Sts1 plays a key role in targeting proteasomes to the nucleus

Abstract

The evidence that nuclear proteins can be degraded by cytosolic proteasomes has received considerable experimental support. However, the presence of proteasome subunits in the nucleus also suggests that protein degradation could occur within this organelle. We determined that Sts1 can target proteasomes to the nucleus and facilitate the degradation of a nuclear protein. Specific sts1 mutants showed reduced nuclear proteasomes at the nonpermissive temperature. In contrast, high expression of Sts1 increased the levels of nuclear proteasomes. Sts1 targets proteasomes to the nucleus by interacting with Srp1, a nuclear import factor that binds nuclear localization signals. Deletion of the NLS in Sts1 prevented 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 rad23Δ rpn10Δ. We show here that Sts1 suppresses a previously undetected proteasome localization defect in this mutant. Taken together, these findings explain the suppression of rad23Δ rpn10Δ by Sts1 and suggest that the degradation of nuclear substrates requires efficient proteasome localization.

FIGURE 1.

Proteasome are assembled and functional in sts1-2. A, protein lysates were resolved in a native polyacrylamide gel and incubated with proteasome substrate LLVY-AMC. The positions of intact 26 S proteasome (RP2CP) and the free CP are indicated on the right. In a control strain, increased proteasome dissociation in rpn11-1 is indicated by the high levels of CP (lane 2). Lysates prepared from STS1 and sts1-2 cells that were grown at 23 and 37 °C were examined (lanes 3–6). B, in vivo stability of FLAG-Sts1 and FLAG-sts1-2 was determined by metabolic labeling with [³⁵S]methionine. Cells were grown at 23 °C and then incubated at 37 °C for 4 h prior to labeling. Following 5 min of labeling, cells were transferred to chase medium, and samples were withdrawn at the times indicated in minutes. Equal amount of TCA-insoluble protein was immunoprecipitated and examined by autoradiography. C, in vitro interaction of GST-Sts1 and GST-sts1-2 with His₆-Srp1 and His₆-Rpn11 was tested by immunoblotting. His₆-Srp1 and His₆-Rpn11 proteins did not bind the control GST protein. Sts1 and sts1-2 interacted with both His₆-Srp1 and His₆-Rpn11.

FIGURE 2.

A nuclear localization defect in sts1-2. Rpn1-GFP was integrated in STS1 and sts1-2 at the chromosomal loci and expressed at physiological levels. A, actively growing cells were diluted into fresh medium and incubated at 23 °C and viewed by immunofluorescence at the times indicated. B, aliquot from the same cultures was transferred to medium at 37 °C, and samples were examined at the times indicated. C, following incubation for 5 h at 37 °C, STS1 and sts1-2 were diluted and incubated for ∼15 h in fresh medium at either 23 or 37 °C to test for reversibility of the defect. D, Zeiss imaging software was used to quantify the pixel density of the images seen in the microscopy images. A minimum of five independent viewing fields were examined, and well separated cells were quantified for both cytosolic (cyto) and nuclear compartment (NC). The numbers (n) represent the sum of individual cells that were viewed. The standard deviation is shown.

FIGURE 3.

Proteasome translocation defect of sts1-2 is fully suppressed by plasmid-encoded Sts1. A, high copy plasmid expressing FLAG-Sts1 was transformed into sts1-2, and the localization of integrated Pre6-GFP was examined at 37 °C. The localization defect is evident in sts1-2. However, this defect was completely rescued by expression of FLAG-Sts1. DAPI staining of the nuclei showed that the Pre6-GFP signal was predominantly co-localized to the nucleus. B, microscopy data were quantified (n, the number of cells quantified), and complete rescue of the trafficking defect of sts1-2 was confirmed.

FIGURE 4.

