Coiled-coil Motif Research Articles

Analyses of the essential C82 subunit uncovered some differences in RNA polymerase III transcription between Trypanosoma brucei and Leishmania major.

The 17-subunit RNA polymerase III (RNAP III) synthesizes essential untranslated RNAs such as tRNAs and 5S rRNA. In yeast and vertebrates, subunit C82 forms a stable subcomplex with C34 and C31 that is necessary for promoter-specific transcription initiation. Little is known about RNAP III transcription in trypanosomatid parasites. To narrow this knowledge gap, we characterized the C82 subunit in Trypanosoma brucei and Leishmania major. Bioinformatic analyses showed that the 4 distinctive extended winged-helix (eWH) domains and the coiled-coil motif are present in C82 in these microorganisms. Nevertheless, C82 in trypanosomatids presents certain unique traits, including an exclusive loop within the eWH1 domain. We found that C82 localizes to the nucleus and binds to RNAP III-dependent genes in the insect stages of both parasites. Knock-down of C82 by RNA interference significantly reduced the levels of tRNAs and 5S rRNA and led to the death of procyclic forms of T. brucei. Tandem affinity purifications with both parasites allowed the identification of several C82-interacting partners, including C34 and some genus-specific putative regulators of transcription. However, the orthologue of C31 was not found in trypanosomatids. Interestingly, our data suggest a strong association of C82 with TFIIIC subunits in T. brucei, but not in L. major.

Ehbp1 orchestrates orderly sorting of Wnt/Wingless to the basolateral and apical cell membranes

Wingless (Wg)/Wnt signaling plays a critical role in both development and adult tissue homeostasis. In the Drosophila larval wing disc epithelium, the orderly delivery of Wg/Wnt to the apical and basal cell surfaces is essential for wing development. Here, we identified Ehbp1 as the switch that dictates the direction of Wg/Wnt polarized intracellular transport: the Adaptor Protein complex 1 (AP-1) delivers Wg/Wnt to the basolateral cell surface, and its sequestration by Ehbp1 redirects Wg/Wnt for apical delivery. Genetic analyses showed that Ehbp1 specifically regulates the polarized distribution of Wg/Wnt, a process that depends on the dedicated Wg/Wnt cargo receptor Wntless. Mechanistically, Ehbp1 competes with Wntless for AP-1 binding, thereby preventing the unregulated basolateral Wg/Wnt transport. Reducing Ehbp1 expression, or removing the coiled-coil motifs within its bMERB domain, leads to basolateral Wg/Wnt accumulation. Importantly, the regulation of polarized Wnt delivery by EHBP1 is conserved in vertebrates. The generality of this switch mechanism for regulating intracellular transport remains to be determined in future studies.

Design of Coiled-Coil Protein Nanostructures for Therapeutics and Drug Delivery.

Coiled-coil protein motifs have become widely employed in the design of biomaterials. Some of these designs have been studied for use in drug delivery due to the unique ability of coiled-coils to impart stability, oligomerization, and supramolecular assembly. To leverage these properties and improve drug delivery, release, and targeting, a variety of nano- to mesoscale architectures have been adopted. Coiled-coil drug delivery and therapeutics have been developed by using the coiled-coil alone, designing for higher-order assemblies such as fibers and hydrogels, and combining coiled-coil proteins with other biocompatible structures such as lipids and polymers. We review the recent development of these structures and the design criteria used to generate functional proteins of varying sizes and morphologies.

Conformational Space of the Translocation Domain of Botulinum Toxin: Atomistic Modeling and Mesoscopic Description of the Coiled-Coil Helix Bundle.

The toxicity of botulinum multi-domain neurotoxins (BoNTs) arises from a sequence of molecular events, in which the translocation of the catalytic domain through the membrane of a neurotransmitter vesicle plays a key role. A recent structural study of the translocation domain of BoNTs suggests that the interaction with the membrane is driven by the transition of an α helical switch towards a β hairpin. Atomistic simulations in conjunction with the mesoscopic Twister model are used to investigate the consequences of this proposition for the toxin-membrane interaction. The conformational mobilities of the domain, as well as the effect of the membrane, implicitly examined by comparing water and water-ethanol solvents, lead to the conclusion that the transition of the switch modifies the internal dynamics and the effect of membrane hydrophobicity on the whole protein. The central two α helices, helix 1 and helix 2, forming two coiled-coil motifs, are analyzed using the Twister model, in which the initial deformation of the membrane by the protein is caused by the presence of local torques arising from asymmetric positions of hydrophobic residues. Different torque distributions are observed depending on the switch conformations and permit an origin for the mechanism opening the membrane to be proposed.

