Dfm1 Forms Distinct Complexes with Cdc48 and the ER Ubiquitin Ligases and Is Required for ERAD

Proteins imported into the endoplasmic reticulum (ER) are scanned for their folding status. Those that do not reach their native conformation are degraded via the ubiquitin‐proteasome system. This process is called ER‐associated degradation (ERAD). Der1 is known to be one of the components required for efficient degradation of soluble ERAD substrates like CPY^* (mutated carboxypeptidase yscY). A homologue of Der1 exists, named Dfm1. No function of Dfm1 has been discovered, although a C‐terminally hemagglutinin (HA)₃‐tagged Dfm1 protein has been shown to interact with the ERAD machinery. In our studies, we found Dfm1‐HA₃ to be an ERAD substrate and therefore not suitable for functional studies of Dfm1 in ERAD. We found cellular, non‐tagged Dfm1 to be a stable protein. We identified Dfm1 to be part of complexes which contain the ERAD‐L ligase Hrd1/Der3 and Der1 as well as the ERAD‐C ligase Doa10. In addition, ERAD of Ste6^*‐HA₃ was strongly dependent on Dfm1. Interestingly, Dfm1 forms a complex with the AAA‐ATPase Cdc48 in a strain lacking the Cdc48 membrane‐recruiting component Ubx2. This complex does not contain the ubiquitin ligases Hrd1/Der3 and Doa10. The existence of such a complex might point to an additional function of Dfm1 independent from ERAD.     

The Cdc20 homolog,FZY-1, and its interacting protein,IFY-1, are required for proper chromosome segregation in Caenorhabditis elegans

Accurate chromosome segregation is achieved by a series of highly regulated processes that culminate in the metaphase-to-anaphase transition of the cell cycle. In the budding yeast Saccharomyces cerevisiae, the degradation of the securin protein Pds1 reverses the binding and inhibition of the separase protein Esp1. Esp1 cleaves Scc1. That cleavage promotes the dissociation of the cohesin complex from the chromosomes and leads the separation of sister chromatids. Proteolysis of Pds1 is regulated by the anaphase-promoting complex (APC), a large multi-subunit E3 ubiquitin ligase whose activity is regulated by Cdc20/Fizzy. We have previously shown that the Caenorhabditis elegans genes mdf-1/MAD1 and mdf-2/MAD2 encode key members of the spindle checkpoint. Loss of function of either gene leads to an accumulation of somatic and heritable defects and ultimately results in death. Here we show that a missense mutation in fzy-1/CDC20/Fizzy suppresses mdf-1 lethality. We identified a FZY-1-interacting protein, IFY-1, a novel destruction-box protein. IFY-1 accumulates in one-cell-arrested emb-30/APC4 embryos and interacts with SEP-1, a C. elegans separase, suggesting that IFY-1 functions as a C. elegans securin.     

Sli15 associates with the ipl1 protein kinase to promote proper chromosome segregation in Saccharomyces cerevisiae.

The conserved Ipl1 protein kinase is essential for proper chromosome segregation and thus cell viability in the budding yeast Saccharomyces cerevisiae. Its human homologue has been implicated in the tumorigenesis of diverse forms of cancer. We show here that sister chromatids that have separated from each other are not properly segregated to opposite poles of ipl1-2 cells. Failures in chromosome segregation are often associated with abnormal distribution of the spindle pole-associated Nuf2-GFP protein, thus suggesting a link between potential spindle pole defects and chromosome missegregation in ipl1 mutant cells. A small fraction of ipl1-2 cells also appears to be defective in nuclear migration or bipolar spindle formation. Ipl1 associates, probably directly, with the novel and essential Sli15 protein in vivo, and both proteins are localized to the mitotic spindle. Conditional sli15 mutant cells have cytological phenotypes very similar to those of ipl1 cells, and the ipl1-2 mutation exhibits synthetic lethal genetic interaction with sli15 mutations. sli15 mutant phenotype, like ipl1 mutant phenotype, is partially suppressed by perturbations that reduce protein phosphatase 1 function. These genetic and biochemical studies indicate that Sli15 associates with Ipl1 to promote its function in chromosome segregation.     