High expression of Sts1 increases the level of nuclear proteasomes. A, empty vector or the same vector overexpressing FLAG-Sts1 was transformed into STS1. Actively growing yeast cells were grown with or without the addition of copper sulfate to the medium. The amount of CuSO₄ added is indicted at the top of each panel. Upper panels show the localization of Pre6-GFP, and the lower panels show a merged image of GFP + differential contrast microscopy. B, fluorescence images were quantified, and the number of cells examined is indicated (n). Dark-shaded columns represent nuclear staining (NC), and light-shaded columns indicate cytosolic GFP levels (cyto). C, expression level of FLAG-Sts1 in the presence of 0, 50, and 200 μm CuSO₄ was determined by immunoprecipitation. The level of proteasome subunit Rpt1 that was co-purified with FLAG-Sts1 was also determined. A faint band detected in the vector lane (V) is a nonspecific reaction against the immunoglobulin heavy chain (asterisk).

FIGURE 5.

NLS in Sts1 contributes to its function. A, interaction between GST-Sts1 and both His₆-Srp1 and His₆-Rpn11 was examined. GST-Sts1 was immobilized on glutathione-Sepharose, and bacterial lysates containing a fixed amount of His₆-Rpn11 and increasing amounts of His₆-Srp1 were added. Following incubation for 4 h at 4 °C, the unbound proteins were removed, and the proteins bound to GST-Sts1 were detected by immunoblotting. Direct interaction between GST-Sts1 and both His₆-Srp1 and His₆-Rpn11 was confirmed (lane 1). No interaction was observed with the control GST beads (lane 5). Addition of increasing amounts of His₆-Srp1 did not affect Sts1 interaction with Rpn11 (lanes 2–4). However, higher amounts of His₆-Srp1 led to increased interaction with GST-Sts1 (compare lanes 1 and 4). B, in a complementary binding study, GST-tagged Sts1 and mutant derivatives were immobilized on glutathione-Sepharose and incubated with His₆-Srp1 and His₆-Rpn11, as described in A. Both Sts1 and sts1-2 formed equivalent interactions with His₆-Srp1 and His₆-Rpn11. Ponceau S staining of the nitrocellulose filter confirmed equal amounts of the GST-Sts1 proteins on the matrix. Removal of the nuclear localization signal from Sts1 (GST-sts1^ΔNLS) prevented interaction with His₆-Srp1 and His₆-Rpn11 (lane 5). As noted previously, we observed no interactions with the GST control beads (lane 2). C, in a reciprocal experiment, GST-Srp1 was immobilized on glutathione-Sepharose and combined with His₆-Sts1 (lane 1), His₆-sts1-2 (lane 2), and His₆-sts1^ΔNLS (lane 3). GST-Srp1 interaction with another mutant, sts1-11,12 was also examined (lane 4). Nonspecific interaction between GST and His6-Sts1 was not observed (lane 5). His₆-sts1^ΔNLS was unable to bind GST-Srp1 (lane 3), consistent with the results in B. The weaker interaction between GST-Srp1 and His₆-sts1-2 is due to lower expression levels of sts1-2 in E. coli (see E). D, we also immobilized GST-Rpn11 on glutathione-Sepharose and examined its interaction with His₆-Sts1 and mutant derivatives. Consistent with previous data, GST-Rpn11 interacted with His₆-tagged Sts1, sts1-2, sts1–11,12, and sts1^ΔNLS, although no interaction was detected with control GST beads. E, expression levels of the various Sts1/sts1 proteins in E. coli is shown.

FIGURE 6.

Growth deficiency of sts1-2 is closely linked to its proteasome targeting defect. A, ability of sts1 mutants to suppress the temperature-sensitive growth defect of sts1-2 was determined. Plasmids expressing Sts1, sts1^ΔNLS, and sts1-11 were transformed into sts1-2. Yeast cultures were adjusted to a density of A₆₀₀ = 1, and 10-fold serial dilutions were spotted onto agar medium that was incubated at either 23 or 37 °C. The temperature sensitivity of sts1-2 is compared with the wild type strain (both expressing vector) in the upper two lanes. B, to examine the requirement of the NLS in Sts1 for targeting proteasomes to the nucleus, we transformed sts1-2 with empty vector or plasmids expressing either Sts1 or sts1^ΔNLS. Proteasome localization was restored by Sts1 but not sts1^ΔNLS, as indicated by the localization of Pre6-GFP. C, suppression of the targeting defect of sts1-2 by Sts1 was quantified. Expression of Sts1 from the pRS vector showed proteasome localization was indistinguishable from the wild type strain (STS1 + vector). sts1^ΔNLS did not overcome the proteasome mislocalization defect of sts1-2.

