Protein domain-domain interactions and requirements for the negative regulation of Arabidopsis CDC48/p97 by the plant ubiquitin regulatory X (UBX) domain-containing protein, PUX1

CDC48/p97 is an essential AAA-ATPase chaperone that functions in numerous diverse cellular activities through its interaction with specific adapter proteins. The ubiquitin regulatory X (UBX)-containing protein, PUX1, functions to regulate the hexameric structure and ATPase activity of AtCDC48. To characterize the biochemical mechanism of PUX1 action on AtCDC48, we have defined domains of both PUX1 and AtCDC48 that are critical for interaction and oligomer disassembly. Binding of PUX1 to AtCDC48 was mediated through a region containing both the UBX domain and the immediate C-terminal flanking amino acids (UBX-C). Like other UBX domains, the primary binding site for the UBX-C of PUX1 is the N(a) domain of AtCDC48. Alternative plant PUX protein UBX domains also bind AtCDC48 through the N terminus but were found not to be able to substitute for the action imparted by the UBX-C of PUX1 in hexamer disassembly, suggesting unique features for the UBX-C of PUX1. We propose that the PUX1 UBX-C domain modulates a second binding site on AtCDC48 required for the N-terminal domain of PUX1 to interact with and promote dissociation of the AtCDC48 hexamer. Utilizing Atcdc48 ATP hydrolysis and binding mutants, we demonstrate that PUX1 binding was not affected but that hexamer disassembly was significantly influenced by the ATP status of AtCDC48. ATPase activity in both the D1 and the D2 domains was critical for PUX1-mediated AtCDC48 hexamer disassembly. Together these results provide new mechanistic insight into how the hexameric status and ATPase activity of AtCDC48 are modulated.     

Plant UBX domain-containing protein 1, PUX1, regulates the oligomeric structure and activity of arabidopsis CDC48

p97/CDC48 is a highly abundant hexameric AAA-ATPase that functions as a molecular chaperone in numerous diverse cellular activities. We have identified an Arabidopsis UBX domain-containing protein, PUX1, which functions to regulate the oligomeric structure of the Arabidopsis homolog of p97/CDC48, AtCDC48, as well as mammalian p97. PUX1 is a soluble protein that co-fractionates with non-hexameric AtCDC48 and physically interacts with AtCDC48 in vivo. Binding of PUX1 to AtCDC48 is mediated through the UBX-containing C-terminal domain. However, disassembly of the chaperone is dependent upon the N-terminal domain of PUX1. These findings provide evidence that the assembly and disassembly of the hexameric p97/CDC48 complex is a dynamic process. This new unexpected level of regulation for p97/CDC48 was demonstrated to be critical in vivo as pux1 loss-of-function mutants display accelerated growth relative to wild-type plants. These results suggest a role for AtCDC48 and PUX1 in regulating plant growth.     

UBX domain-containing proteins are involved in lipid homeostasis and stress responses in Pichia pastoris

Ubiquitin regulatory X (UBX) domain-containing proteins constitute a family of proteins and are substrate adaptors of AAA ATPase Cdc48. UBX proteins can bind to the N-terminal region of Cdc48 to perform endoplasmic reticulum associated protein degradation (ERAD). In this study, we identified two UBX domain-containing proteins, Ubx1 and Ubx2, in Pichia pastoris and found that the two proteins could recover the growth defect of Saccharomyces cerevisiae in ubx2Δ. Our results revealed that Ubx1 and Ubx2 play critical roles in synthesis of unsaturated fatty acids by affecting Spt23. In addition, the results demonstrated that both Ubx1 and Ubx2 are involved in lipid droplet formation and protein degradation. Deletion of UBX1 led to increased sensitivity to oxidative stress and disruption of UBX2 impaired cell viability under osmotic stress. The phenotypes of ubx1Δ+UBX2, ubx2Δ+UBX1 and ubx1Δubx2Δ and RNA-seq data suggested that Ubx1 and Ubx2 play different roles in cell functions, and the roles of Ubx1 may be more numerous than Ubx2. In summary, our findings provide new insights into the relationship between lipid homeostasis and cell functions in the oil-producing organism P. pastoris.     

