Yeast Importin-α (Srp1) Performs Distinct Roles in the Import of Nuclear Proteins and in Targeting Proteasomes to the Nucleus

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.     

Sts1 plays a key role in targeting proteasomes to the nucleus

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.     

Evidence for separable functions of Srp1p, the yeast homolog of importin alpha (Karyopherin alpha): role for Srp1p and Sts1p in protein degradation

Srp1p (importin alpha) functions as the nuclear localization signal (NLS) receptor in Saccharomyces cerevisiae. The srp1-31 mutant is defective in this nuclear localization function, whereas an srp1-49 mutant exhibits defects that are unrelated to this localization function, as was confirmed by intragenic complementation between the two mutants. RPN11 and STS1 (DBF8) were identified as high-dosage suppressors of the srp1-49 mutation but not of the srp1-31 mutation. We found that Sts1p interacts directly with Srp1p in vitro and also in vivo, as judged by coimmunoprecipitation and two-hybrid analyses. Mutants of Sts1p that cannot interact with Srp1p are incapable of suppressing srp1-49 defects, strongly suggesting that Sts1p functions in a complex with Srp1p. STS1 also interacted with the second suppressor, RPN11, a subunit of the 26S proteasome, in the two-hybrid system. Further, degradation of Ub-Pro-beta-galactosidase, a test substrate for the ubiquitin-proteasome system, was defective in srp1-49 but not in srp1-31. This defect in protein degradation was alleviated by overexpression of either RPN11 or STS1 in srp1-49. These results suggest a role for Srp1p in regulation of protein degradation separate from its well-established role as the NLS receptor.     

Spg5 Protein Regulates the Proteasome in Quiescence

The ubiquitin-proteasome system is the major pathway for selective protein degradation in eukaryotes. Despite extensive study of this system, the mechanisms by which proteasome function and cell growth are coordinated remain unclear. Here, we identify Spg5 as a novel component of the ubiquitin-proteasome system. Spg5 binds the regulatory particle of the proteasome and the base subassembly in particular, but it is excluded from mature proteasomes. The SPG5 gene is strongly induced in the stationary phase of budding yeast, and spg5Δ mutants show a progressive loss of viability under these conditions. Accordingly, during logarithmic growth, Spg5 appears largely dispensable for proteasome function, but during stationary phase the proteasomes of spg5Δ mutants show both structural and functional defects. This loss of proteasome function is reflected in the accumulation of oxidized proteins preferentially in stationary phase in spg5Δ mutants. Thus, Spg5 is a positive regulator of the proteasome that is critical for survival of cells that have ceased to proliferate due to nutrient limitation.     

Intracellular localization of the proteasome in response to stress conditions

The ubiquitin–proteasome system fulfills an essential role in regulating protein homeostasis by spatially and temporally controlling proteolysis in an ATP- and ubiquitin-dependent manner. However, the localization of proteasomes is highly variable under diverse cellular conditions. In yeast, newly synthesized proteasomes are primarily localized to the nucleus during cell proliferation. Yeast proteasomes are transported into the nucleus through the nuclear pore either as immature subcomplexes or as mature enzymes via adapter proteins Sts1 and Blm10, while in mammalian cells, postmitotic uptake of proteasomes into the nucleus is mediated by AKIRIN2, an adapter protein essentially required for nuclear protein degradation. Stressful growth conditions and the reversible halt of proliferation, that is quiescence, are associated with a decline in ATP and the reorganization of proteasome localization. Cellular stress leads to proteasome accumulation in membraneless granules either in the nucleus or in the cytoplasm. In quiescence, yeast proteasomes are sequestered in an ubiquitin-dependent manner into motile and reversible proteasome storage granules in the cytoplasm. In cancer cells, upon amino acid deprivation, heat shock, osmotic stress, oxidative stress, or the inhibition of either proteasome activity or nuclear export, reversible proteasome foci containing polyubiquitinated substrates are formed by liquid–liquid phase separation in the nucleus. In this review, we summarize recent literature revealing new links between nuclear transport, ubiquitin signaling, and the intracellular organization of proteasomes during cellular stress conditions.     

