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

Abstract

The proteasome is responsible for degradation of substrates of the ubiquitin pathway. 20S proteasomes are cylindrical particles with subunits arranged in a stack of four heptameric rings. The outer rings are composed of alpha subunits, and the inner rings are composed of beta subunits. A well-characterized archaeal proteasome has a single type of each subunit, and the N-terminal threonine of the beta subunit is the active-site nucleophile. Yeast proteasomes have seven different beta subunits and exhibit several distinct peptidase activities, which were proposed to derive from disparate active sites. We show that mutating the N-terminal threonine in the yeast Pup1 beta subunit eliminates cleavage after basic residues in peptide substrates, and mutating the corresponding threonine of Pre3 prevents cleavage after acidic residues. Surprisingly, neither mutation has a strong effect on cell growth, and they have at most minor effects on ubiquitin-dependent proteolysis. We show that Pup1 interacts with Pup3 in each beta subunit ring. Our data reveal that different proteasome active sites contribute very differently to protein breakdown in vivo, that contacts between particular subunits in each beta subunit ring are critical for active-site formation, and that active sites in archaea and different eukaryotes are highly similar.

Figure 1

Effect of mutating the putative active-site Thr residues in Pup1 and Pre3 on proteasome function. (A) Peptidase activities of glycerol-gradient-purified proteasomes. Values are the mean of three measurements; error bars indicate standard deviations. (B) Anti-HA immunoblot analysis of spheroplast extracts of cells expressing HA-tagged proteins. Cells expressing untagged Pup1 were used as a control. The expected sizes for HA-proPup1 and HA-Pup1 are 33 kDa and 30 kDa, respectively, consistent with the sizes observed in this 10% gel. In higher percentage gels (e.g., C and D), the mobilities of the Pup1 derivatives suggested apparent masses slightly higher than predicted; the reason for this behavior is unknown. Asterisks mark apparent proteolytic fragments of HA-tagged proteins. Whether the protein that comigrates with the Pup1-sized species in the mutant is correctly processed is unknown (see D). Size standards are marked (in kDa). (C) Anti-HA immunoblot analysis of glycerol gradient-fractionated extract from HA-PUP1 cells. Positions of size standards in the gradient are indicated (in kDa) above the gel. Twelve 1-ml fractions were collected; the 20S proteasome activity peak is in fraction 4. (D) Same as C except the extract was derived from HA-pup1-T30A cells. The majority of HA-reactive protein migrated below the size of the full-length protein in fractions 7–10, and these species were also seen in HA-PUP1 extracts; they appear to be proteolytic fragments derived from full-length Pup1. It is not known if any of this proteolysis results from the HA insertion. The Pup1 in these fractions may be in proteasome assembly intermediates.

Figure 2

Ubiquitin-dependent proteolysis in pup1-T30A, pup1-K58E pup3-E151K, and pre3-T20A cells. (A) MATα2 degradation in pup1-T30A cells at 30°C. The half-life for α2 was ≈4 min in both wild-type (MHY1066) and mutant (MHY1073) cells. (B) Leu-βgal degradation in pup1-T30A cells. (C) Deg1_α2-βgal degradation in pup1-T30A and pup1-K58E pup3-E151K cells. (D) MATα2 degradation in pre3-T20A cells. The half-life for α2 was slightly under 5 min in both wild-type (MHY1156) and mutant (MHY1157) cells.

Figure 3

Phenotypes of proteasome mutants. Exponentially growing cultures (in YPD) were plated in 1:10 serial dilutions on the indicated medium and incubated for 2 days (A) or 3 days (B–D). The yeast strains used were (from the top) MHY952, MHY973, MHY1066, MHY1073, MHY1072, and MHY1071. In strains MHY952 and MHY973, the Doa3 propeptide is expressed separately from the mature domain to circumvent the near-lethality associated with a failure to process proDoa3 with a T76A mutation (6). The MHY952 and MHY1073 strains both show an increase in Cd resistance.

Figure 4

Putative Pup1–Pup3 interaction sites and effect of NEM on peptidase activities of purified proteasomes. (A) Pup1 and Pup3 β subunits in the regions surrounding the residues involved in the putative salt bridge (double arrow) and the region of Pup3 with the conserved Cys residue (arrowhead) whose modification by NEM in bovine C10 inhibits trypsin-like activity. Secondary structures expected based on the Thermoplasma structure (5) are also shown: s, β-strand; h, helix. (B) Inhibition by NEM of peptidase activities of purified yeast 20S proteasomes. Values are the mean of three measurements. Purified proteasomes were incubated with 5 mM NEM for 20 min at 30°C.

Figure 5

Structure-based pseudoreversion analysis of the interaction between Pup1 and Pup3. MHY1004 cells transformed with the indicated plasmid-borne alleles were streaked on 5-fluoroorotic acid plates and grown at 30°C.