Complex of Fas-associated Factor 1 (FAF1) with Valosin-containing Protein (VCP)-Npl4-Ufd1 and Polyubiquitinated Proteins Promotes Endoplasmic Reticulum-associated Degradation (ERAD)

Fas-associated factor 1 (FAF1) is a ubiquitin receptor containing multiple ubiquitin-related domains including ubiquitin-associated (UBA), ubiquitin-like (UBL) 1, UBL2, and ubiquitin regulatory X (UBX). We previously showed that N-terminal UBA domain recognizes Lys⁴⁸-ubiquitin linkage to recruit polyubiquitinated proteins and that a C-terminal UBX domain interacts with valosin-containing protein (VCP). This study shows that FAF1 interacts only with VCP complexed with Npl4-Ufd1 heterodimer, a requirement for the recruitment of polyubiquitinated proteins to UBA domain. Intriguingly, VCP association to C-terminal UBX domain regulates ubiquitin binding to N-terminal UBA domain without direct interaction between UBA and UBX domains. These interactions are well characterized by structural and biochemical analysis. VCP-Npl4-Ufd1 complex is known as the machinery required for endoplasmic reticulum-associated degradation. We demonstrate here that FAF1 binds to VCP-Npl4-Ufd1 complex via UBX domain and polyubiquitinated proteins via UBA domain to promote endoplasmic reticulum-associated degradation.     

AtCDC48A is involved in the turnover of an NLR immune receptor

Plants rely on different immune receptors to recognize pathogens and defend against pathogen attacks. Nucleotide‐binding domain and leucine‐rich repeat (NLR) proteins play a major role as intracellular immune receptors. Their homeostasis must be maintained at optimal levels in order to effectively recognize pathogens without causing autoimmunity. Previous studies have shown that the activity of the ubiquitin‐proteasome system is essential to prevent excessive accumulation of NLR proteins such as Suppressor of NPR1, Constitutive 1 (SNC1). Attenuation of the ubiquitin E3 ligase SCF^CPR1 (Constitutive expressor of Pathogenesis Related genes 1) or the E4 protein MUSE3 (Mutant, SNC1‐Enhancing 3) leads to NLR accumulation and autoimmunity. In the current study, we report the identification of AtCDC48A as a negative regulator of NLR‐mediated immunity. Plants carrying Atcdc48A‐4, a partial loss‐of‐function allele of AtCDC48A, exhibit dwarf morphology and enhanced disease resistance to the oomycete pathogen Hyaloperonospora arabidopsidis (H.a.) Noco2. The SNC1 level is increased in Atcdc48A‐4 plants and AtCDC48A interacts with MUSE3 in co‐immunoprecipitation experiments, supporting a role for AtCDC48A in NLR turnover. While Arabidopsis contains four other paralogs of AtCDC48A, knockout mutants of these genes do not show obvious immunity‐related phenotypes, suggesting functional divergence within this family. As an AAA‐ATPase, AtCDC48A likely serves to process the poly‐ubiquitinated NLR substrate for final protein degradation by the 26S proteasome.     

Crystal structure of human FAF1 UBX domain reveals a novel FcisP touch-turn motif in p97/VCP-binding region

UBX domain is a general p97/VCP-binding module found in an increasing number of proteins including FAF1, p47, SAKS1 and UBXD7. FAF1, a multi-functional tumor suppressor protein, binds to the N domain of p97/VCP through its C-terminal UBX domain and thereby inhibits the proteasomal protein degradation in which p97/VCP acts as a co-chaperone. Here we report the crystal structure of human FAF1 UBX domain at 2.9 Å resolution. It reveals that the conserved FP sequence in the p97/VCP-binding region adopts a rarely observed cis-Pro touch-turn structure. We call it an FcisP touch-turn motif and suggest that it is the conserved structural element of the UBX domain. Four FAF1 UBX molecules in an asymmetric unit of the crystal show two different conformations of the FcisP touch-turn motif. The phenyl ring of F⁶¹⁹ in the motif stacks partly over cis-Pro⁶²⁰ in one conformation, whereas it is swung out from cis-P⁶²⁰, in the other conformation, and forms hydrophobic contacts with the residues of the neighboring molecule. In addition, the entire FcisP touch-turn motif is pulled out in the second conformation by about 2 Å in comparison to the first conformation. Those conformational differences observed in the p97/VCP-binding motif caused by the interaction with neighboring molecules presumably represent the conformational change of the UBX domain on its binding to the N domain of p97/VCP.     