RNF20 Regulates Oocyte Meiotic Spindle Assembly by Recruiting TPM3 to Centromeres and Spindle Poles.

Previously a ring finger protein 20 (RNF20) is found to be essential for meiotic recombination and mediates H2B ubiquitination during spermatogenesis. However, its role in meiotic division is still unknown. Here, it is shown that RNF20 is localized at both centromeres and spindle poles, and it is required for oocyte acentrosomal spindle organization and female fertility. RNF20-depleted oocytes exhibit severely abnormal spindle and chromosome misalignment caused by defective bipolar organization. Notably, it is found that the function of RNF20 in spindle assembly is not dependent on its E3 ligase activity. Instead, RNF20 regulates spindle assembly by recruiting tropomyosin3 (TPM3) to both centromeres and spindle poles with its coiled-coil motif. The RNF20-TPM3 interaction is essential for acentrosomal meiotic spindle assembly. Together, the studies uncover a novel function for RNF20 in mediating TPM3 recruitment to both centromeres and spindle poles during oocyte spindle assembly.

DNA-Mediated Peptide Assembly into Protein Mimics.

The design of new protein structures is challenging due to their vast sequence space and the complexity of protein folding. Here, we report a new modular DNA-templated strategy to construct protein mimics. We achieve the spatial control of multiple peptide units by conjugation with DNA and hybridization to a branched DNA trimer template followed by covalent stapling of the preorganized peptides into a single unit. A library of protein mimics with different lengths, sequences, and heptad registers has been efficiently constructed. DNA-templated protein mimics show an α-helix or coiled-coil motif formation even when they are constructed from weakly interacting peptide units. Their attached DNA handles can be used to exert dynamic control over the protein mimics' secondary and tertiary structures. This modular strategy will facilitate the development of DNA-encoded protein libraries for the rapid discovery of new therapeutics, enzymes, and antibody mimics.

The influence of coiled-coil motif of serine recombinase toward the directionality regulation

Serine integrases promote the recombination of two complementary DNA sequences, attP and attB, to create hybrid sequences, attL and attR. The reaction is unidirectional in the absence of an accessory protein called recombination directionality factor. We utilized tethered particle motion (TPM) experiments to investigate the reaction behaviors of two model serine integrases from Listeria innocua phage LI and Streptomyces coelicolor phage C31. Detailed kinetic analyses of wild-type and mutant proteins were carried out to verify the mechanisms of recombination directionality. In particular, we assessed the influence of a coiled-coil motif (CC) that is conserved in the C-terminal domain of serine integrases and is an important prerequisite for efficient recombination. Compared to wild type, we found that CC deletions in both serine integrases reduced the overall abundance of integrase (Int) att-site complexes and favored the formation of nonproductive complexes over recombination-competent complexes. Furthermore, the rate at which CC mutants formed productive synaptic complexes and disassembled aberrant nonproductive complexes was significantly reduced. It is notable that while the φC31 Int CC is essential for recombination, the LI Int CC plays an auxiliary role for recombination to stabilize protein-protein interactions and to control the directionality of the reaction.

Peptide-Assembled Single-Chain Atomic Crystal Enhances Pluripotent Stem Cell Differentiation to Neurons.

Nanomaterials hybridized with biological components have widespread applications. among many candidates, peptides are attractive in that their peptide sequences can self-assemble with the surface of target materials with high specificity without perturbing the intrinsic properties of nanomaterials. Here, a 1D hybrid nanomaterial was developed through self-assembly of a designed peptide. A hexagonal coiled-coil motif geometrically matched to the diameter of the inorganic nanomaterial was fabricated, whose hydrophobic surface was wrapped along the axis of the hydrophobic core of the coiled coil. Our morphological and spectroscopic analyses revealed rod-shaped, homogeneous peptide-inorganic nanomaterial complexes. Culturing embryonic stem cells on surfaces coated with this peptide-assembled single-chain atomic crystal increased the growth and adhesion of the embryonic stem cells. The hybridized nanomaterial also served as an ECM for brain organoids, accelerating the maturation of neurons. New methods to fabricate hybrid materials through peptide assembly can be applied.

