The C-terminal Extension of the β7 Subunit and Activator Complexes Stabilize Nascent 20 S Proteasomes and Promote Their Maturation

Abstract

The eukaryotic 20 S proteasome is formed by dimerization of two precursor complexes containing the maturation factor Ump1. ␤7/Pre4 is the only one of the 14 subunits forming the 20 S proteasome that is absent from these precursor complexes in Saccharomyces cerevisiae. Increased expression of Pre4 leads to a reduction in the level of precursor complex, indicating that Pre4 incorporation into these complexes is rate-limiting for their dimerization. When we purified these precursor complexes, we observed co-purification of Blm10, a large protein known to attach to the ␣ ring surface of proteasomes. In contrast to single mutants lacking either Blm10 or the C-terminal extension of Pre4, a mutant lacking both grew extremely poorly, accumulated very high levels of precursor complexes, and was impaired in ␤ subunit maturation. The effect of blm10⌬ on proteasome biogenesis is modest, apparently because the 19 S regulatory particle is capable of substituting for Blm10, as long as precursor complex dimers are stabilized by the Pre4 C terminus. We found that a mutation (sen3/rpn2) affecting the Rpn2 subunit inhibits attachment of the 19 S activator to the 20 S particle or its precursors. Although the sen3 mutation alone had no apparent effect on precursor complex dimerization and active site maturation, the sen3 blm10 double mutant was impaired in these processes. Together these data demonstrate that Blm10 and the 19 S activator have a partially redundant function in stabilizing nascent 20 S proteasomes and in promoting their activation.

Involvement of Bag6 and the TRC pathway in proteasome assembly

“…Half-CPs then dimerize on the incorporation of b7 to form mature CPs, accompanied by cleavage of b-subunit propeptides and degradation of UMP1 and PAC1-PAC2 ( Supplementary Fig. S1) 14,26,[31][32][33][34][35][36] .…”

Proteasome assembly from 15S precursors involves major conformational changes and recycling of the Pba1–Pba2 chaperone

“…The abundance of the 15S intermediate in the proteasome biogenesis pathway is very low in wild-type cells. In order to enrich the cellular 15S precursor population and to enable selective affinity purification, we used a yeast strain lacking the C-terminal extension of b7 (b7DCTE) 18 and expressing an N-terminally FLAG-6 Â His (FH)-tagged version of Ump1. The b7DCTE mutation does not affect the structure of the 15S intermediate because the b7 subunit is absent from this complex 18 ; however, lack of the CTE causes an accumulation of the 15S PC because it inhibits 20S CP formation by complex dimerization 20 .…”

The Ubiquitin–Proteasome System of Saccharomyces cerevisiae

“…The b5 propeptide also interacts physically with Ump1 (Heink et al 2005), a chaperone that suppresses halfmer dimerization until the b ring is complete (Li et al 2007c). The b ring is completed with the addition of the b7 subunit (Marques et al 2007). This subunit has a C-terminal tail that reaches to the neighboring b ring and inserts into the interface between b1 and b2.…”

“…Perhaps in this way, Blm10 promotes degradation of Sfp1, a transcriptional activator of ribosomal protein genes Lopez et al 2011). Blm10 also participates in assembly of the CP (Fehlker et al 2003;Marques et al 2007).…”