Substrate Binding Promotes Formation of the Skp1-Cul1-Fbxl3 (SCFFbxl3) Protein Complex

The Skp1–Cul1–F-box protein (SCF) complex is one of the most well characterized types of ubiquitin ligase (E3), with the E3 activity of the complex being regulated in part at the level of complex formation. Fbxl3 is an F-box protein that is responsible for the ubiquitylation and consequent degradation of cryptochromes (Crys) and thus regulates oscillation of the circadian clock. Here we show that formation of the SCF^Fbxl3 complex is regulated by substrate binding in vivo. Fbxl3 did not associate with Skp1 and Cul1 to a substantial extent in transfected mammalian cells. Unexpectedly, however, formation of the SCF^Fbxl3 complex was markedly promoted by forced expression of its substrate Cry1 in these cells. A mutant form of Fbxl3 that does not bind to Cry1 was unable to form an SCF complex, suggesting that interaction of Cry1 with Fbxl3 is essential for formation of SCF^Fbxl3. In contrast, recombinant Fbxl3 associated with recombinant Skp1 and Cul1 in vitro even in the absence of recombinant Cry1. Domain-swap analysis revealed that the COOH-terminal leucine-rich repeat domain of Fbxl3 attenuates the interaction of Skp1, suggesting that a yet unknown protein associated with the COOH-terminal domain of Fbxl3 and inhibited SCF complex formation. Our results thus provide important insight into the regulation of both SCF ubiquitin ligase activity and circadian rhythmicity.     

NADH Binds and Stabilizes the 26S Proteasomes Independent of ATP

The 26S proteasome is the end point of the ubiquitin- and ATP-dependent degradation pathway. The 26S proteasome complex (26S PC) integrity and function has been shown to be highly dependent on ATP and its homolog nucleotides. We report here that the redox molecule NADH binds the 26S PC and is sufficient in maintaining 26S PC integrity even in the absence of ATP. Five of the 19S proteasome complex subunits contain a putative NADH binding motif (GxGxxG) including the AAA-ATPase subunit, Psmc1 (Rpt2). We demonstrate that recombinant Psmc1 binds NADH via the GxGxxG motif. Introducing the ΔGxGxxG Psmc1 mutant into cells results in reduced NADH-stabilized 26S proteasomes and decreased viability following redox stress induced by the mitochondrial inhibitor rotenone. The newly identified NADH binding of 26S proteasomes advances our understanding of the molecular mechanisms of protein degradation and highlights a new link between protein homeostasis and the cellular metabolic/redox state.     

Cse1p Is Involved in Export of Yeast Importin α from the Nucleus

Proteins bearing a nuclear localization signal (NLS) are targeted to the nucleus by the heterodimeric transporter importin. Importin α binds to the NLS and to importin β, which carries it through the nuclear pore complex (NPC). Importin disassembles in the nucleus, evidently by binding of RanGTP to importin β. The importin subunits are exported separately. We investigated the role of Cse1p, the Saccharomyces cerevisiae homologue of human CAS, in nuclear export of Srp1p (yeast importin α). Cse1p is located predominantly in the nucleus but also is present in the cytoplasm and at the NPC. We analyzed the in vivo localization of the importin subunits fused to the green fluorescent protein in wild-type and cse1-1 mutant cells. Srp1p but not importin β accumulated in nuclei of cse1-1 mutants, which are defective in NLS import but not defective in NLS-independent import pathways. Purified Cse1p binds with high affinity to Srp1p only in the presence of RanGTP. The complex is dissociated by the cytoplasmic RanGTP-binding protein Yrb1p. Combined with the in vivo results, this suggests that a complex containing Srp1p, Cse1p, and RanGTP is exported from the nucleus and is subsequently disassembled in the cytoplasm by Yrb1p. The formation of the trimeric Srp1p-Cse1p-RanGTP complex is inhibited by NLS peptides, indicating that only NLS-free Srp1p will be exported to the cytoplasm.     