CCDC85C suppresses colorectal cancer cells proliferation and metastasis through activating GSK-3β and promoting β-catenin degradation

Coiled-coil domain-containing 85C (CCDC85C) is a member of the DIPA family and contains a pair of conserved coiled-coil motifs, which was found to be related to a therapeutic target for colorectal cancer, however, its biological effects require further elucidation. This study aimed to determine the effect of CCDC85C on Colorectal Cancer (CRC) progression and to explore the related mechanism. pLV-PURO plasmid was used to construct CCDC85C-overexpressing cells while CRISPR-CasRx was used to construct CCDC85C knockdown cells. Effects of CCDC85C on cell proliferation, cycle and migration were examined using cell counting kit-8 assay, flow cytometry, wound healing assay and transwell assay. Immunofluorescence staining, immunoprecipitation, Western blot, co-immunoprecipitation and qPCR were performed to explore the mechanism. The overexpression of CCDC85C inhibited the proliferation and migration of HCT-116 and RKO cells in vitro and in vivo, but its knockdown promoted the proliferation of HCT-116 and RKO cells in vitro. Moreover, co-immunoprecipitation experiment confirmed that CCDC85C binding with GSK-3β in RKO cells. Excess CCDC85C promoted phosphorylation and ubiquitination of β-catenin. Our results suggested that CCDC85C binds to GSK-3β to promote its activity and facilitates ubiquitination of β-catenin. β-catenin degradation is responsible for the inhibitory effect of CCDC85C on CRC cell proliferation and migration.

Recurrent TRAK1::RAF1 Fusions in pediatric low-grade gliomas.

Fusions involving CRAF (RAF1) are infrequent oncogenic drivers in pediatric low-grade gliomas, rarely identified in tumors bearing features of pilocytic astrocytoma, and involving a limited number of known fusion partners. We describe recurrent TRAK1::RAF1 fusions, previously unreported in brain tumors, in three pediatric patients with low-grade glial-glioneuronal tumors. We present the associated clinical, histopathologic and molecular features. Patients were all female, aged 8 years, 15 months, and 10 months at diagnosis. All tumors were located in the cerebral hemispheres and predominantly cortical, with leptomeningeal involvement in 2/3 patients. Similar to previously described activating RAF1 fusions, the breakpoints in RAF1 all occurred 5' of the kinase domain, while the breakpoints in the 3' partner preserved the N-terminal kinesin-interacting domain and coiled-coil motifs of TRAK1. Two of the three cases demonstrated methylation profiles (v12.5) compatible with desmoplastic infantile ganglioglioma (DIG)/desmoplastic infantile astrocytoma (DIA) and have remained clinically stable and without disease progression/recurrence after resection. The remaining tumor was non-classifiable; with focal recurrence 14 months after initial resection; the patient remains symptom free and without further recurrence/progression (5 months post re-resection and 19 months from initial diagnosis). Our report expands the landscape of oncogenic RAF1 fusions in pediatric gliomas, which will help to further refine tumor classification and guide management of patients with these alterations.

Self-Assembly of Hybrid Peptide-DNA Nanostructures using Homotrimeric Coiled-Coil/Nucleic Acid Building Blocks.

Peptides and DNA are two of the most commonly used self-assembling biological molecules for the construction of nanomaterials. However, there are only a few examples that combine these two self-assembly motifs as key structural elements in a nanostructure. We report on the synthesis of a peptide-DNA conjugate that self-assembles into a stable homotrimer based on the coiled-coil motif. The hybrid peptide-DNA trimer was then used as a novel three-way junction to link together either small DNA tile nanostructures, or to close up a triangular wireframe DNA structure. The resulting nanostructures were characterized by atomic force microscopy, and compared with a scrambled, non-assembling peptide as a control. These hybrid nanostructures enable the integration of peptide motifs and potentially bio-functionality with DNA nanostructures, and open the door to novel nano-materials that have the advantages of both molecules.

A trimeric coiled-coil motif binds bacterial lipopolysaccharides with picomolar affinity.

