Yeast importin-α (Srp1) performs distinct roles in the import of nuclear proteins and in targeting proteasomes to the nucleus

Abstract

Srp1 (importin-α) can translocate proteins that contain a nuclear localization signal (NLS) into the nucleus. The loss of Srp1 is lethal, although several temperature-sensitive mutants have been described. Among these mutants, srp1-31 displays the characteristic nuclear import defect of importin-α mutants, whereas srp1-49 shows a defect in protein degradation. We characterized these and additional srp1 mutants to determine whether distinct mechanisms were required for intracellular proteolysis and the import of NLS-containing proteins. We determined that srp1 mutants that failed to import NLS-containing proteins (srp1-31 and srp1-55) successfully localized proteasomes to the nucleus. In contrast, srp1 mutants that did not target proteasomes to the nucleus (srp1-49 and srp1-E402Q) were able to import NLS-containing proteins. The proteasome targeting defect of specific srp1 mutants caused stabilization of nuclear substrates and overall accumulation of multiubiquitylated proteins. Co-expression of a member of each class of srp1 mutants corrected both the proteasome localization defect and the import of NLS-containing proteins. These findings indicate that the targeting of proteasomes to the nucleus occurs by a mechanism distinct from the Srp1-mediated import of nuclear proteins.

Keywords: Proteasome; Protein Sorting; Protein Stability; Proteolysis; Ubiquitin-dependent Protease.

FIGURE 1.

Domain structure of Srp1/importin-α. A, the structure of the array of 10 ARM repeats in the central portion of Srp1 is shown (12), and the position of each repeat motif is indicated. The concave surface contains two NLS binding surfaces. The major NLS pocket is generated by ARM-3 and -4, and the minor NLS is formed by helix-3 residues in ARM-7 and -8. Small blue lines represent NLS peptides. The side chains of Ser-116, Glu-145, and Glu-402 are shown in yellow. The image in A was derived from crystallographic data of Conti et al. (12) (Protein Data Bank entry 1BK6) and was prepared using PyMOL (version 1.5.0.4, Schrödinger, LLC, New York). B, three key domains in Srp1 are shown in this schematic of the full-length Srp1. Solid lines demarcate the region of the protein that is represented in A. The amino-terminal IBB domain contains an engineered mutation, R55A, which causes an import defect. The ARM repeat motifs are indicated by shaded ovals. The darker shaded ovals represent the major (ARM-3 and -4) and minor (ARM-7 and -8) NLS-binding pockets. The carboxyl terminus interacts with Cse1. C, the growth defects associated with key mutants are shown. srp1-31 and srp1-49 are unable to grow at 37 °C. srp1-55 showed a pronounced cold temperature growth defect (16 °C). The srp1-E402Q mutant showed poor growth at both high and low temperatures.

FIGURE 2.

Proteasome mislocalization in srp1-49. A and B, 19 and 20 S proteasome subunits (Rpn11-GFP and Pup1-RFP, respectively) were expressed at physiological levels in SRP1, srp1-31, and srp1-49. The localization of both proteins was examined in the same field of cells, at the permissive (A, 23 °C) and non-permissive (B, 37 °C) temperatures. A merged image that includes DAPI and differential interference contrast (DIC) is shown. C, protein extracts were prepared from cultures examined in A and B to measure Rpn11-GFP and Pup1-RFP protein levels. Antibody reaction against Rpn12 and Rad23 is also shown. D, the fluorescence signal from Rpn11-GFP and Pup1-RFP in A and B was quantified using Zeiss imaging software. Multiple fields were examined, and the total number of cells sampled is indicated within each bar. The graph shows the pixel intensity in the nucleus and cytoplasm. An equal intensity in the nucleus and cytosol would be consistent with proteasome mislocalization.

FIGURE 3.

srp1-E402Q has a proteasome localization defect. A, the nuclear targeting of Rpt1-GFP was investigated in srp1^E402Q and srp1-55. The temperature-sensitive srp1^E402Q mutant was examined at 23 and 37 °C, whereas the cold-sensitive srp1-55 mutant was examined at 23 and 18 °C. B, the fluorescence intensity in A was quantified. Both srp1-49 and srp1-E402Q showed reduced levels of nuclear proteasomes at 37 °C (top). The levels of proteasome subunits Rpt1-GFP and Rpn12 and the shuttle factor Rad23 were determined at the permissive and restrictive temperatures.