Lipid-regulated degradation of HMG-CoA reductase and Insig-1 through distinct mechanisms in insect cells

In mammalian cells, levels of the integral membrane proteins 3-hydroxy-3-methylglutaryl-CoA reductase and Insig-1 are controlled by lipid-regulated endoplasmic reticulum-associated degradation (ERAD). The ERAD of reductase slows a rate-limiting step in cholesterol synthesis and results from sterol-induced binding of its membrane domain to Insig-1 and the highly related Insig-2 protein. Insig binding bridges reductase to ubiquitin ligases that facilitate its ubiquitination, thereby marking the protein for cytosolic dislocation and proteasomal degradation. In contrast to reductase, Insig-1 is subjected to ERAD in lipid-deprived cells. Sterols block this ERAD by inhibiting Insig-1 ubiquitination, whereas unsaturated fatty acids block the reaction by preventing the protein''s cytosolic dislocation. In previous studies, we found that the membrane domain of mammalian reductase was subjected to ERAD in Drosophila S2 cells. This ERAD was appropriately accelerated by sterols and required the action of Insigs, which bridged reductase to a Drosophila ubiquitin ligase. We now report reconstitution of mammalian Insig-1 ERAD in S2 cells. The ERAD of Insig-1 in S2 cells mimics the reaction that occurs in mammalian cells with regard to its inhibition by either sterols or unsaturated fatty acids. Genetic and pharmacologic manipulations coupled with subcellular fractionation indicate that Insig-1 and reductase are degraded through distinct mechanisms that are mediated by different ubiquitin ligase complexes. Together, these results establish Drosophila S2 cells as a model system to elucidate mechanisms through which lipid constituents of cell membranes (i.e., sterols and fatty acids) modulate the ERAD of Insig-1 and reductase.     

Regulation of the Polycomb protein RING1B ubiquitination by USP7

The E3 ubiquitin ligase RING1B plays an important role in Polycomb-mediated gene silencing by monoubiquitinating histone H2A. Both the activity and stability of RING1B are controlled by ubiquitination in two distinct manners. Self ubiquitination of RING1B generates K6, K27 and K48-based mixed polyubiquitin chain, and is required for its activity as a ligase. On the other hand, its proteasomal degradation is mediated by another ligase; E6-AP catalyzes the formation of K48-based chains. Since these two modes of ubiquitination target the same lysine residues and are therefore mutually exclusive, an important mode of regulation of RING1B should be at the level of deubiquitination. Here we identify USP7 as a deubiquitinating enzyme that regulates the ubiquitination state of RING1B. RING1B interacts with USP7, which is mediated in part by its RING domain. In addition, USP7 was found in a complex with other Polycomb proteins, suggesting a broad role in regulating these complexes. Although, USP7 directly and specifically deubiquitinates RING1B in vitro and in vivo, it does not discriminate between the activating and proteolysis-targeting modes of ubiquitination, and therefore has a stabilizing effect on RING1B.     

A Cdc48p-associated factor modulates endoplasmic reticulum-associated degradation, cell stress, and ubiquitinated protein homeostasis

The hexameric AAA-ATPase, Cdc48p, catalyzes an array of cellular activities, including endoplasmic reticulum (ER)-associated degradation (ERAD), ER/Golgi membrane dynamics, and DNA replication. Accumulating data suggest that unique Cdc48p partners, such as Npl4p-Ufd1p and Ubx1p/Shp1p (p47 in vertebrates), target Cdc48p for these diverse functions. Other Cdc48p-associated proteins have been identified, but the interplay among these factors and their activities is largely cryptic. We now report on a previously uncharacterized Cdc48p-associated protein, Ydr049p, also known as Vms1p, which binds Cdc48p at both the ER membrane and in the cytosol under non-stressed conditions. Loss of YDR049 modestly slows the degradation of the cystic fibrosis transmembrane conductance regulator but does not impede substrate ubiquitination, suggesting that Ydr049p acts at a postubiquitination step in the ERAD pathway. Consistent with Ydr049p playing a role in Cdc48p substrate release, ydr049 mutant cells accumulate Cdc48p-bound ubiquitinated proteins at the ER membrane. Moreover, YDR049 interacts with genes encoding select UBX (ubiquitin regulatory X) and UFD (ubiquitin fusion degradation) proteins, which are Cdc48p partners. Exacerbated growth defects are apparent in some of the mutant combinations, and synergistic effects on the degradation of cystic fibrosis transmembrane conductance regulator and CPY*, which is a soluble ERAD substrate, are evident in specific ydr049-ufd and -ubx mutants. These data suggest that Ydr049p acts in parallel with Cdc48p partners to modulate ERAD and other cellular activities.     