α-helical coiled-coils are ubiquitous protein structures in all living organisms. For decades, modified coiled-coils sequences have been used in biotechnology, vaccine development, and biochemical research to induce protein oligomerization, and form self-assembled protein scaffolds. A prominent model for the versatility of coiled-coil sequences is a peptide derived from the yeast transcription factor, GCN4. In this work, we show that its trimeric variant, GCN4-pII, binds bacterial lipopolysaccharides (LPS) from different bacterial species with picomolar affinity. LPS molecules are highly immunogenic, toxic glycolipids that comprise the outer leaflet of the outer membrane of Gram-negative bacteria. Using scattering techniques and electron microscopy, we show how GCN4-pII breaks down LPS micelles in solution. Our findings suggest that the GCN4-pII peptide and derivatives thereof could be used for novel LPS detection and removal solutions with high relevance to the production and quality control of biopharmaceuticals and other biomedical products, where even minuscule amounts of residual LPS can be lethal.

Coiled-coil structure mediated inhibition of the cytotoxic huntingtin amyloid fibrils by an IP3 receptor fragment

Disruption of cellular homeostasis by the aggregation of polyglutamine (polyQ) in the huntingtin protein (Htt) leads Huntington's disease (HD). Effective drugs for treating HD have not been developed, as the molecular mechanism underlying HD pathogenesis remains unclear. To develop strategies for inhibiting HD pathogenesis, the intermolecular interaction of Htt with IP3 receptor 1 (IP3R1) was investigated. Peptide (termed ICT60) corresponding to a coiled-coil motif in the C-terminus of IP3R1 was designed. Several biophysical approaches revealed the strong and specific binding of ICT60 to the N-terminal part of HttEx1. ICT60 inhibited not only amyloid formation by HttEx1, but also the cytotoxicity and cell-penetration ability of the amyloid fibrils of HttEx1. The importance of coiled–coil structure was verified by charge-manipulated variants. The coiled-coil structures of ICT60-KK and -EE were partially and largely disrupted, respectively. ICT60 wild-type and -KK inhibited amyloid formation by HttEx1-46Q, whereas ICT60-EE did not block amyloidogenesis. Similarly, the cytotoxicity and cell-penetration ability of the amyloid fibrils of HttEx1-46Q were efficiently inhibited by ICT60 wild-type and ICT60-KK, but not by ICT60-EE. We propose a mechanical model explaining how an IP3 receptor-inspired molecule can modulate cytotoxic amyloid formation by Htt, providing a molecular basis for developing therapeutics to treat HD.

A non-catalytic N-terminus domain of WRN prevents mitotic telomere deprotection

Telomeric ends form a loop structure (T-loop) necessary for the repression of ATM kinase activation throughout the normal cell cycle. However, cells undergoing a prolonged mitotic arrest are prone to lose the T-loop, resulting in Aurora B kinase-dependent mitotic telomere deprotection, which was proposed as an anti-tumor mechanism that eliminates precancerous cells from the population. The mechanism of mitotic telomere deprotection has not been elucidated. Here, we show that WRN, a RECQ helicase family member, can suppress mitotic telomere deprotection independently of its exonuclease and helicase activities. Truncation of WRN revealed that N-terminus amino acids 168–333, a region that contains a coiled-coil motif, is sufficient to suppress mitotic telomere deprotection without affecting both mitotic Aurora B-dependent spindle checkpoint and ATM kinase activity. The suppressive activity of the WRN168–333 fragment is diminished in cells partially depleted of TRF2, while WRN is required for complete suppression of mitotic telomere deprotection by TRF2 overexpression. Finally, we found that phosphomimetic but not alanine mutations of putative Aurora B target sites in the WRN168–333 fragment abolished its suppressive effect. Our findings reveal a non-enzymatic function of WRN, which may be regulated by phosphorylation in cells undergoing mitotic arrest. We propose that WRN enhances the protective function of TRF2 to counteract the hypothetical pathway that resolves the mitotic T-loop.