A Functional GTPase Domain,but not its Transmembrane Domain,is Required for Function of the SRP Receptor β-subunit

The signal recognition particle and its receptor (SR) target nascent secretory proteins to the ER. SR is a heterodimeric ER membrane protein whose subunits, SRα and SRβ, are both members of the GTPase superfamily. Here we characterize a 27-kD protein in Saccharomyces cerevisiae (encoded by SRP102) as a homologue of mammalian SRβ. This notion is supported (a) by Srp102p''s sequence similarity to SRβ; (b) by its disposition as an ER membrane protein; (c) by its interaction with Srp101p, the yeast SRα homologue; and (d) by its role in SRP-dependent protein targeting in vivo. The GTP-binding site in Srp102p is surprisingly insensitive to single amino acid substitutions that inactivate other GTPases. Multiple mutations in the GTP-binding site, however, inactivate Srp102p. Loss of activity parallels a loss of affinity between Srp102p and Srp101p, indicating that the interaction between SR subunits is important for function. Deleting the transmembrane domain of Srp102p, the only known membrane anchor in SR, renders SR soluble in the cytosol, which unexpectedly does not significantly impair SR function. This result suggests that SR functions as a regulatory switch that needs to associate with the ER membrane only transiently through interactions with other components.     

The ATP Costs and Time Required to Degrade Ubiquitinated Proteins by the 26 S Proteasome

The degradation of ubiquitinated proteins by 26 S proteasomes requires ATP hydrolysis. To investigate if the six proteasomal ATPases function independently or in a cyclic manner, as proposed recently, we used yeast mutants that prevent ATP binding to Rpt3, Rpt5, or Rpt6. Although proteasomes contain six ATPase subunits, each of these single mutations caused a 66% reduction in basal ATP hydrolysis, and each blocked completely the 2–3-fold stimulation of ATPase activity induced by ubiquitinated substrates. Therefore, the ATPase subunits must function in a ordered manner, in which each is required for the stimulation of ATPase activity by substrates. Although ATP is essential for multiple steps in proteasome function, when the rate of ATP hydrolysis was reduced incrementally, the degradation of Ub₅-DHFR (where Ub is ubiquitin and DHFR is dihydrofolate reductase) decreased exactly in parallel. This direct proportionality implies that a specific number of ATPs is consumed in degrading a ubiquitinated protein. When the ubiquitinated DHFR was more tightly folded (upon addition of the ligand folate), the rate of ATP hydrolysis was unchanged, but the time to degrade a Ub₅-DHFR molecule (∼13 s) and the energy expenditure (50–80 ATPs/Ub₅-DHFR) both increased by 2-fold. With a mutation in the ATPase C terminus that reduced gate opening into the 20 S proteasome, the energy costs and time required for conjugate degradation also increased. Thus, different ubiquitin conjugates activate similarly the ATPase subunit cycle that drives proteolysis, but polypeptide structure determines the time required for degradation and thus the energy cost.     

Structure of a Proteasome Pba1-Pba2 Complex: IMPLICATIONS FOR PROTEASOME ASSEMBLY,ACTIVATION, AND BIOLOGICAL FUNCTION*

The 20S proteasome is an essential, 28-subunit protease that sequesters proteolytic sites within a central chamber, thereby repressing substrate degradation until proteasome activators open the entrance/exit gate. Two established activators, Blm10 and PAN/19S, induce gate opening by binding to the pockets between proteasome α-subunits using C-terminal HbYX (hydrophobic-tyrosine-any residue) motifs. Equivalent HbYX motifs have been identified in Pba1 and Pba2, which function in proteasome assembly. Here, we demonstrate that Pba1-Pba2 proteins form a stable heterodimer that utilizes its HbYX motifs to bind mature 20S proteasomes in vitro and that the Pba1-Pba2 HbYX motifs are important for a physiological function of proteasomes, the maintenance of mitochondrial function. Other factors that contribute to proteasome assembly or function also act in the maintenance of mitochondrial function and display complex genetic interactions with one another, possibly revealing an unexpected pathway of mitochondrial regulation involving the Pba1-Pba2 proteasome interaction. Our determination of a proteasome Pba1-Pba2 crystal structure reveals a Pba1 HbYX interaction that is superimposable with those of known activators, a Pba2 HbYX interaction that is different from those reported previously, and a gate structure that is disrupted but not sufficiently open to allow entry of even small peptides. These findings extend understanding of proteasome interactions with HbYX motifs and suggest multiple roles for Pba1-Pba2 interactions throughout proteasome assembly and function.     

