase complex can act on such tightly folded ubiquitinated proteins and facilitate their degradation by the 26S (Beskow et al., 2009
). In this role p97 with its associated cofactors may function to expose domains that allow their tighter binding to the proteasome. In any case, highly stable ubiquitinated proteins are probably continually being released from the 26S in vivo with Ub chains attached for further destabilization by the p97 complex or deubiquitination in the cytosol.
It is noteworthy that after the transition to the tightly bound state, the Ub chain on the substrate is still largely intact, but it does not appear to be essential for the tighter binding which, unlike the initial binding, is not blocked by an excess of the UIM domains. Furthermore, inactivating the 19S-associated DUBs does not inhibit this transition (Fig. S3B, C
). Therefore, conjugate disassembly must occur at a later step in the degradation process. Recently, we showed that the binding of Ub conjugates to Usp14/Ubp6 activates gate-opening in the 20S and thus facilitates polypeptide degradation (Peth et al. 2009
). When conjugates were initially bound at 4°C and then switched to 37°C, the time lag until tight binding and gate-opening reached their maximum were very similar (about 20 - 25 minutes)(). Presumably, during this period, there is an ATP-dependent structural rearrangement in the 26S (or the Ub conjugate) allowing the 26S to hold the substrate more tightly and to avoid its release when the Ub chain is removed. Because this tighter binding precedes deubiquitination, it may even be a prerequisite for possible editing of the Ub chain by DUBs (e.g. Usp14) or E3s (e.g. Hul5), which have been shown to regulate substrate degradation (Crosas et al., 2006
). The existence of this further editing step would also imply that this commitment step does not necessarily lead to proteolysis. Such a commitment step that follows initial binding of a Ub conjugate and precedes its deubiquitination has been hypothesized previously (Verma et al., 2002
). The present findings confirm this key biochemical transition and directly link it to the requirement of loosely folded domains in the substrate for degradation.
It is very likely that during the transition to the committed state, the easily unfolded domains bind directly to the ATPases, since the addition of casein or ligand-free DHFR inhibited this step. The 19S ATPase ring, like its evolutionary precursor, the PAN ATPase, has an intrinsic affinity for unfolded proteins, such as casein, which can be degraded in an ATP-stimulated process without ubiquitination (Tanaka et al., 1983
). Unlike the initial binding of conjugates, which is supported better by ATPγS than by ATP, the transition to tighter binding requires ATP and does not occur at low temperatures. Thus, it seems most likely that this step involves an association of the easily unfolded domain with the ATPases and probably a partial ATPase-driven translocation of the polypeptide into the ATPase ring.
In summary, our findings have dissociated a specific sequence of events occurring on the 26S during the degradation of a ubiquitinated protein. 1) The Ub chain first binds to a site formed by Rpn10/Rpn13, when these proteins are in their high affinity conformation, which requires ATP binding to the ATPases. 2) Subsequently, a loosely folded domain of the polypeptide interacts with a site probably on the ATPase subunits, which has an affinity for unstructured proteins. ATP-hydrolysis then leads to a tighter association with the 26S and further commits the substrate on the path for degradation. 3) During this process, a transfer of the Ub conjugate to a lower affinity site seems likely, leading in turn to interaction with the DUBs and perhaps chain editing. The association of the Ub chain with the Usp14 active site leads to either shortening or removal of the chain, and simultaneously enhances gate opening in the 20S, facilitating polypeptide entry. 5) Concomitantly, the ATPase subunits unfold and translocate the protein through the fully opened gate into the 20S particle for processive degradation.