Self-regulated Plk1 recruitment to kinetochores by the Plk1-PBIP1 interaction is critical for proper chromosome segregation

The polo-box domain (PBD) of mammalian polo-like kinase 1 (Plk1) is essential in targeting its catalytic activity to specific subcellular structures critical for mitosis. The mechanism underlying Plk1 recruitment to the kinetochores and the role of Plk1 at this site remain elusive. Here, we demonstrate that a PBD-binding protein, PBIP1, is crucial for recruiting Plk1 to the interphase and mitotic kinetochores. Unprecedentedly, Plk1 phosphorylated PBIP1 at T78, creating a self-tethering site that specifically interacted with the PBD of Plk1, but not Plk2 or Plk3. Later in mitosis, Plk1 also induced PBIP1 degradation in a T78-dependent manner, thereby enabling itself to interact with other components critical for proper kinetochore functions. Absence of the p-T78-dependent Plk1 localization induced a chromosome congression defect and compromised the spindle checkpoint, ultimately leading to aneuploidy. Thus, Plk1 self-regulates the Plk1-PBIP1 interaction to timely localize to the kinetochores and promote proper chromosome segregation.     

Kremen proteins interact with Dickkopf1 to regulate anteroposterior CNS patterning

A gradient of Wnt/beta-catenin signalling formed by posteriorising Wnts and anteriorising Wnt antagonists regulates anteroposterior (AP) patterning of the central nervous system (CNS) during Xenopus gastrulation. In this process, the secreted Wnt antagonist Dkk1 functions in the Spemann organiser and its anterior derivatives by blocking Wnt receptors of the lipoprotein receptor-related protein (LRP) 5 and 6 class. In addition to LRP6, Dkk1 interacts with another recently identified receptor class, the transmembrane proteins Kremen1 (Krm1) and Kremen2 (Krm2) to synergistically inhibit LRP6. We have investigated the role of Krm1 and Krm2 during early Xenopus embryogenesis. Consistent with a role in zygotic Wnt inhibition, overexpressed Krm anteriorises embryos and rescues embryos posteriorised by Wnt8. Antisense morpholino oligonucleotide (Mo) knockdown of Krm1 and Krm2 leads to deficiency of anterior neural development. In this process, Krm proteins functionally interact with Dkk1: (1) in axis duplication assays krm2 synergises with dkk1 in inhibiting Wnt/LRP6 signalling; (2) krm2 rescues microcephalic embryos induced by injection of inhibitory anti-Dkk1 antibodies; and (3) injection of krm1/2 antisense Mo enhances microcephaly induced by inhibitory anti-Dkk1 antibodies. The results indicate that Krm proteins function in a Wnt inhibition pathway regulating early AP patterning of the CNS.     

PP2A is required for centromeric localization of Sgo1 and proper chromosome segregation

Loss of sister-chromatid cohesion triggers chromosome segregation in mitosis and occurs through two mechanisms in vertebrate cells: (1) phosphorylation and removal of cohesin from chromosome arms by mitotic kinases, including Plk1, during prophase, and (2) cleavage of centromeric cohesin by separase at the metaphase-anaphase transition. Bub1 and the MEI-S332/Shugoshin (Sgo1) family of proteins protect centromeric cohesin from mitotic kinases during prophase. We show that human Sgo1 binds to protein phosphatase 2A (PP2A). PP2A localizes to centromeres in a Bub1-dependent manner. The Sgo1-PP2A interaction is required for centromeric localization of Sgo1 and proper chromosome segregation in human cells. Depletion of Plk1 by RNA interference (RNAi) restores centromeric localization of Sgo1 and prevents chromosome missegregation in cells depleted of PP2A_Aalpha. Our findings suggest that Bub1 targets PP2A to centromeres, which in turn maintains Sgo1 at centromeres by counteracting Plk1-mediated chromosome removal of Sgo1.     