Slippery Substrates Impair ATP-dependent Protease Function by Slowing Unfolding

ATP-dependent proteases are responsible for most energy-dependent protein degradation across all species. Proteases initially bind an unstructured region on a substrate and then translocate along the polypeptide chain, unfolding and degrading protein domains as they are encountered. Although this process is normally processive, resulting in the complete degradation of substrate proteins to small peptides, some substrates are released prematurely. Regions of low sequence complexity within the substrate such as the glycine-rich region (GRR) from p105 or glycine-alanine repeats (GAr) from the EBNA1 (Epstein-Barr virus nuclear antigen-1) protein, can trigger partial degradation and fragment release. Loss of processivity could be due to inability to hold on to the substrate (faster release) or inability to unfold and degrade a substrate domain (slower unfolding). I previously showed that the GRR slows domain unfolding by the proteasome (Kraut, D. A., Israeli, E., Schrader, E. K., Patil, A., Nakai, K., Nanavati, D., Inobe, T., and Matouschek, A. (2012) ACS Chem. Biol. 7, 1444–1453). In contrast, a recently published study concluded that GArs increase the rate of substrate release from ClpXP, a bacterial ATP-dependent protease (Too, P. H., Erales, J., Simen, J. D., Marjanovic, A., and Coffino, P. (2013) J. Biol. Chem. 288, 13243–13257). Here, I show that these apparently contradictory results can be reconciled through a reanalysis of the ClpXP GAr data. This reanalysis shows that, as with the proteasome, low complexity sequences in substrates slow their unfolding and degradation by ClpXP, with little effect on release rates. Thus, despite their evolutionary distance and limited sequence identity, both ClpXP and the proteasome share a common mechanism by which substrate sequences regulate the processivity of degradation.     

MDM2 Promotes Proteasomal Degradation of p21Waf1 via a Conformation Change

MDM2 plays a major role in cancer development and progression via both p53-dependent and -independent functions. One of its p53-independent functions is the induction of the ubiquitin-independent proteasomal degradation of p21^Waf1. The present study was designed to characterize the mechanism(s) by which MDM2 induces p21^Waf1 degradation. We first determined the regions of MDM2 required for p21^Waf1 degradation using pulldown assays and Western blotting and then examined the mechanisms using limited proteolysis and fluorescence resonance energy transfer assays. We found that the MDM2-p21^Waf1 interaction depended on the central domain of MDM2 and that nuclear localization of both proteins was necessary for p21^Waf1 degradation. Specifically, amino acids 226–250 of MDM2 were required for p21^Waf1 binding and degradation, and amino acids 251–260 were necessary for p21^Waf1 degradation. The latter region induced a conformation change in p21^Waf1, increasing its interaction with the C8 subunit of the proteasome, leading to its degradation. When MDM2 lacked either segment (aa 226–250 or aa 251–260), its capacity to promote p21^Waf1 degradation and cell cycle progression was significantly reduced. In summary, the present study elucidated a previously unknown mechanism by which MDM2 promotes the degradation of an intact protein (p21^Waf1) through an ubiquitin-independent proteasomal degradation pathway. Because MDM2 also increases the degradation of other proteins in a ubiquitin-independent manner, this mechanism may underlie part of its tumorigenic properties.     

