The evidence that nuclear proteins can be degraded by cytosolic proteasomes has received considerable experimental support. its interaction with Srp1 and caused proteasome mislocalization. In agreement with this observation a mutation in Srp1 that weakened its interaction with Sts1 also reduced nuclear targeting of proteasomes. We reported that Sts1 could suppress growth and proteolytic defects of mutant (C194Y) was defective in protein degradation and accumulated high levels of ubiquitinated proteins. Significantly the interaction between multiubiquitinated proteins and proteasomes was reduced in (17) is most likely due to reduced levels of proteasomes at the nucleus. We determined that proteasomes are also mislocalized in mutant (mutant that is unable Presapogenin CP4 to bind Srp1 has reduced levels of nuclear proteasomes. As expected a nuclear localization signal (NLS) in Sts1 is required for binding Srp1 and promoting nuclear trafficking of proteasomes. In a reciprocal study we found that a mutation in Srp1 that reduced its interaction with Sts1 was also deficient in recruiting proteasomes to the nucleus. Thus protein degradation appears to be affected by the level of nuclear proteasomes which is a consequence of the interaction between Srp1 and Sts1. Taken together these genetic and biochemical studies offer insight into the mechanism of proteasome translocation to the nucleus and demonstrate that a failure causes cell death. EXPERIMENTAL PROCEDURES Yeast Strains and Plasmids Yeast strains harboring mutations in were provided by Drs. P. Tongaonkar and M. Nomura (University of California Irvine). DNA templates were sequenced and the mutations were verified (S116F; E145K). Strains containing mutations in were also provided by Dr. F. Wyers. Plasmids for generating integrated derivatives of GFP-tagged proteasome subunits were generously provided by Dr. C. Enenkel (Humboldt University). All the amplified DNAs were verified by sequencing both strands. A list of yeast strains and plasmids is shown in Tables 1 and ?and2 2 respectively. TABLE 1 TABLE 2 Growth Assays and Sensitivity to Temperature Yeast cultures were grown in selective medium and normalized to an absorbance at and expressing FLAG-Sts1 and FLAG-sts1-2 Rabbit polyclonal to PFKFB3. respectively. Following incubation for 5 min at 30 °C labeling was terminated by the addition of cycloheximide and aliquots were withdrawn at 0 10 30 and 60 min. Equal amounts of trichloroacetic acid-insoluble material was incubated with anti-FLAG M2-agarose beads. The samples were resolved by SDS-PAGE and exposed to x-ray film. Native PAGE Measurement of peptidase activity of proteasomes was examined in a native polyacrylamide gel as described previously (20). Protein lysates (50 μg) were Presapogenin CP4 separated in a native gel that was overlaid with buffer containing LLVY-AMC2 in the presence of 0.05% SDS. The fluorescence signal was detected with Kodak GelLogic Imager. Purification of Recombinant Proteins and in Vitro Binding Assay Expression of proteins from pGEX and pET28 vectors was achieved in BL21S cells in the presence of 1 mm isopropyl 1-thio-β-d-galactopyranoside. Cells were lysed (in 50 mm Tris-HCl pH 7.5 150 mm NaCl 5 mm Na-EDTA 1 Triton X-100 and protease inhibitor mixture) and total protein lysate was applied to glutathione-Sepharose to purify GST-tagged proteins. Protein expression levels were determined before performing binding studies. Bacterial lysates that contained His6-tagged proteins were mixed with purified GST proteins for 4 h at 4 °C. The unbound proteins were removed by four washes in lysis buffer. The bound proteins were released in SDS gel loading buffer separated in 10 or 12% SDS-Tricine/PAGE and examined by immunoblotting. Immunoprecipitation/Immunoblotting Yeast cells were suspended in buffer A (50 mm HEPES pH 7.5 150 mm Presapogenin CP4 NaCl 5 mm EDTA and 1% Triton X-100) containing protease Presapogenin CP4 inhibitors (Roche Applied Science) and lysed by glass bead disruption. Protein extracts were normalized using the Bradford method (Bio-Rad) and incubated with anti-FLAG-M2-agarose. The bound proteins were released in SDS gel loading buffer separated in 10 or 12% SDS-Tricine/PAGE and characterized by immunoblotting. The signals were quantified using Kodak GelLogic Imaging software. Fluorescence Microscopy 500 μl of yeast cells were pelleted washed with 1 ml of phosphate-buffered saline (PBS) and suspended.