Assembly Chaperone Research Articles

Xeno-interactions between MHC-I proteins and molecular chaperones enable ligand exchange on a broad repertoire of HLA allotypes

Abstract Immunological chaperones Tapasin and TAPBPR play key roles in antigenic peptide optimization and quality control of nascent MHC-I molecules. The polymorphic nature of MHC-I proteins leads to a range of allelic dependencies on chaperones for assembly and cell-surface expression, limiting chaperone-mediated peptide exchange to a restricted set of human HLA allotypes. Here, we demonstrate and characterize xeno-interactions between a chicken TAPBPR ortholog and a complementary repertoire of HLA allotypes, relative to its human counterpart. We find that TAPBPR orthologs recognize empty MHC-I with broader allele specificity and facilitate peptide exchange by maintaining a reservoir of receptive molecules. Deep mutational scanning of human TAPBPR further identifies gain-of-function mutants, resembling the chicken sequence, which can enhance HLA-A* 01:01 expression in situ and promote peptide exchange in vitro. These results highlight that polymorphic sites on MHC-I and chaperone surfaces can be engineered to manipulate their interactions, enabling chaperone-mediated peptide exchange on disease-relevant HLA alleles. This work was supported by NIAID (5R01AI143997), NIDDK (5U01DK112217), NIGMS (5R35GM125034), and NIGMS (R35GM142505).

UBE4A catalyzes NRF1 ubiquitination and facilitates DDI2-mediated NRF1 cleavage

The transcription factor nuclear factor erythroid 2 like 1 (NFE2L1 or NRF1) regulates constitutive and inducible expression of proteasome subunits and assembly chaperones. The precursor of NRF1 is integrated into the endoplasmic reticulum (ER) and can be retrotranslocated from the ER to the cytosol where it is processed by ubiquitin-directed endoprotease DDI2. DDI2 cleaves and activates NRF1 only when NRF1 is highly polyubiquitinated. It remains unclear how retrotranslocated NRF1 is primed with large amount of ubiquitin and/or very long polyubiquitin chain for subsequent processing. Here, we report that E3 ligase UBE4A catalyzes ubiquitination of retrotranslocated NRF1 and promotes its cleavage. Depletion of UBE4A reduces the amount of ubiquitin modified on NRF1, shortens the average length of polyubiquitin chain, decreases NRF1 cleavage efficiency and causes accumulation of non-cleaved, inactivated NRF1. Expression of a UBE4A mutant lacking ligase activity impairs the cleavage, likely due to a dominant negative effect. UBE4A interacts with NRF1 and the recombinant UBE4A can promote ubiquitination of retrotranslocated NRF1 in vitro. In addition, knocking out UBE4A reduces transcription of proteasomal subunits in cells. Our results indicate that UBE4A primes NRF1 for DDI2-mediated activation to facilitate expression of proteasomal genes.

Xeno interactions between MHC-I proteins and molecular chaperones enable ligand exchange on a broad repertoire of HLA allotypes.

Immunological chaperones tapasin and TAP binding protein, related (TAPBPR) play key roles in antigenic peptide optimization and quality control of nascent class I major histocompatibility complex (MHC-I) molecules. The polymorphic nature of MHC-I proteins leads to a range of allelic dependencies on chaperones for assembly and cell-surface expression, limiting chaperone-mediated peptide exchange to a restricted set of human leukocyte antigen (HLA) allotypes. Here, we demonstrate and characterize xeno interactions between a chicken TAPBPR ortholog and a complementary repertoire of HLA allotypes, relative to its human counterpart. We find that TAPBPR orthologs recognize empty MHC-I with broader allele specificity and facilitate peptide exchange by maintaining a reservoir of receptive molecules. Deep mutational scanning of human TAPBPR further identifies gain-of-function mutants, resembling the chicken sequence, which can enhance HLA-A*01:01 expression in situ and promote peptide exchange in vitro. These results highlight that polymorphic sites on MHC-I and chaperone surfaces can be engineered to manipulate their interactions, enabling chaperone-mediated peptide exchange on disease-relevant HLA alleles.

