Autocatalytic Subunit Processing Couples Active Site Formation in the 20S Proteasome to Completion of Assembly

Abstract

The eukaryotic 20S proteasome is responsible for the degradation of many cellular proteins, but how it is assembled and how its distinct active sites are formed are not understood. Like other proteasome subunits, the yeast Doa3 protein is synthesized in precursor form. We show that the N-terminal propeptide is required for Doa3 incorporation into the proteasome and, remarkably, that the propeptide functions in trans, suggesting it serves a chaperone-like function in proteasome biogenesis. Propeptide processing is not required for proteasome assembly but is needed for maturation of a specific subset of active sites. The likely nucleophile for these sites is provided by the N-terminal threonine of mature Doa3. Additional data indicate that precursor processing is autocatalytic and requires association of the two halves of the proteasome particle, thereby preventing formation of proteolytic sites until the central hydrolytic chamber has been sealed off from the rest of the cell.

Identification of the yeast 20S proteasome catalytic centers and subunit interactions required for active-site formation

“…Moreover, mutation of the putative active site Thr of Pre3 specifically blocks PGPH activity and has no obvious effect on growth. This contrasts with the major contribution of the chymotrypsin-like active sites to ubiquitin-dependent proteolysis and growth (6).…”

“…This model received strong support from both the Thermoplasma proteasome-inhibitor structure (4) and recent experiments in yeast (6). However, it seems likely that specific interactions between heterologous ␤ subunits within each ␤ subunit ring will also be important for active site formation (8).…”

“…Each ␤ subunit is synthesized with a short N-terminal propeptide; the hydroxyl of the N-terminal Thr residue of the processed ␤ subunit is responsible for nucleophilic attack on substrates (4,5). Recently, it has been shown that the yeast Saccharomyces cerevisiae 20S proteasome is likely to use the same catalytic mechanism (6). Consistent with this, when mammalian proteasomes are treated with lactacystin, an irreversible proteasome inhibitor, one of the two rapidly modified sites in the ␤ subunit X͞MB1 is the N-terminal Thr (7).…”

Induction of protein catabolism in myotubes by 15(S)-hydroxyeicosatetraenoic acid through increased expression of the ubiquitin–proteasome pathway

“…Since the effect of PIF on protein catabolism in myotubes has been shown to be mediated through an upregulation of the ubiquitin -proteasome proteolytic pathway , if 15(S)-HETE was acting as an intracellular transducer of PIF action then the effect on protein degradation would also be mediated by this pathway. Accordingly 15(S)-HETE was shown to increase the 'chymotrypsin-like' enzyme activity of the b subunits of the proteasome, suggested to be the rate-determining step in protein catabolism (Chen and Hochstrasser, 1996;Rock et al, 1994), in the same concentration range as that stimulating total protein degradation. Further confirmation that 15(S)-HETE stimulated expression of the rate-limiting components of the ubiquitin -proteasome pathway was provided by an increased mRNA level for proteasome subunits C2 and C5, as well as E2 14k , and increased protein levels of proteasome subunit, p42, over the same concentration range as that inducing total protein degradation.…”

The proteasome: a protein‐destroying machine

“…Most, but not all, ␤-type proteasomal subunits are synthesized as proproteins and processed to their mature forms by the removal of their N-terminal prosequences to become an active assembly, and this precursor processing occurs via an autocatalytic mechanism (Chen & Hochstrasser 1996;Schmidtke et al 1996). In addition, maturation of certain catalytically inactive ␤-type subunits appears to be exerted by other active ␤-type subunits, forming the fully assembled 20S particle (Heinemeyer et al 1997).…”