Identification of Pep4p as the protease responsible for formation of the SAGA-related SLIK protein complex

Abstract

The Saccharomyces cerevisiae Spt-Ada-Gcn5 acetyltransferase (SAGA) protein complex is a coactivator for transcription by RNA polymerase II and has various activities, including acetylation and deubuiqitination of histones and recruitment of TATA-binding protein to promoters. The Spt7p subunit is subject to proteolytic cleavage at its C terminus resulting in removal of the Spt8p-binding domain and generation of the SAGA-related SALSA/SAGA-like (SLIK) protein complex. Here, we report identification of the protease responsible for this cleavage. Screening of a protease knock-out collection revealed PEP4 to be required for cleavage of Spt7p within SAGA in vitro. Endogenous formation of truncated Spt7p was abolished in cells lacking PEP4. Purified Pep4p but not catalytic dead mutant Pep4p or unrelated Prc1p protease specifically cleaved Spt7p within SAGA into SLIK-related Spt7p. Interestingly, SAGA lacking Spt8p was more sensitive to Pep4p-mediated truncation of Spt7p, suggesting that Spt8p counteracted its own release from SAGA. Strains mimicking constitutive SLIK formation showed increased resistance to rapamycin treatment, suggesting a role for SLIK in regulating cellular responses to nutrient stress.

FIGURE 1.

Aspartic enzymatic activity cleaves Spt7p^SAGA into Spt7p^SLIK. A, purified SAGA and SLIK complexes were stained with Coomassie. SPT20-C-TAP was used to purify both SAGA and SLIK (left lane). N-HA-SPT7-C-TAP was used to purify SAGA-only (right lane). Positions of full-length (Spt7p^SAGA) and C-terminally truncated (Spt7p^SLIK) forms of Spt7p as well as other SAGA/SLIK subunits are indicated. Identification of SAGA/SLIK subunits by mass spectrometry has been reported by us previously (31). B, activity present in yeast WCE cleaves Spt7p^SAGA into Spt7p^SLIK. Purified SAGA-only was incubated with yeast WCE for 30 min at 30 °C, and Spt7p processing was monitored by immunoblotting using an anti-HA antibody. Anti-tubulin antibody was used as a control for the amount of WCE added. FY2031 (N-HA-SPT7-C-TAP) WCE was used as positive control for in vivo generated Spt7p^SLIK. FY2032 (29) immunoprecipitate (IP) was prepared by TAP purification and represents a genetic mimic of Spt7p^SLIK. C, Spt7p-processing activity present in WCE belongs to the aspartic protease family. In vitro cleavage assay was as in B, but after inactivation of the WCE at 95 °C or with addition of protease inhibitors as indicated.

FIGURE 2.

Aspartic protease Pep4p is required to cleave Spt7p^SAGA. A, WCEs from Δpep4 and Δprb1 lack or have a reduced activity for generating Spt7p^SLIK from Spt7p^SAGA, respectively. WT, Δpep4, or Δprb1 yeast WCE was incubated with purified SAGA for 30 min at 30 °C, and cleavage was monitored by anti-HA immunoblot analysis. Total protein content of WCE was monitored by anti-tubulin immunoblotting. B, residual protease activity in Δprb1 WCE is inhibited by pepstatin A. In vitro cleavage assay was performed as in A but with a 30-min preincubation of WCE with aspartic (10–100-1000 ng/ml pepstatin), serine (1 mm AESBF), or cysteine (10 μm E64) protease inhibitor (*, background band). C, Pep4p is required for endogenous Spt7p^SLIK formation. N-TAP-SPT7 WT or Δpep4, Δprb1 or Δprc1 WCEs were analyzed for the presence of Spt7p^SAGA and Spt7p^SLIK. Immunoblot detection was performed using anti-TAP antibody. Anti-tubulin antibody was used to monitor the total protein content.

FIGURE 3.

Purified Pep4p directly cleaves Spt7p^SAGA to Spt7p^SLIK. A, purified Pep4p cleaves Spt7p^SAGA into Spt7p^SLIK. Purified Pep4p along with control protease purifications (Pep4p D294A, Prc1p) were tested for in vitro cleavage of N-HA-Spt7p^SAGA for 30 min at 30 °C. Detection of cleavage was obtained using HA antibody. FY2032 immunoprecipitation (IP) was used as size control for the Spt7p^SLIK generated. B, protease activity of Pep4p on Spt7p^SAGA is pepstatin-sensitive. In vitro cleavage assay was carried out as in A but with a 30-min preincubation on ice with the aspartic protease inhibitor pepstatin. FY2032 IP was used as size control for the cleavage product. C, Pep4p specifically cleaves Spt7p^SAGA within the SAGA complex. In vitro cleavage assay was performed as in A, and the reaction was analyzed on 4–12% acrylamide gel by Coomassie staining.

FIGURE 4.

Spt8p interferes with Spt7p^SLIK formation. Pep4p protease activity is more efficient on SAGA-only complex lacking Spt8p (SAGA complex w/o Spt8p) compared with SAGA-only complex (SAGA complex). Purified complexes were incubated with purified Pep4p protease at 30 °C for 30 min, and truncation of Spt7p^SAGA into Spt7p^SLIK was detected by HA immunoblotting.

FIGURE 5.

Pep4p-mediated SLIK formation is involved in rapamycin resistance. Rapamycin (rapa) sensitivity of strains deleted for PEP4 or mimicking constitutive presence of SLIK using FY2032 (SLIK only; lacking the C terminus of Spt7p as in SLIK (29)) or by deletion of SPT8. Cells were spotted in a dilution series from 10⁶ to 10⁴ cells/ml.