DnaK and DnaJ proteins from Hsp70/40 family are involved in Rubisco biosynthesis in Synechocystis sp. PCC6803 and sustain the enzyme assembly in a heterologous system

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the first step of carbon fixation performed by photosynthetic organisms. Form I of this enzyme found in plants and cyanobacteria is composed of eight large (RbcL) and eight small (RbcS) subunits. To form a functional enzyme, Rubisco subunits need to be properly folded, with the assistance of cellular chaperone machinery, and consecutively assembled in a strictly orchestrated manner, with the help of multiple auxiliary factors. In recent years, multiple Rubisco assembly chaperones and their function in enzyme biogenesis have been extensively characterized. Little is known about the potential specialized factors involved in Rubisco subunits folding at the pre-chaperonin stage, yet this knowledge is greatly needed for the fast and efficient testing of new Rubisco variants.Synechococcus sp. PCC 6803 Rubisco shows limited solubility and a lack of assembly in the Escherichia coli expression system. In this study, we aim to identify which additional chaperones are necessary and sufficient in sustaining the heterologous assembly of native Rubisco. Our findings prove that upon the introduction of Synechocystis DnaK2 to the E. coli system, RbcL is produced in soluble form. The addition of specific DnaJ (Sll1384) enhances this effect. We explain these combined effects based on binding constancies, measured for particular partners in vitro, as well as our analysis of the putative tertiary structure of the proteins. Our results have potential implications for Rubisco engineering.

Elucidating the role of the Hsc70 chaperone in SNARE complex assembly.

The complex mechanism of synaptic vesicle fusion with the plasma membrane for neurotransmitter release is initiated by the formation of the SNARE complex at the presynaptic terminal. SNARE is a proteinaceous complex composed of four helices contributed by three proteins: one helix from syntaxin, two from SNAP-25 (both at the plasma membrane) and, one helix from synaptobrevin (at the vesicle). SNAP-25 is an intrinsically disordered protein that is prone to aggregation. The fusion process is tightly regulated and requires the constitutively expressed Hsp70 chaperone (Hsc70) and its co-chaperones CSPα (J protein) and Hsp110 (nucleotide exchange factor). However, the precise molecular details by which the Hsc70 system participates in the SNARE complex formation remain unknown. SNAP-25 must neither aggregate nor fold prematurely before SNARE formation. We hypothesize that Hsc70 and CSPα cooperate to chaperone SNAP-25 to keep it in folding competent state for SNARE complex formation and facilitates the bringing of the membranes to proximity for exocytosis. To test this hypothesis, we employed a bottom-up approach and studied the interaction between Hsc70 and CSPα with SNAP-25 in vitro. Using a peptide array that spans the sequence of SNAP-25, we identified three potential Hsc70-interacting sequences and designed peptides containing these sequences to test binding in solution. We characterized the interaction of SNAP-25 peptides with Hsc70 and CSPα using a combination of biochemical and biophysical techniques, such as native-PAGE, binding affinity by fluorescence anisotropy, ATP-hydrolysis activity of Hsc70, and NMR. Our peptide binding data show different affinities of SNAP-25 peptides with Hsc70 in solution. We have tentatively identified an Hsc70 binding site within SNAP-25 that is likely to represent the site used in the cell. Importantly, we showed that the aggregation of SNAP-25 is delayed in the presence of Hsc70 and CSPα.

Assembly chaperone Nas6 selectively destabilizes 26S proteasomes with defective regulatory particle-core particle interfaces