In planta analysis of the cell cycle-dependent localization of AtCDC48A and its critical roles in cell division, expansion, and differentiation

CDC48/p97 is a conserved homohexameric AAA-ATPase chaperone required for a variety of cellular processes but whose role in the development of a multicellular model system has not been examined. Here, we have used reverse genetics, visualization of a functional Arabidopsis (Arabidopsis thaliana) CDC48 fluorescent fusion protein, and morphological analysis to examine the subcellular distribution and requirements for AtCDC48A in planta. Homozygous Atcdc48A T-DNA insertion mutants arrest during seedling development, exhibiting decreased cell expansion and displaying pleiotropic defects in pollen and embryo development. Atcdc48A insertion alleles show significantly reduced male transmission efficiency due to defects in pollen tube growth. Yellow fluorescent protein-AtCDC48A, a fusion protein that functionally complements the insertion mutant defects, localizes in the nucleus and cytoplasm and is recruited to the division mid-zone during cytokinesis. The pattern of nuclear localization differs according to the stage of the cell cycle and differentiation state. Inducible expression of an Atcdc48A Walker A ATPase mutant in planta results in cytokinesis abnormalities, aberrant cell divisions, and root trichoblast differentiation defects apparent in excessive root hair emergence. At the biochemical level, our data suggest that the endogenous steady-state protein level of AtCDC48A is dependent upon the presence of ATPase-active AtCDC48A. These results demonstrate that CDC48A/p97 is critical for cytokinesis, cell expansion, and differentiation in plants.     

Shp1 and Ubx2 are adaptors of Cdc48 involved in ubiquitin-dependent protein degradation

Known activities of the ubiquitin-selective AAA ATPase Cdc48 (p97) require one of the mutually exclusive cofactors Ufd1/Npl4 and Shp1 (p47). Whereas Ufd1/Npl4 recruits Cdc48 to ubiquitylated proteins destined for degradation by the 26S proteasome, the UBX domain protein p47 has so far been linked exclusively to nondegradative Cdc48 functions in membrane fusion processes. Here, we show that all seven UBX domain proteins of Saccharomyces cerevisiae bind to Cdc48, thus constituting an entire new family of Cdc48 cofactors. The two major yeast UBX domain proteins, Shp1 and Ubx2, possess a ubiquitin-binding UBA domain and interact with ubiquitylated proteins in vivo. Deltashp1 and Deltaubx2 strains display defects in the degradation of a ubiquitylated model substrate, are sensitive to various stress conditions and are genetically linked to the 26S proteasome. Our data suggest that Shp1 and Ubx2 are adaptors for Cdc48-dependent protein degradation through the ubiquitin/proteasome pathway.     

Regulation of RNF144A E3 Ubiquitin Ligase Activity by Self-association through Its Transmembrane Domain

RNF144A, an E3 ubiquitin ligase for DNA-dependent protein kinase catalytic subunit (DNA-PKcs), can promote DNA damage-induced cell apoptosis. Here we characterize an important regulation of RNF144A through its transmembrane (TM) domain. The TM domain of RNF144A is highly conserved among species. Deletion of the TM domain abolishes its membrane localization and also significantly reduces its ubiquitin ligase activity. Further evidence shows that the TM domain is required for RNF144A self-association and that the self-association may be partially mediated through a classic GXXXG interaction motif. A mutant RNF144A-G252L/G256L (in the G²⁵²XXXG²⁵⁶ motif) preserves membrane localization but is defective in self-association and ubiquitin ligase activity. On the other hand, a membrane localization loss mutant of RNF144A still retains self-association and E3 ligase activity, which can be blocked by additional G252L/G256L mutations. Therefore, our data demonstrate that the TM domain of RNF144A has at least two independent roles, membrane localization and E3 ligase activation, to regulate its physiological function. This regulatory mechanism may be applicable to other RBR (RING1-IBR-RING2) E3 ubiquitin ligases because, first, RNF144B also self-associates. Second, all five TM-containing RBR E3 ligases, including RNF144A and RNF144B, RNF19A/Dorfin, RNF19B, and RNF217, have the RBR-TM(GXXXG) superstructure. Mutations of the GXXXG motifs in RNF144A and RNF217 have also be found in human cancers, including a G252D mutation of RNF144A. Interestingly, RNF144A-G252D still preserves self-association and ubiquitin ligase activity but loses membrane localization and is turned over rapidly. In conclusion, both proper membrane localization and self-association are important for RNF144A function.     