PSMC3 promotes RNAi by maintaining AGO2 stability through USP14

BackgroundArgonaute 2 (AGO2), the only protein with catalytic activity in the human Argonaute family, is considered as a key component of RNA interference (RNAi) pathway. Here we performed a yeast two-hybrid screen using the human Argonaute 2 PIWI domain as bait to screen for new AGO2-interacting proteins and explored the specific mechanism through a series of molecular biology and biochemistry experiments.MethodsThe yeast two-hybrid system was used to screen for AGO2-interacting proteins. Co-immunoprecipitation and immunofluorescence assays were used to further determine interactions and co-localization. Truncated plasmids were constructed to clarify the interaction domain. EGFP fluorescence assay was performed to determine the effect of PSMC3 on RNAi. Regulation of AGO2 protein expression and ubiquitination by PSMC3 and USP14 was examined by western blotting. RT-qPCR assays were applied to assess the level of AGO2 mRNA. Rescue assays were also performed.ResultsWe identified PSMC3 (proteasome 26S subunit, ATPase, 3) as a novel AGO2 binding partner. Biochemical and bioinformatic analysis demonstrates that this interaction is performed in an RNA-independent manner and the N-terminal coiled-coil motif of PSMC3 is required. Depletion of PSMC3 impairs the activity of the targeted cleavage mediated by small RNAs. Further studies showed that depletion of PSMC3 decreased AGO2 protein amount, whereas PSMC3 overexpression increased the expression of AGO2 at a post-translational level. Cycloheximide treatment indicated that PSMC3 depletion resulted in a decrease in cytoplasmic AGO2 amount due to an increase in AGO2 protein turnover. The absence of PSMC3 promoted ubiquitination of AGO2, resulting in its degradation by the 26S proteasome. Mechanistically, PSMC3 assists in the interaction of AGO2 with the deubiquitylase USP14(ubiquitin specific peptidase 14) and facilitates USP14-mediated deubiquitination of AGO2. As a result, AGO2 is stabilized, which then promotes RNAi.ConclusionOur findings demonstrate that PSMC3 plays an essential role in regulating the stability of AGO2 and thus in maintaining effective RNAi.

Genome-Wide Identification of WRKY Gene Family in Pitaya Reveals the Involvement of HmoWRKY42 in Betalain Biosynthesis.

The WRKY gene family is a plant-specific transcription factor (TF) that regulates many physiological processes and (a) biotic stress responses. Despite this, little is known about the molecular properties and roles of WRKY TFs in pitaya betalain biosynthesis. Here we report the identification of 70 WRKY in Hylocereus undatus, their gene structure, locations on each chromosome, systematic phylogenetic analysis, conserved motif analysis, and synteny of HuWRKY genes. HmoWRKY42 is a Group IIb WRKY protein and contains a coiled-coil motif, a WRKY domain and a C2H2 zinc-finger motif (CX5CX23HXH). Results from yeast one-hybrid and transient dual-luciferase assays showed that HmoWRKY42 was a transcriptional repressor and could repress HmocDOPA5GT1 expression by binding to its promoter. Yeast two-hybrid assays showed that HmoWRKY42 could interact with itself to form homodimers. Knocking out the coiled-coil motif of HmoWRKY42 prevented its self-interaction and prevented it from binding to the HmocDOPA5GT1 promoter. Knocking out the WRKY domain and C2H2 zinc-finger motif sequence of HmoWRKY42 also prevented it from binding to the HmocDOPA5GT1 promoter. The coiled-coil motif, the WRKY domain and the C2H2 zinc finger motif are key motifs for the binding of HmoWRKY42 to the HmocDOPA5GT1 promoter. HmoWRKY42 is localized in the nucleus and possesses trans-activation ability responsible for pitaya betalain biosynthesis by repressing the transcription of HmocDOPA5GT1. As far as we know, no reports are available on the role of HmoWRKY42 in pitaya betalain biosynthesis. The results provide an important foundation for future analyses of the regulation and functions of the HuWRKY gene family.

Identification and characterization of coiled-coil motifs across Autographa californica multiple nucleopolyhedrovirus genome

Coiled coils (CCs) are protein structural motifs universally found in proteins and mediate a plethora of biological interactions, and thus their reliable annotation is crucial for studies of protein structure and function. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a large double-stranded DNA (dsDNA) virus and encodes 154 proteins. In this study, genome-wide scans of previously uncharacterized CC motifs throughout AcMNPV was conducted using CC prediction software. In total, 24 CC motifs in 19 CC proteins with high confidence were identified. The characteristic of viral CC motifs were analyzed. The CC proteins could be divided into 12 viral structural proteins and 7 non-structural proteins, including viral membrane fusion proteins, enzymes, and transcription factors. Moreover, CC motifs are conserved in the baculoviral orthologs of 14 of the 19 proteins. It is noted that five CC proteins, including Ac51, Ac66, Exon0, Ac13, and GP16, were previously identified to function in the nuclear egress of nucleocapsids, and Ac66 contains multiple CC motifs, the longest of which comprises 252 amino acids, suggesting a role of CC motifs in this process. Taken together, the CC motifs identified in this study are valuable resource for studying protein function and protein interaction networks during virus replication.