The 26S proteasome is a 66-subunit-chambered protease present in all eukaryotes that maintains organismal health by degrading unneeded or defective proteins. Defects in proteasome function or assembly are known to contribute to the development of various cancers, neurodegeneration, and diabetes. During proteasome biogenesis, a family of evolutionarily conserved chaperones assembles a hexameric ring of AAA+ family ATPase subunits contained within the proteasomal regulatory particle (RP) and guide their docking onto the surface of the proteolytic core particle (CP). This RP-CP interaction couples the substrate capture and unfolding process to proteolysis. We previously reported a mutation in the proteasome that promoted dissociation of the RP and CP by one of these chaperones, Nas6. However, the nature of the signal for Nas6-dependent proteasome disassembly and the generality of this postassembly proteasome quality control function for Nas6 remain unknown. Here, we use structure-guided mutagenesis and invitro proteasome disassembly assays to demonstrate that Nas6 more broadly destabilizes 26S proteasomes with a defective RP-CP interface. We show that Nas6 can promote dissociation of mature proteasomes into RP and CP in cells harboring defects on either side of the RP-CP interface. This function is unique to Nas6 and independent from other known RP assembly chaperones. Further biochemical experiments suggest that Nas6 may exploit a weakened RP-CP interface to dissociate the RP from the CP. We propose that this postassembly role of Nas6 may fulfill a quality control function in cells by promoting the recycling of functional subcomplexes contained within defective proteasomes.

The penultimate step of proteasomal ATPase assembly is mediated by a switch dependent on the chaperone Nas2

The proteasome holoenzyme is a complex molecular machine that degrades most proteins. In the proteasome holoenzyme, six distinct ATPase subunits (Rpt1 through Rpt6) enable protein degradation by injecting protein substrates into it. Individual Rpt subunits assemble into a heterohexameric "Rpt ring" in a stepwise manner, by binding to their cognate chaperones. Completion of the heterohexameric Rpt ring correlates with release of a specific chaperone, Nas2; however, it is unclear whether and how this event may ensure proper Rpt ring assembly. Here, we examined the action of Nas2 by capturing the poorly characterized penultimate step of heterohexameric Rpt ring assembly. For this, we used a heterologous Escherichia coli system coexpressing all Rpt subunits and assembly chaperones as well as Saccharomyces cerevisiae to track Nas2 actions during endogenous Rpt ring assembly. We show that Nas2 uses steric hindrance to block premature progression of the penultimate step into the final step of Rpt ring assembly. Importantly, Nas2 can activate an assembly checkpoint via its steric activity, when the last ATPase subunit, Rpt1, cannot be added in a timely manner. This checkpoint can be relieved via Nas2 release, when Nas2 recognizes proper addition of Rpt1 to one side of its cognate Rpt5, and ATP hydrolysis by Rpt4 on the other side of Rpt5, allowing completion of Rpt ring assembly. Our findings reveal dual criteria for Nas2 release, as a mechanism to ensure both the composition and functional competence of a newly assembled proteasomal ATPase, to generate the proteasome holoenzyme.

Oligomer-to-monomer transition underlies the chaperone function of AAGAB in AP1/AP2 assembly

Assembly of protein complexes is facilitated by assembly chaperones. Alpha and gamma adaptin-binding protein (AAGAB) is a chaperone governing the assembly of the heterotetrameric adaptor complexes 1 and 2 (AP1 and AP2) involved in clathrin-mediated membrane trafficking. Here, we found that before AP1/2 binding, AAGAB exists as a homodimer. AAGAB dimerization is mediated by its C-terminal domain (CTD), which is critical for AAGAB stability and is missing in mutant proteins found in patients with the skin disease punctate palmoplantar keratoderma type 1 (PPKP1). We solved the crystal structure of the dimerization-mediating CTD, revealing an antiparallel dimer of bent helices. Interestingly, AAGAB uses the same CTD to recognize and stabilize the γ subunit in the AP1 complex and the α subunit in the AP2 complex, forming binary complexes containing only one copy of AAGAB. These findings demonstrate a dual role of CTD in stabilizing resting AAGAB and binding to substrates, providing a molecular explanation for disease-causing AAGAB mutations. The oligomerization state transition mechanism may also underlie the functions of other assembly chaperones.