The proteasome regulator PTRE1 contributes to the turnover of SNC1 immune receptor

Plants have evolved a sophisticated immune system in order to recognize and respond to microbes in their environments. Nucleotide-binding leucine-rich repeat (NLR) proteins detect the presence of specific effector molecules delivered into host cells by pathogens and activate strong defence responses. However, as excessive accumulation of NLRs can result in inappropriate immune responses, their abundance must be tightly regulated. Targeted degradation of NLRs through the ubiquitin proteasome pathway is an important mechanism to limit NLR accumulation. Mutations that perturb NLR degradation can cause autoimmune phenotypes. In this study, we show that the proteasome regulator PTRE1 also contributes to NLR degradation. ptre1 mutant plants exhibit increased defence marker gene expression and enhanced disease resistance against virulent pathogens. The stability of the NLR, SUPPRESSOR OF npr1-1 CONSTITUTIVE 1 (SNC1) is also increased in the ptre1 mutant. Although the mouse homologue of PTRE1 was reported to interact with a Cell Division Control protein 48 (CDC48) homologue in vitro (Clemen et al., 2015), we only observed interaction between PTRE1 and AtCDC48A in a split luciferase assay, but not in co-immunoprecipitation. In addition, a related Arabidopsis protein PTRE1h shares partial redundancy with PTRE1. Together, PTRE1 acts as a negative regulator of plant immunity partly by facilitating the degradation of immune receptors such as SNC1.     

Affinity makes the difference: nonselective interaction of the UBA domain of Ubiquilin-1 with monomeric ubiquitin and polyubiquitin chains

Ubiquilin/PLIC proteins belong to the family of UBL-UBA proteins implicated in the regulation of the ubiquitin-dependent proteasomal degradation of cellular proteins. A human presenilin-interacting protein, ubiquilin-1, has been suggested as potential therapeutic target for treating Huntington's disease. Ubiquilin's interactions with mono- and polyubiquitins are mediated by its UBA domain, which is one of the tightest ubiquitin binders among known ubiquitin-binding domains. Here we report the three-dimensional structure of the UBA domain of ubiquilin-1 (UQ1-UBA) free in solution and in complex with ubiquitin. UQ1-UBA forms a compact three-helix bundle structurally similar to other known UBAs, and binds to the hydrophobic patch on ubiquitin with a K_d of 20 μM. To gain structural insights into UQ1-UBA's interactions with polyubiquitin chains, we have mapped the binding interface between UQ1-UBA and Lys48- and Lys63-linked di-ubiquitins and characterized the strength of UQ1-UBA binding to these chains. Our NMR data show that UQ1-UBA interacts with the individual ubiquitin units in both chains in a mode similar to its interaction with mono-ubiquitin, although with an improved binding affinity for the chains. Our results indicate that, in contrast to UBA2 of hHR23A that has strong binding preference for Lys48-linked chains, UQ1-UBA shows little or no binding selectivity toward a particular chain linkage or between the two ubiquitin moieties in the same chain. The structural data obtained in this study provide insights into the possible structural reasons for the diversity of polyubiquitin chain recognition by UBA domains.     

The neuralized homology repeat 1 domain of Drosophila neuralized mediates nuclear envelope association and delta-dependent inhibition of nuclear import

Signaling by the Notch (N) pathway is critical for many developmental processes and requires complex trafficking of both the N receptor and its transmembrane ligands, Delta (Dl) and Serrate. neuralized encodes an E3 ubiquitin ligase required for N ligand internalization. Neuralized (Neur) is conserved from worms to humans and comprises two Neur homology repeat (NHR) domains, NHR1 and NHR2, and a carboxyl-terminal RING domain. We have previously shown that the RING domain is required for ubiquitin ligase activity and that NHR1 mediates binding to Dl, a ubiquitination target. In Drosophila, Neur associates with the plasma membrane and hepatocyte responsive serum phosphoprotein-positive endosomes. Here we demonstrate that Neur also exhibits nuclear envelope localization. We have determined that Neur subcellular localization is regulated by nuclear trafficking and that inhibition of chromosome region maintenance 1, a nuclear export receptor, interferes with Neur nuclear export, trapping Neur in the nucleus. Moreover, we demonstrate that nuclear envelope localization is mediated by the Neur NHR1 domain. Interestingly, Dl expression in Schneider cells is sufficient to inhibit Neur nuclear import and inhibition occurs in an NHR1-dependent manner, suggesting that Neur nuclear localization occurs in contexts where Dl expression is either low or absent. Consistent with this, we found that Neur exhibits nuclear trafficking and associates with the nuclear envelope in the secretory cells of the larval salivary gland and that overexpression of Dl can reduce Neur localization to the nucleus. Altogether, our data demonstrate that Neur localizes to the nuclear envelope and that this localization can be negatively regulated by Dl, suggesting a possible nuclear function for Neur in Drosophila.     

