Many short-lived and damaged proteins are degraded by the ubiquitin-proteasome system (UPS) (1
). The UPS is comprised of the 26S proteasome, the small protein ubiquitin, and the protein machinery used to attach ubiquitin to target proteins. Most proteins are targeted for degradation by attachment of a poly-ubiquitin chain containing multiple ubiquitin monomers linked together through lysine 48. Poly-ubiquitin chains containing four or more monomers of ubiquitin are efficiently recognized by proteins in the regulatory particle (RP) of proteasome. The RP then removes the ubiquitin chain and the six proteasomal ATPases (Rpt 1–6) assist in translocation of the target protein into the interior of the barrel shaped core particle (CP) where the proteolytic active sites are located. The CP can be capped on either end by the RP.
The proteolytic activity of the UPS is intimately involved in RNA polymerase II transcription at many levels. It has long been known that the UPS can negatively regulate transcription by proteolysis of activators, thus keeping their level too low to drive gene transcription (2–4
). On the other hand, proteasome-mediated proteolysis has been found to have a stimulatory effect on the transcription of many genes, for example, through the degradation of repressor proteins such as IκB (5
). It has also been shown that proteasome-mediated turnover of activators, coactivators and other promoter-bound transcription factors are essential for the expression of some genes, though the mechanistic basis of this phenomenon is not clear. Finally, the proteasome is involved in the efficient termination of transcription and clearance of the RNAP II from sites of DNA damage (6
The UPS also affects transcription through non-proteolytic mechanisms. Chromatin immunoprecipitation followed by microarrays (ChIP-chip protocol) have revealed that proteasomal proteins are associated with DNA throughout the yeast genome (7
). This suggests that the proteasomal proteins played a role in nucleic acid metabolism and, in agreement with this view, several roles of the proteasome have been found at different stages in transcriptional regulation. These roles include chromatin modification (9
) and transcriptional elongation (11–14
), both of which occur independent of proteolytic activity. Studies of the GAL
and heat shock genes in yeast have shown that the proteasomal ATPases, but not the 20S CP, are required for efficient elongation in vitro
and in vivo
). It was shown that the transactivator Gal4 binds directly to two of the Rpt proteins (Rpt4 and Rpt6) and acts to recruit a fragment of the proteasome that includes the six ATPases (Rpts 1–6), Rpn1 and Rpn2, and perhaps other proteins, but excludes the 20CP as well as the 19S RP lid sub-complex to GAL promoters in vivo
). The mechanism by which this sub-complex of the 19S RP stimulates elongation is unclear, but it has been speculated to be involved in the remodeling of initiation complexes into elongation complexes and in the partial disassembly of nucleosomes in the pathway of the elongating polymerase.
More recently, a second non-proteolytic activity of the proteasomal ATPase complex was discovered, which is the ATP-dependent destabilization of activator-promoter complexes (18
). This destabilization activity requires physical contact between the activation domain (AD) of the Gal4 transactivator and two of the proteasomal proteins, Rpt4 and Rpt6, and probably involves the unfolding of the activator by the proteasomal ATPases, though this has not been shown conclusively (19
). This potent activity can strongly repress GAL
transcription by preventing stable association of the activator with the promoter in vivo
Interestingly, however, this activity is manifest only in the context of certain Gal4 mutants, whereas the wild-type protein is immune to this activity in vivo
. Recent investigations have revealed that the mutations that render Gal4 sensitive to this ‘stripping’ activity also prevent it from being mono-ubiquitylated within the DNA-binding domain, suggesting that mono-ubiquitylation of the activator serves to protect it from the proteasomal APTase complex (20
). This provides a potential explanation for the stimulatory effect that mono-ubiquitylation has been shown to have on some activators [also see ref. (20
) for another possible mechanism].
In agreement with this idea, it was found that high levels of soluble Ub blocks the destabilization of Gal4–DNA complexes in vitro
, arguing that Ub contacts with either the activator or proteasomal ATPases down-regulate the stripping reaction and that these interactions can be driven in trans by high levels of Ub (19
). There are several known ubiquitin receptors present in the proteasome. Rpn10 is a known poly-ubiquitin chain receptor, but deletion of the protein doesn’t grossly inhibit degradation of poly-ubiquitylated substrates in vivo
). The ATPase Rpt5 is known to interact with lysine-48-linked tetra-ubiquitin chains using a cross-linking strategy, but monomeric ubiquitin did not demonstrate any detectable interaction by the same cross-linking methodology (23
). Finally, Rpn13 has recently been demonstrated to bind to both monomeric and lysine 48-linked ubiquitin chains (24
). To determine the identity of the ubiquitin receptor in the context of the Gal4 system, a novel chemical cross-linking and label transfer strategy was used (26
). Ubiquitin was found to bind directly to Rpn1 and Rpt1 in the proteasomal ATPase complex and that these interactions disrupt the AD-Rpt6/Rpt4 interactions, causing the dissociation of the Gal4-proteasomal ATPase complex and terminating the stripping reaction.
One of the interesting observations from the previous study was that while mono-ubiquitylation added in trans
would prevent destabilization of the transactivators from DNA, lysine-48-linked tetra-ubiquitin did not display a protective effect even at much higher concentrations. This difference between monomeric and chain forms of ubiquitin was also seen for interaction of ubiquitin to the ATPase Rpt5 shown by Pickart and co-workers (23
). The explanation for this difference between forms of ubiquitin was not apparent in either of the prior studies.
In the current study, we set out to determine what surfaces of ubiquitin were important for its protective function. Proteasomal-mediated destabilization assays were used as a tool to isolate surfaces of ubiquitin for further study. These studies reveal that ‘exposed’ versions of ubiquitin, but not versions that form higher order packed quantanary structures, would effectively inhibit destabilization of Gal4/DNA complexes. Linkages of poly-ubiquitin chains that bury a hydrophobic patch, centered around isoleucine 44, do not effectively prevent destabilization. Further, mutation of the hydrophobic patch abrogates the ability of ubiquitin to interact with the proteasomal subunit Rpt1. In vivo, ubiquitin with the I44A mutation is no longer able to prevent proteasomal-mediated destabilization when fused to a mutant form of Gal4. We conclude that the hydrophobic patch of ubiquitin is important for the inhibition of destabilization of activator–DNA complexes.