FIGURE 7.

Rapid release of a GFP-tagged proteasome subunit from sts1-2 following ultrasonic disruption. A, STS1 expressing GFP or Nup49-GFP, and sts1-2 co-expressing Rpn1-GFP + FLAG-Sts1, or Rpn1-GFP + empty vector were exposed to 23-kHz ultrasonic pulses (for 5, 10, 20, and 30 s). Strong nuclear localization of Rpn1-GFP is evident in sts1-2 expressing FLAG-Sts1, even after brief ultrasonic treatment (5 s). Similarly, the nuclear localization of Nup49-GFP (seen as a fluorescent ring circumscribing the nucleus) was unaffected after a 5-s pulse. The mislocalization of proteasomes (Rpn1-GFP) is sts1-2 resembles the cytosolic localization of GFP in STS1 (compare the two lower rows). Cells were also imaged at all time points to monitor cell morphology and integrity. Prolonged sonication (>60 s) resulted in complete cell lysis and release of both nuclear (Nup49-GFP/Rpn1-GFP) and cytosolic proteins (GFP; data not shown). B, cells were pelleted after sonication, and duplicate aliquots of the supernatant were withdrawn, and GFP fluorescence was measured using a fluorescence plate reader. Rpn1-GFP, Nup49-GFP, and GFP were detected in the extracellular medium following sonication. The data representing duplicate measurements, from four independent experiments, are plotted. The error bars represent standard deviation measurements (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

FIGURE 8.

srp1-49 shows reduced binding to Sts1. A, GST-tagged Srp1 and mutant derivatives were immobilized and incubated with E. coli extracts containing His₆-Sts1. Binding was examined by immunoblotting. Both Srp1 and srp1-31 formed equivalent interactions with His₆-Sts1, although srp1-49 showed reduced binding. No interaction was detected with the control GST beads. B, binding results in A were quantified by densitometry, and ∼3-fold lower amounts of His₆-Sts1 were co-precipitated with GST-srp1-49. C, in a reciprocal assay GST-Sts1 was immobilized and incubated with His₆-tagged forms of Srp1/srp1. Lanes 1–3 show that equivalent amounts of Srp1, srp1-31, and srp1-49 were present in E. coli extracts. However, following incubation with GST-Sts1, lower amounts of His₆-srp1-49 were purified, consistent with the previous results. No interaction was detected with GST control beads. D, binding results in C were quantified by densitometry, and lower interaction between Sts1 and srp1-49 was observed. E, protein extracts were prepared from SRP1, srp1-31, and srp1-49 and separated in a native polyacrylamide gel. The gel was incubated with proteasome substrate Suc-LLVY-AMC, and the fluorescence signal from AMC (released by chymotryptic activity of the proteasome) was detected. The positions of the intact 26 S proteasome (RP2CP) and the catalytic core particle (CP) are shown at both 23 and 37 °C. The slight increase in the levels of free CP in srp1-49 at 23 °C is not significant.

FIGURE 9.

srp1 mutant displays defective proteasome localization. A, Rpn1-GFP was detected by immunofluorescence, and defective nuclear targeting of proteasomes was observed in srp1-49. The defect was less severe in srp1-31. B, fluorescence signals were quantified, and significant decreases in nuclear staining (p < 0.001) were evident in srp1-49 compared with the SRP1 wild type strain.

FIGURE 10.