The intronic region of Fbxl12 functions as an alternative promoter regulated by UV irradiation

The ubiquitin ligases, SCF complexes, consist of Cul1, Skp1, Rbx1 and the substrate recognition components F-box proteins. Previous studies have reported that one of these F-box proteins, Fbl12, which is produced by Fbxl12 gene, regulates both cell cycle and differentiation. In this paper, we show that the intronic region of Fbxl12 gene acts as an alternative promoter and induces expression of a short form of Fbl12 that lacks F-box domain (Fbl12ΔF). We also found that UV irradiation increases Fbl12ΔF mRNA in cells. Finally, Fbl12ΔF may promote the subcellular localization of Fbl12 from nucleus to cytoplasm through their binding. Our data provide the possibility that Fbl12ΔF induced by alternative promoter controls the SCF^Fbl12 activity in response to UV stimulation.     

Hierarchical binding of cofactors to the AAA ATPase p97

The hexameric AAA ATPase p97 is involved in several human proteinopathies and mediates ubiquitin-dependent protein degradation among other essential cellular processes. Via its N-terminal domain (N domain), p97 interacts with multiple regulatory cofactors including the UFD1/NPL4 heterodimer and members of the "ubiquitin regulatory X" (UBX) domain protein family; however, the principles governing cofactor selectivity remain to be deciphered. Our crystal structure of the FAS-associated factor 1 (FAF1)UBX domain in complex with the p97N domain reveals that the signature Phe-Pro-Arg motif known to be crucial for interactions of UBX domains with p97 adopts a cis-proline configuration, in contrast to a cis-trans mixture we derive for the isolated FAF1UBX domain. Biochemical studies confirm that binding critically depends on a proline at this position. Furthermore, we observe that the UBX proteins FAF1 and UBXD7 only bind to p97-UFD1/NPL4, but not free p97, thus demonstrating for the first time a hierarchy in p97-cofactor interactions.     

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.     

Conserved CDC20 cell cycle functions are carried out by two of the five isoforms in Arabidopsis thaliana

Background

The CDC20 and Cdh1/CCS52 proteins are substrate determinants and activators of the Anaphase Promoting Complex/Cyclosome (APC/C) E3 ubiquitin ligase and as such they control the mitotic cell cycle by targeting the degradation of various cell cycle regulators. In yeasts and animals the main CDC20 function is the destruction of securin and mitotic cyclins. Plants have multiple CDC20 gene copies whose functions have not been explored yet. In Arabidopsis thaliana there are five CDC20 isoforms and here we aimed at defining their contribution to cell cycle regulation, substrate selectivity and plant development.

Methodology/Principal Findings

Studying the gene structure and phylogeny of plant CDC20s, the expression of the five AtCDC20 gene copies and their interactions with the APC/C subunit APC10, the CCS52 proteins, components of the mitotic checkpoint complex (MCC) and mitotic cyclin substrates, conserved CDC20 functions could be assigned for AtCDC20.1 and AtCDC20.2. The other three intron-less genes were silent and specific for Arabidopsis. We show that AtCDC20.1 and AtCDC20.2 are components of the MCC and interact with mitotic cyclins with unexpected specificity. AtCDC20.1 and AtCDC20.2 are expressed in meristems, organ primordia and AtCDC20.1 also in pollen grains and developing seeds. Knocking down both genes simultaneously by RNAi resulted in severe delay in plant development and male sterility. In these lines, the meristem size was reduced while the cell size and ploidy levels were unaffected indicating that the lower cell number and likely slowdown of the cell cycle are the cause of reduced plant growth.

Conclusions/Significance

The intron-containing CDC20 gene copies provide conserved and redundant functions for cell cycle progression in plants and are required for meristem maintenance, plant growth and male gametophyte formation. The Arabidopsis-specific intron-less genes are possibly “retrogenes” and have hitherto undefined functions or are pseudogenes.