MTCL2 promotes asymmetric microtubule organization by crosslinking microtubules on the Golgi membrane.

The Golgi complex plays an active role in organizing asymmetric microtubule arrays, which are essential for polarized vesicle transport. The coiled-coil protein MTCL1 stabilizes microtubules nucleated from the Golgi membrane. Here, we report an MTCL1 paralog, MTCL2, which preferentially acts on the perinuclear microtubules accumulated around the Golgi. MTCL2 associates with the Golgi membrane through the N-terminal coiled-coil region and directly binds microtubules through the conserved C-terminal domain without promoting microtubule stabilization. Knockdown of MTCL2 significantly impaired microtubule accumulation around the Golgi, as well as the compactness of the Golgi ribbon assembly structure. Given that MTCL2 forms parallel oligomers through homo-interaction of the central coiled-coil motifs, our results indicate that MTCL2 promotes asymmetric microtubule organization by crosslinking microtubules on the Golgi membrane. Results of in vitro wound healing assays further suggest that this function of MTCL2 enables integration of the centrosomal and Golgi-associated microtubules on the Golgi membrane, supporting directional migration. Additionally, the results demonstrated the involvement of CLASPs and giantin in mediating the Golgi association of MTCL2.

The Medicago truncatula IEF Gene Is Crucial for the Progression of Bacterial Infection During Symbiosis.

Legumes are able to meet their nitrogen need by establishing nitrogen-fixing symbiosis with rhizobia. Nitrogen fixation is performed by rhizobia, which has been converted to bacteroids, in newly formed organs, the root nodules. In the model legume Medicago truncatula, nodule cells are invaded by rhizobia through transcellular tubular structures called infection threads (ITs) that are initiated at the root hairs. Here, we describe a novel M. truncatula early symbiotic mutant identified as infection-related epidermal factor (ief), in which the formation of ITs is blocked in the root hair cells and only nodule primordia are formed. We show that the function of MtIEF is crucial for the bacterial infection in the root epidermis but not required for the nodule organogenesis. The IEF gene that appears to have been recruited for a symbiotic function after the duplication of a flower-specific gene is activated by the ERN1-branch of the Nod factor signal transduction pathway and independent of the NIN activity. The expression of MtIEF is induced transiently in the root epidermal cells by the rhizobium partner or Nod factors. Although its expression was not detectable at later stages of symbiosis, complementation experiments indicate that MtIEF is also required for the proper invasion of the nodule cells by rhizobia. The gene encodes an intracellular protein of unknown function possessing a coiled-coil motif and a plant-specific DUF761 domain. The IEF protein interacts with RPG, another symbiotic protein essential for normal IT development, suggesting that combined action of these proteins plays a role in nodule infection.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Etp1 confers arsenite resistance by affecting ACR3 expression.

In a high-throughput yeast two-hybrid screen of predicted coiled-coil motif interactions in the Saccharomyces cerevisiae proteome, the protein Etp1 was found to interact with the yeast AP-1-like transcription factors Yap8, Yap1 and Yap6. Yap8 plays a crucial role during arsenic stress since it regulates expression of the resistance genes ACR2 and ACR3. The function of Etp1 is not well understood but the protein has been implicated in transcription and protein turnover during ethanol stress, and the etp1∆ mutant is sensitive to ethanol. In this current study, we investigated whether Etp1 is implicated in Yap8-dependent functions. We show that Etp1 is required for optimal growth in the presence of trivalent arsenite and for optimal expression of the arsenite export protein encoded by ACR3. Since Yap8 is the only known transcription factor that regulates ACR3 expression, we investigated whether Etp1 regulates Yap8. Yap8 ubiquitination, stability, nuclear localization and ACR3 promoter association were unaffected in etp1∆ cells, indicating that Etp1 affects ACR3 expression independently of Yap8. Thus, Etp1 impacts gene expression under arsenic and other stress conditions but the mechanistic details remain to be elucidated.