The assembly of the base seems to occur by initial pairing of Rpt proteins (). The Rpt1 and Rpt2 ATPases together with Rpn1 and Hsm3 have been repeatedly observed in cell lysates. The stability of this complex depends on Hsm321–25
. Rpt5 might associate with this complex as well21
. This entity, dubbed BP1, is a true assembly intermediate and not a dead-end product as it disappears in a chase experiment21
. Most Rpt1-bound Hsm3 seems to associate with BP1 although some Hsm3 is found associated with base and RP23, 25
. A second pair formed by Rpt4 and Rpt5 with the chaperone Nas2 has also been characterized in detail22, 23
. Nas2 is important for the presence of this complex but dissociates upon further assembly. Rpt3 and Rpt6, the third pair of Rpt proteins, have been found in a complex with Nas6 and Rpn14, although these proteins have been more readily detected when associated with the base and/or RP23–25
. Each pair interacts with at least three other subcomplexes in assembled proteasomes as indicated by the green arrows in . In what order these interactions are established is not well understood.
Some models suggest that these complexes, together with Rpn2 and Rpn13, form the base22–24
. In this process Hsm3 and Nas2 might dissociate. Upon association with the lid, RP is formed. When the RP associates with CP the Rpn14 and Nas6 proteins leave the RP, although Rpn14 dissociation might also occur at an earlier step. These models, however, do not address all observations. Mutants defective in CP assembly have shown defects in RP assembly14
. Furthermore, deletion of the C-terminal amino acid of Rpt 4 and Rpt6 causes dramatic assembly defects21
. The tails of the Rpt proteins dock into pockets in the CP40–42
and deleting the C-terminal amino acid disturbs this CP-Rpt interaction. Thus, there is a clear role for the CP in RP assembly (). To ensure proper ordering of the Rpt proteins scaffolding proteins might play a role in assembly, however, such a role has not yet been shown for the chaperones. Rpn1 and Rpn2 are potential scaffolds21, 25
. Rpn1 can bind to Rpt1 and Rpt2, thus it might act as a scaffold for the BP1. Rpn2, Rpt6 and Rpt3 interact14, 43, 44
but these proteins do not appear to form a precursor complex together23, 24
. The CP itself might form another scaffold21, 25
with the pockets of the CP presumably having Rpt tail specificity40, 41
and thus directing Rpt proteins to distinct positions in the ring. With CP as a scaffold, the chaperones could regulate the incorporation of the different precursors. However, if the precursor complexes assemble on the CP, free base and free RP observed in cell lysates must have dissociated from the CP and rebound to some of the chaperones. Two other recent papers report insights into the assembly process without using the chaperones as starting points19, 45
. Hendil, et al. used pulse chase in Hela cells to nicely show a rapid formation of an Rpt3-Rpt6 pair45
. Furthermore, they argue for the formation of a precursor complex consisting of CP plus Rpn2, Rpn10, Rpn11, Rpn13 and Txnl1 (the last protein has recently been identified as associating with the proteasome) based on pulse chase and immunoprecipitation experiments. This proposed precursor complex has, however, not been biochemically identified. Their suggestion that pairs of Rpt proteins would subsequently join this assembly fits nicely with an important role for CP as a scaffold in RP assembly and a function for chaperones in regulating CP-Rpt interaction.
The second paper by Thompson, et al. identified three RP sub-complexes from red blood cells19
. Consistent with the other studies, each sub-complex contains one Rpt pair. Ps-1 contains Rpt3 and Rpt6 together PAAF-1 and almost all RP subunits, except for Rpt1, 2, 4 and 5. Ps-2 is similar to BP-1, containing Rpn1, Rpt1, Rpt2 and S5b. Ps-3 is formed by Rpt4, Rpt5 and Nas2. In vitro
these complexes can be reconstituted efficiently to form RP. While the role of these sub-complexes in assembly in vivo
remains to be determined, it is very interesting that, apart from gankyrin, the RP chaperones are present in the different complexes. The role of the chaperones in reconstitution unfortunately has not been studied, but this study shows that CP is not required for this in vitro
In summary, it seems plausible that more than one pathway can lead to assembled 26S proteasomes. In demanding conditions, such as the high temperature used in one of the studies23
, a single preferred 26S proteasome assembly pathway might be used. Although the proteasome assembly pathways appear largely conserved from yeast to man, there are probably subtle differences in the process between different organisms. Clearly, more detailed mechanistic and structural studies are needed to completely understand the assembly process.
In addition to CP and chaperones the role of other molecular players in assembly need to be characterized. Hsp90 stimulates lid complex and 26S maintenance and assembly, but it is unclear how46
. Nob1 has a controversial role associating CP with RP in the nucleus47, 48
. Ecm29 could tether CP and RP as it stabilizes their association even in the absence of ATP18, 49
. The proteasome activator Blm10/PA200 has been reported to promote CP assembly and activation50
. There is no reason to assume this list is complete and there are likely to be more chaperones or modulators of the assembly process.