Prognostic and Immune Infiltration Value of Proteasome Assembly Chaperone (PSMG) Family Genes in Lung Adenocarcinoma

The complexity of lung adenocarcinoma (LUAD) including many interacting biological processes makes it difficult to find therapeutic biomarkers for treatment. Previous studies demonstrated that PSMG (proteasome assembly chaperone) family members regulate the degradation of abnormal proteins. However, transcript expressions of this gene family in LUAD still need to be more fully investigated. Therefore, we used a holistic bioinformatics approach to explore PSMG genes involved in LUAD patients by integrating several high-throughput databases and tools including The Cancer Genome Atlas (TCGA), and Kaplan-Meier plotter database. These data demonstrated that PSMG3 and PSMG4 were expressed at significantly higher levels in neoplastic cells than in normal lung tissues. Notably, increased expressions of these proteins were correlated with poor prognoses of lung cancer patients, which probably confirmed their fundamental roles in the staging of LUAD tumors. Meanwhile, it was also indicated that there were positive correlations between PSMG family genes and the immune response, metabolism of ubiquinone, cell cycle regulatory pathways, and heat shock protein 90 (HSP90)/phosphatidylinositol 3-kinase (PI3K)/Wnt signaling. Experimental data also confirmed that the knockdown of PSMG4 in LUAD cell lines decreased cell proliferation and influenced expressions of downstream molecules. Collectively, this study revealed that PSMG family members are novel prognostic biomarkers for LUAD progression, which also provide new therapeutic targets of LUAD patients.

Hsp70 and Hsp110 Chaperones Promote Early Steps of Proteasome Assembly

Whereas assembly of the 20S proteasome core particle (CP) in prokaryotes apparently occurs spontaneously, the efficiency of this process in eukaryotes relies on the dedicated assembly chaperones Ump1, Pba1-Pba2, and Pba3-Pba4. For mammals, it was reported that CP assembly initiates with formation of a complete α-ring that functions as a template for β subunit incorporation. By contrast, we were not able to detect a ring composed only of a complete set of α subunits in S. cerevisiae. Instead, we found that the CP subunits α1, α2, and α4 each form independent small complexes. Purification of such complexes containing α4 revealed the presence of chaperones of the Hsp70/Ssa and Hsp110/Sse families. Consistently, certain small complexes containing α1, α2, and α4 were not formed in strains lacking these chaperones. Deletion of the SSE1 gene in combination with deletions of PRE9 (α3), PBA3, or UMP1 genes resulted in severe synthetic growth defects, high levels of ubiquitin-conjugates, and an accumulation of distinct small complexes with α subunits. Our study shows that Hsp70 and Hsp110 chaperones cooperate to promote the folding of individual α subunits and/or their assembly with other CP subunits, Ump1, and Pba1-Pba4 in subsequent steps.

Beyond Chaperoning: UCS Proteins Emerge as Regulators of Myosin-Mediated Cellular Processes.

The UCS (UNC-45/CRO1/She4p) family of proteins has emerged as chaperones specific for the folding, assembly, and function of myosin. UCS proteins participate in various myosin-dependent cellular processes including myofibril organization and muscle functions, cell differentiation, striated muscle development, cytokinesis, and endocytosis. Mutations in the genes that code for UCS proteins cause serious defects in myosin-dependent cellular processes. UCS proteins that contain an N-terminal tetratricopeptide repeat (TPR) domain are called UNC-45. Vertebrates usually possess two variants of UNC-45, the ubiquitous general-cell UNC-45 (UNC-45A) and the striated muscle UNC-45 (UNC-45B), which is exclusively expressed in skeletal and cardiac muscles. Except for the TPR domain in UNC-45, UCS proteins comprise of several irregular armadillo (ARM) repeats that are organized into a central domain, a neck region, and the canonical C-terminal UCS domain that functions as the chaperoning module. With or without TPR, UCS proteins form linear oligomers that serve as scaffolds that mediate myosin folding, organization into myofibrils, repair, and motility. This chapter reviews emerging functions of these proteins with a focus on UNC-45 as a dedicated chaperone for folding, assembly, and function of myosin at protein and potentially gene levels. Recent experimental evidences strongly support UNC-45 as an absolute regulator of myosin, with each domain of the chaperone playing different but complementary roles during the folding, assembly, and function of myosin, as well as recruiting Hsp90 as a co-chaperone to optimize key steps. It is becoming increasingly clear that UNC-45 also regulates the transcription of several genes involved in myosin-dependent cellular processes.