FIGURE 4.

An NLS-containing protein is successfully imported in srp1-49. A, SV40-NLS-GFP was detected in the nucleus of SRP1, srp1-31, and srp1-49 at the permissive temperature (23 °C). However, after transfer to the non-permissive temperature (37 °C), the level of nuclear SV40-NLS-GFP was significantly reduced in srp1-31 but not in SRP1 or srp1-49. B, the nuclear localization of SV40-NLS-GFP was also examined in srp1^E402Q and srp1-55. C, the nuclear import of a reporter protein bearing a bipartite NLS (BPSV40T3-NLS-GFP) was examined in srp1-31 and srp1-49, as described in A.

FIGURE 5.

Intragenic complementation by srp1 alleles. SRP1, srp1-31, and srp1-49 were transformed with plasmids expressing Srp1, srp1-31, srp1-49, srp1-E402Q, and srp1-55. A, yeast cells were plated on agar medium and incubated at either 23 or 37 °C. The growth defect of srp1-31 and srp1-49 (37 °C; second panel) was suppressed by wild type Srp1. The expression of srp1-31 protein restored growth in all mutants except for srp1-31. Similarly, only srp1-49 failed to suppress the growth defect of srp1-49. The expression of srp1-E402Q and srp1-55 suppressed the temperature-sensitive growth defects of both srp1-31 and srp1-49. B, the inability of srp1-31 to import SV40-NLS-GFP was suppressed by all srp1 mutant proteins except for srp1-31. C, the proteasome targeting defect of srp1-49 was restored by all srp1 mutant proteins except for srp1-49. As expected, wild type Srp1 suppressed the NLS import and proteasome targeting defects of srp1-31 and srp1-49, respectively.

FIGURE 6.

Proteasome mislocalization in srp1-49 stabilizes Matα2-GFP. A, Matα2-GFP was examined in SRP1, srp1-31, and srp1-49 at 23 and 37 °C. High level of Matα2-GFP was detected in srp1-49 at 23 °C. Matα2-GFP levels increased significantly at 37 °C. B, extracts were prepared from the cultures examined in A and characterized by immunoblotting. Anti-GFP antibodies confirmed elevated levels of Matα2-GFP in srp1-49. The levels of proteasome subunits Pre10 and Rpn12 were unaffected.

FIGURE 7.

Proteasomes mislocalization inhibits the turnover of Matα2-GFP in srp1-E402Q. A, Matα2-GFP was expressed in srp1-E402Q and srp1-49, and dramatic nuclear accumulation was observed at both 23 and 37 °C. A reduced exposure (1/2 exposure) is shown. The degradation of Matα2-GFP was not affected in srp1-55 (at 18 °C). B, extracts were prepared from the cultures described in A, and Matα2-GFP levels were measured by immunoblotting. Matα2-GFP protein levels decreased rapidly in SRP1, srp1-31 (at 37 °C), and srp1-55 (at 18 °C), but were stable in srp1-49 and srp1-E402Q. The elimination of multiubiquitylated proteins was delayed in both srp1-49 and srp1-E402Q. Rad23 levels were similar in all strains. C, the level of Matα2-GFP in A was quantified in multiple fields, and the number of cells characterized in each strain is indicated. (The level of Matα2-GFP in srp1-E402Q was not determined due to the high signal.) D, the level of Matα2-GFP protein in B was quantified by densitometry, and the zero time for each strain was set to an arbitrary value of 10. CHX, cycloheximide.

FIGURE 8.

Proteasome mislocalization stabilizes DNA repair and cell-cycle factors. A, the turnover of the nucleotide excision repair factor Rad4-HA was determined in SRP1, srp1-31, and srp1-49, following the addition of cycloheximide. The reactions to antibodies against the HA epitope, ubiquitin (Ub), Rad23, and proteasome subunit Rpn12 are shown. B, Rad4-HA levels were quantified by densitometry, and the level in each strain was compared with its individual zero time point. C, Clb2-HA was expressed from a galactose-inducible promoter (P_GAL1) in SRP1, srp1-31, and srp1-49. Protein levels were examined after the cells were transferred from inducing (Gal) to repressive (Glu) medium at 37 °C and quantified (D) as described above.