Sts1 can suppress the proteasome localization defect of rad23Δ rpn10Δ. A, Rpt1-GFP was integrated in wild type and rad23Δ rpn10Δ strains and grown at a semi-permissive temperature (18 °C). Fluorescence imaging showed defective nuclear targeting of proteasomes in rad23Δ rpn10Δ. Expression of P_GAL1::STS1 in rad23Δ rpn10Δ restored nuclear localization of Rpt1-GFP, showing full suppression of this targeting defect. DAPI staining of nuclei in rad23Δ rpn10Δ is difficult due to the highly aberrant cell morphology of this mutant. However, a merged image of GFP + differential interference contrast microscopy shows clear co-localization of the Rpt1-GFP signal with DAPI. B, proteolytic defects of rad23Δ rpn10Δ include stabilization of substrates and accumulation of multiubiquitinated proteins (2nd lane). Overexpression of Sts1 restored normal levels of ubiquitinated (Ub) proteins in rad23Δ rpn10Δ (3rd lane) to the levels detected in the wild type strain (1st lane). Overexpression of Sts1 did not affect the abundance of proteasomes, as indicated by the levels of Rpn12 (lower panel).

FIGURE 11.

Stabilization of a ubiquitinated protein in sts1-2. A, gene expressing Clb2-HA was integrated in STS1 and sts1-2, and extracts were prepared at 23, 30, and 37 °C. Total protein extract was examined by immunoblotting. Clb2-HA levels were elevated in sts1-2 at all three temperatures. In contrast, the abundance of proteasome subunit Rpn12 was essentially unchanged. B, abundance of Clb2-HA was standardized to the level of Rpn12 by densitometry and plotted. The most significant increase was observed at the nonpermissive temperature (37 °C), consistent with the proteasome mislocalization defect of this mutant. C, extracts described above were incubated with antibodies against the HA epitope to purify Clb2-HA. The purified protein was examined by immunoblotting and incubation with anti-HA antibodies. Consistent with the results in A, we detected higher levels of Clb2-HA in sts1-2 (lanes 2, 4, and 6). D, longer exposure of the image shown in C reveals higher molecular weight forms of Clb2-HA, consistent with multiubiquitination. A temperature-specific accumulation is evident, because extracts prepared from cultures grown at 37 °C showed the highest levels (compare lanes 5 and 6). E, filter shown in D was stripped and reprobed with antibodies against ubiquitin, and a strong reaction was detected. As noted above, the intensity of this modification was higher at 37 °C (lane 6), when the proteasome trafficking defect of sts1-2 is most severe.

FIGURE 12.

Cytoplasmic protein degradation is not impaired in sts1-2. Yeast cells that stabilize Ura3-SL17 can grow on medium lacking uracil. A, STS1 and sts1-2 expressing Ura3-SL17 were unable to grow on SM-uracil at a semi-permissive temperature (30 °C). However, a pre1-1 pre2-2 proteasome mutant formed colonies on SM-ura, in agreement with previous studies. Deletion of the E2 enzymes that target Ura3-SL17 for degradation (ubc6Δ ubc7Δ) also allowed growth on SM-ura medium. B, we examined Ura3-SL17 abundance to verify that growth on SM-ura was the result of its stabilization. Ura3-SL17 was detected at very low levels in STS1 and sts1-2, although high levels were detected in ubc6Δ ubc7Δ. The abundance of Rad23 and proteasome subunit Rpn12 was essentially similar in all the strains characterized. The filter was stripped and reprobed with antibodies against ubiquitin, and higher levels of multiubiquitinated proteins were seen in pre1-1 pre2-2 and sts1-2. The stabilization of Ura3-SL17 in ubc6Δ ubc7Δ is consistent with the ability of this strain to grow on SM-ura. C, to improve detection, Ura3-SL17 was immunoprecipitated with antibodies against HA epitope. Ura3-SL17 was detected in STS1 but was present at significantly lower levels in sts1-2, suggesting that its degradation might be accelerated by the higher levels of cytosolic proteasomes. This filter was stripped and reprobed with antibodies against ubiquitin, and a broad smear of multiubiquitin cross-reacting signal was detected in pre1-1 pre2-2.