Strategies for manipulating Rubisco and creating photorespiratory bypass to boost C3 photosynthesis: Prospects on modern crop improvement.

Photosynthesis is a process that uses solar energy to fix CO2 in the air and converts it into sugar, and ultimately powers almost all life activities on the earth. C3 photosynthesis is the most common form of photosynthesis in crops. Current efforts of increasing crop yields in response to growing global food requirement are mostly focused on improving C3 photosynthesis. In this review, we summarized the strategies of C3 photosynthesis improvement in terms of Rubisco properties and photorespiratory limitation. Potential engineered targets include Rubisco subunits and their catalytic sites, Rubisco assembly chaperones, and Rubisco activase. In addition, we reviewed multiple photorespiratory bypasses built by strategies of synthetic biology to reduce the release of CO2 and ammonia and minimize energy consumption by photorespiration. The potential strategies are suggested to enhance C3 photosynthesis and boost crop production.

Producing fast and active Rubisco in tobacco to enhance photosynthesis.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) performs most of the carbon fixation on Earth. However, plant Rubisco is an intrinsically inefficient enzyme given its low carboxylation rate, representing a major limitation to photosynthesis. Replacing endogenous plant Rubisco with a faster Rubisco is anticipated to enhance crop photosynthesis and productivity. However, the requirement of chaperones for Rubisco expression and assembly has obstructed the efficient production of functional foreign Rubisco in chloroplasts. Here, we report the engineering of a Form 1A Rubisco from the proteobacterium Halothiobacillus neapolitanus in Escherichia coli and tobacco (Nicotiana tabacum) chloroplasts without any cognate chaperones. The native tobacco gene encoding Rubisco large subunit was genetically replaced with H. neapolitanus Rubisco (HnRubisco) large and small subunit genes. We show that HnRubisco subunits can form functional L8S8 hexadecamers in tobacco chloroplasts at high efficiency, accounting for ∼40% of the wild-type tobacco Rubisco content. The chloroplast-expressed HnRubisco displayed a ∼2-fold greater carboxylation rate and supported a similar autotrophic growth rate of transgenic plants to that of wild-type in air supplemented with 1% CO2. This study represents a step toward the engineering of a fast and highly active Rubisco in chloroplasts to improve crop photosynthesis and growth.

Rvb1/Rvb2 proteins couple transcription and translation during glucose starvation.

During times of unpredictable stress, organisms must adapt their gene expression to maximize survival. Along with changes in transcription, one conserved means of gene regulation during conditions that quickly repress translation is the formation of cytoplasmic phase-separated mRNP granules such as P-bodies and stress granules. Previously, we identified that distinct steps in gene expression can be coupled during glucose starvation as promoter sequences in the nucleus are able to direct the subcellular localization and translatability of mRNAs in the cytosol. Here, we report that Rvb1 and Rvb2, conserved ATPase proteins implicated as protein assembly chaperones and chromatin remodelers, were enriched at the promoters and mRNAs of genes involved in alternative glucose metabolism pathways that we previously found to be transcriptionally upregulated but translationally downregulated during glucose starvation in yeast. Engineered Rvb1/Rvb2-binding on mRNAs was sufficient to sequester mRNAs into mRNP granules and repress their translation. Additionally, this Rvb tethering to the mRNA drove further transcriptional upregulation of the target genes. Further, we found that depletion of Rvb2 caused decreased alternative glucose metabolism gene mRNA induction, but upregulation of protein synthesis during glucose starvation. Overall, our results point to Rvb1/Rvb2 coupling transcription, mRNA granular localization, and translatability of mRNAs during glucose starvation. This Rvb-mediated rapid gene regulation could potentially serve as an efficient recovery plan for cells after stress removal.