FIGURE 9.

A proteasome substrate accumulates in the nucleus in sts1-2. A, Matα2-GFP was expressed in STS1 and sts1-2 at 23 °C. The cultures were resuspended in fresh medium containing cycloheximide and incubated at 37 °C. The GFP signal was lost rapidly in STS1 but remained elevated in sts1-2. B, Matα2-GFP fluorescence was measured in multiple fields, and the number of cells examined is indicated. C, protein extracts were prepared from the same samples described in A and characterized by immunoblotting (top). Matα2-GFP levels were quantified by densitometry and standardized to the level detected at time 0.

FIGURE 10.

A proteolytic substrate accumulates in the nucleus in proteasome mutants. A, Matα2-GFP was expressed in RPN11 and rpn11-1, and its localization was examined at 23 and 37 °C. B, the same yeast cultures were transferred from 23 to 37 °C for 1 h, after which cycloheximide (CHX) was added to the medium, and aliquots were withdrawn at the times indicated. C, extracts were prepared from the cultures examined in B and characterized by immunoblotting. The stabilization of Matα2-GFP in rpn11-1 was accompanied by dramatic accumulation of high molecular weight ubiquitylated species. D, Matα2-GFP was stabilized in other proteasome mutants (rpt1/cim5-1 and pre1-1 pre2-2) and accumulated in the nucleus.

FIGURE 11.

Srp1 does not affect the proteasome-mediated degradation of a cytosolic protein. FLAG-Deg1-Sec62 was expressed in srp1 mutants, and protein extracts were prepared at the times indicated. An immunoblot was incubated with antibodies against the FLAG epitope and Rad23. FLAG-Deg1-Sec62 stability was also examined in wild type (PRE1 PRE2) and pre1-1 pre2-2 proteasome mutant.

FIGURE 12.

Srp1/Sts1 interaction is required for suppression of srp1-49. A, Sts1 and ^ΔNLSsts1 were expressed from the galactose-inducible P_GAL1 promoter in SRP1, srp1-31, and srp1-49. 10-fold serial dilutions were spotted on agar medium, and growth was examined on galactose medium at 23 and 37 °C. B, Rpt1-GFP localization was examined at 37 °C on glucose (left) and galactose (right) media in SRP1, srp1-31, and srp1-49 overexpressing Sts1. C, Matα2-GFP and P_GAL1::STS1 were co-expressed in SRP1, srp1-31, and srp1-49. Overexpression of Sts1 (galactose) resumed degradation of Matα2-GFP, as indicated by the loss of GFP signal. (A reduced exposure is also shown.)

FIGURE 13.

A model for Srp1 function. A, we propose that in wild type cells, Sts1 binds Srp1 to translocate proteasomes to the nucleus. Proteasomes at the nuclear surface may receive nuclear substrates that are exported. A separate activity of Srp1 is to import NLS-containing proteins (cargo), which we presume occurs concurrently with the nuclear targeting of proteasomes. NLS-mediated import requires Srp1 interactions with other import factors (such as Kap85/importin-β). B, a mutation in Sts1 that prevents interaction with Srp1 (^ΔNLSsts1) prevents the translocation of proteasomes to the nucleus and causes stabilization and nuclear accumulation of proteins (28). However, the nuclear import of other NLS-containing proteins is not affected by Sts1. C, certain mutations in Srp1 (srp1-49 and srp1-E402Q) prevent nuclear targeting of proteasomes. However, srp1-49 and srp1-E402Q mutants are proficient in importing cNLS-containing proteins. Proteolytic substrates accumulate inside the nucleus in these mutants. One interpretation of this result is that nuclear substrates are not exported unless proteasomes are available at the nuclear surface. D, the prototypical srp1 mutants, embodied by srp1-31, failed to import NLS-containing proteins. However, these mutants could target proteasomes to the nucleus, allowing successful degradation of nuclear substrates.