A Caspase Cleaved Form of Tau Is Preferentially Degraded through the Autophagy Pathway

The microtubule-associated protein tau plays a central role in the pathogenesis of Alzheimer disease (AD) and abnormally accumulates as neurofibrillary tangles; therefore, the pathways by which tau is degraded have been examined extensively. In AD brain tau is abnormally truncated at Asp⁴²¹ (tauΔC), which increases its fibrillogenic properties and results in compromised neuronal function. Given the fact that the accumulation of tauΔC is a pathogenic process in AD, in this study we examined whether full-length tau and tauΔC are degraded through similar or different mechanisms. To this end a tetracycline-inducible model was used to show that tauΔC was degraded significantly faster than full-length tau (FL-tau). Pharmacological inhibition of the proteasome or autophagy pathways demonstrated that although FL-tau is degraded by the proteasome, tauΔC is cleared predominantly by macroautophagy. We also found that tauΔC binds C terminus of Hsp70-interacting protein more efficiently than tau. This interaction leads to an increased ubiquitylation of tauΔC in a reconstituted in vitro assay, but surprisingly, tau (full-length or truncated) was not ubiquitylated in situ. The finding that tauΔC and FL-tau are differentially processed by these degradation systems provides important insights for the development of therapeutic strategies, which are focused on modulating degradation systems to preferentially clear pathological forms of the proteins.     

Proteasome Control of [URE3] Prion Propagation by Degradation of Anti-Prion Proteins Cur1 and Btn2 in Saccharomyces cerevisiae

[URE3] is a prion of the nitrogen catabolism controller, Ure2p, and [PSI+] is a prion of the translation termination factor Sup35p in S. cerevisiae. Btn2p cures [URE3] by sequestration of Ure2p amyloid filaments. Cur1p, paralogous to Btn2p, also cures [URE3], but by a different (unknown) mechanism. We find that an array of mutations impairing proteasome assembly or MG132 inhibition of proteasome activity result in loss of [URE3]. In proportion to their prion—curing effects, each mutation affecting proteasomes elevates the cellular concentration of the anti-prion proteins Btn2 and Cur1. Of >4,600 proteins detected by SILAC, Btn2p was easily the most overexpressed in a pre9Δ (α3 core subunit) strain. Indeed, deletion of BTN2 and CUR1 prevents the prion—curing effects of proteasome impairment. Surprisingly, the 15 most unstable yeast proteins are not increased in pre9Δ cells suggesting altered proteasome specificity rather than simple inactivation. Hsp42, a chaperone that cooperates with Btn2 and Cur1 in curing [URE3], is also necessary for the curing produced by proteasome defects, although Hsp42p levels are not substantially altered by a proteasome defect. We find that pre9Δ and proteasome chaperone mutants that most efficiently lose [URE3], do not destabilize [PSI+] or alter cellular levels of Sup35p. A tof2 mutation or deletion likewise destabilizes [URE3], and elevates Btn2p, suggesting that Tof2p deficiency inactivates proteasomes. We suggest that when proteasomes are saturated with denatured/misfolded proteins, their reduced degradation of Btn2p and Cur1p automatically upregulates these aggregate-handling systems to assist in the clean-up.     

Dual Positive Feedback Regulation of Protein Degradation of an Extra-cytoplasmic Function σ Factor for Cell Differentiation in Streptomyces coelicolor

Here we report that in Streptomyces coelicolor, the protein stability of an ECF σ factor SigT, which is involved in the negative regulation of cell differentiation, was completely dependent on its cognate anti-σ factor RstA. The degradation of RstA caused a ClpP/SsrA-dependent degradation of SigT during cell differentiation. This was consistent with the delayed morphological development or secondary metabolism in the ΔclpP background after rstA deletion or sigT overexpression. Meanwhile, SigT negatively regulated clpP/ssrA expression by directly binding to the clpP promoter (clpPp). The SigT-clpPp interaction could be disrupted by secondary metabolites, giving rise to the stabilized SigT protein and retarded morphological development in a non-antibiotic-producing mutant. Thus a novel regulatory mechanism was revealed that the protein degradation of the ECF σ factor was initiated by the degradation of its anti-σ factor, and was accelerated in a dual positive feedback manner, through regulation by secondary metabolites, to promote rapid and irreversible development of the secondary metabolism. This ingenious cooperation of intracellular components can ensure economical and exquisite control of the ECF σ factor protein level for the proper cell differentiation in Streptomyces.