TORC1 regulates actin dynamics to control proteasome homeostasis

The conserved protein kinase complex TORC1 is a central regulator of protein homeostasis. TORC1 is active under nutrient replete, unstressed conditions and promotes bulk protein synthesis, cell growth, and proliferation. Many stresses inactivate TORC1, including nutrient starvation. When TORC1 is inactivated, bulk protein synthesis is inhibited, and stress-responsive protein synthesis activated. Degradation of damaged or unwanted proteins is increased through autophagy activation and enhanced proteasome assembly. The 26S proteasome is composed of the 20S core particle (CP), capped with one or two 19S regulatory particles (RP). Following TORC1 inhibition, the Mpk1 kinase is activated. Mpk1 facilitates increased translation of proteasome regulatory particle assembly chaperones (RPACs), leading to increased proteasome assembly. The mechanisms underlying the RPAC translation increase remain unclear, however. Here, we show that actin depolarisation upon TORC1 inhibition causes RPAC mRNAs to associate more with actin patch structures. Increasing this interaction enhances translation further. We identified Ede1 as a protein which binds translating RPAC mRNA and show that Ede1 is required to increase RPAC mRNA association with actin patches, RPAC translation and proteasome assembly upon stress.

Actin remodelling controls proteasome homeostasis upon stress

When cells are stressed, bulk translation is often downregulated to reduce energy demands while stress-response proteins are simultaneously upregulated. To promote proteasome assembly and activity and maintain cell viability upon TORC1 inhibition, 19S regulatory-particle assembly chaperones (RPACs) are selectively translated. However, the molecular mechanism for such selective translational upregulation is unclear. Here, using yeast, we discover that remodelling of the actin cytoskeleton is important for RPAC translation following TORC1 inhibition. mRNA of the RPAC ADC17 is associated with actin cables and is enriched at cortical actin patches under stress, dependent upon the early endocytic protein Ede1. ede1∆ cells failed to induce RPACs and proteasome assembly upon TORC1 inhibition. Conversely, artificially tethering ADC17 mRNA to cortical actin patches enhanced its translation upon stress. These findings suggest that actin-dense structures such as cortical actin patches may serve as a translation platform for a subset of stress-induced mRNAs including regulators of proteasome homeostasis.

Actin dynamics regulate proteasome homeostasis

Cells require thousands of unique proteins to be in the right place, at the right time, in the right amounts and with the right modifications. They do this through several processes collectively known as protein homeostasis. TORC1 is a principal regulator of protein homeostasis, coordinating protein synthesis and degradation. The proteasome, composed of a core particle and one or two regulatory particles, degrades unwanted protein. Diverse stresses cause a protein homeostasis imbalance: inhibiting TORC1 and the misfolded/damaged protein load. Following TORC1 inhibition, proteasome regulatory particle assembly chaperone (RPAC) translation is increased and thus cells assemble more proteasomes to degrade the damaged and misfolded proteins, thereby restoring protein homeostasis. Using yeast, we identify an endocytic protein, Ede1, that interacts with and is critical for translation of RPAC mRNA following TORC1 inhibition. We find two further endocytic proteins important for RPAC translation regulation. Mutants of these proteins cause altered Arp2/3 activity, and hence altered formation of actin patches/endocytic sites. We show that RPAC mRNA is transported on actin cables and interacts with actin patches. TORC1 inhibition depolarises the actin cytoskeleton, causing RPAC mRNA accumulation on actin patches concurrent with translation. We demonstrate Ede1 is essential for RPAC mRNA localisation regulation following rapamycin treatment. This work shows that, upon actin depolarisation, RPAC mRNA is recruited to actin patches, likely by Ede1, and translation occurs. Actin regulation is therefore a key element of proteasome (and therefore protein) homeostasis.

Mechanism of proteasome gate modulation by assembly chaperones Pba1 and Pba2

The active sites of the proteasome are housed within its central core particle (CP), a barrel-shaped chamber of four stacked heptameric rings, and access of substrates to the CP interior is mediated by gates at either axial end. These gates are constitutively closed and may be opened by the regulatory particle (RP), which binds the CP and facilitates substrate degradation. We recently showed that the heterodimeric CP assembly chaperones Pba1/2 also mediate gate opening through an unexpected structural arrangement that facilitates the insertion of the N terminus of Pba1 into the CP interior; however, the full mechanism of Pba1/2-mediated gate opening is unclear. Here, we report a detailed analysis of CP gate modulation by Pba1/2. The clustering of key residues at the interface between neighboring α-subunits is a critical feature of RP-mediated gate opening, and we find that Pba1/2 recapitulate this strategy. Unlike RP, which inserts at six α-subunit interfaces, Pba1/2 insert at only two α-subunit interfaces. Nevertheless, Pba1/2 are able to regulate six of the seven interfacial clusters, largely through direct interactions. The N terminus of Pba1 also physically interacts with the center of the gate, disrupting the intersubunit contacts that maintain the closed state. This novel mechanism of gate modulation appears to be unique to Pba1/2 and therefore likely occurs only during proteasome assembly. Our data suggest that release of Pba1/2 at the conclusion of assembly is what allows the nascent CP to assume its mature gate conformation, which is primarily closed, until activated by RP.

Interaction with the Assembly Chaperone Ump1 Promotes Incorporation of the β7 Subunit into Half-Proteasome Precursor Complexes Driving Their Dimerization.

Biogenesis of the eukaryotic 20S proteasome core particle (PC) is a complex process assisted by specific chaperones absent from the active complex. The first identified chaperone, Ump1, was found in a precursor complex (PC) called 15S PC. Yeast cells lacking Ump1 display strong defects in the autocatalytic processing of β subunits, and consequently have lower proteolytic activity. Here, we dissect an important interaction of Ump1 with the β7 subunit that is critical for proteasome biogenesis. Functional domains of Ump1 and the interacting proteasome subunit β7 were mapped, and the functional consequences of their deletion or mutation were analyzed. Cells in which the first sixteen Ump1 residues were deleted display growth phenotypes similar to ump1∆, but massively accumulate 15S PC and distinct proteasome intermediate complexes containing the truncated protein. The viability of these cells depends on the transcription factor Rpn4. Remarkably, β7 subunit overexpression re-established viability in the absence of Rpn4. We show that an N-terminal domain of Ump1 and the propeptide of β7 promote direct interaction of the two polypeptides in vitro. This interaction is of critical importance for the recruitment of β7 precursor during proteasome assembly, a step that drives dimerization of 15S PCs and the formation of 20S CPs.

Histone Chaperone Nrp1 Mutation Affects the Acetylation of H3K56 in Tetrahymena thermophila.

Histone modification and nucleosome assembly are mainly regulated by various histone-modifying enzymes and chaperones. The roles of histone-modification enzymes have been well analyzed, but the molecular mechanism of histone chaperones in histone modification and nucleosome assembly is incompletely understood. We previously found that the histone chaperone Nrp1 is localized in the micronucleus (MIC) and the macronucleus (MAC) and involved in the chromatin stability and nuclear division of Tetrahymena thermophila. In the present work, we found that truncated C-terminal mutant HA-Nrp1TrC abnormally localizes in the cytoplasm. The truncated-signal-peptide mutants HA-Nrp1TrNLS1 and HA-Nrp1TrNLS2 are localized in the MIC and MAC. Overexpression of Nrp1TrNLS1 inhibited cellular proliferation and disrupted micronuclear mitosis during the vegetative growth stage. During sexual development, Nrp1TrNLS1 overexpression led to abnormal bouquet structures and meiosis arrest. Furthermore, Histone H3 was not transported into the nucleus; instead, it formed an abnormal speckled cytoplastic distribution in the Nrp1TrNLS1 mutants. The acetylation level of H3K56 in the mutants also decreased, leading to significant changes in the transcription of the genome of the Nrp1TrNLS1 mutants. The histone chaperone Nrp1 regulates the H3 nuclear import and acetylation modification of H3K56 and affects chromatin stability and genome transcription in Tetrahymena.