Kaposi's sarcoma-associated herpesvirus-encoded LANA can interact with the nuclear mitotic apparatus protein to regulate genome maintenance and segregation

Kaposi's sarcoma-associated herpesvirus (KSHV) genomes are tethered to the host chromosomes and partitioned faithfully into daughter cells with the host chromosomes. The latency-associated nuclear antigen (LANA) is important for segregation of the newly synthesized viral genomes to the daughter nuclei. Here, we report that the nuclear mitotic apparatus protein (NuMA) and LANA can associate in KSHV-infected cells. In synchronized cells, NuMA and LANA are colocalized in interphase cells and separate during mitosis at the beginning of prophase, reassociating again at the end of telophase and cytokinesis. Silencing of NuMA expression by small interfering RNA and expression of LGN and a dominant-negative of dynactin (P150-CC1), which disrupts the association of NuMA with microtubules, resulted in the loss of KSHV terminal-repeat plasmids containing the major latent origin. Thus, NuMA is required for persistence of the KSHV episomes in daughter cells. This interaction between NuMA and LANA is critical for segregation and maintenance of the KSHV episomes through a temporally controlled mechanism of binding and release during specific phases of mitosis.     

Cdc37 is essential for chromosome segregation and cytokinesis in higher eukaryotes

Cdc37 has been shown to be required for the activity and stability of protein kinases that regulate different stages of cell cycle progression. However, little is known so far regarding interactions of Cdc37 with kinases that play a role in cell division. Here we show that the loss of function of Cdc37 in Drosophila leads to defects in mitosis and male meiosis, and that these phenotypes closely resemble those brought about by the inactivation of Aurora B. We provide evidence that Aurora B interacts with and requires the Cdc37/Hsp90 complex for its stability. We conclude that the Cdc37/Hsp90 complex modulates the function of Aurora B and that most of the phenotypes brought about by the loss of Cdc37 function can be explained by the inactivation of this kinase. These observations substantiate the role of Cdc37 as an upstream regulatory element of key cell cycle kinases.     

Deregulated human Cdc14A phosphatase disrupts centrosome separation and chromosome segregation

We show that human Cdc14A phosphatase interacts with interphase centrosomes, and that this interaction is independent of microtubules and Cdc14A phosphatase activity, but requires active nuclear export. Disrupting the nuclear export signal (NES) led to Cdc14A being localized in nucleoli, which in unperturbed cells selectively contain Cdc14B (ref. 1). Conditional overproduction of Cdc14A, but not its phosphatase-dead or NES-deficient mutants, or Cdc14B, resulted in premature centrosome splitting and formation of supernumerary mitotic spindles. In contrast, downregulation of endogenous Cdc14A by short inhibitory RNA duplexes (siRNA) induced mitotic defects including impaired centrosome separation and failure to undergo productive cytokinesis. Consequently, both overexpression and downregulation of Cdc14A caused aberrant chromosome partitioning into daughter cells. These results indicate that Cdc14A is a physiological regulator of the centrosome duplication cycle, which, when disrupted, can lead to genomic instability in mammalian cells.     

Cdc48/p97 and Shp1/p47 regulate autophagosome biogenesis in concert with ubiquitin-like Atg8

The molecular details of the biogenesis of double-membraned autophagosomes are poorly understood. We identify the Saccharomyces cerevisiae AAA–adenosine triphosphatase Cdc48 and its substrate-recruiting cofactor Shp1/Ubx1 as novel components needed for autophagosome biogenesis. In mammals, the Cdc48 homologue p97/VCP and the Shp1 homologue p47 mediate Golgi reassembly by extracting an unknown monoubiquitinated fusion regulator from a complex. We find no requirement of ubiquitination or the proteasome system for autophagosome biogenesis but detect interaction of Shp1 with the ubiquitin-fold autophagy protein Atg8. Atg8 coupled to phosphatidylethanolamine (PE) is crucial for autophagosome elongation and, in vitro, mediates tethering and hemifusion. Interaction with Shp1 requires an FK motif within the N-terminal non–ubiquitin-like Atg8 domain. Based on our data, we speculate that autophagosome formation, in contrast to Golgi reassembly, requires a complex in which Atg8 functionally substitutes ubiquitin. This, for the first time, would give a rationale for use of the ubiquitin-like Atg8 during macroautophagy and would explain why Atg8-PE delipidation is necessary for efficient macroautophagy.     

Transcription factors interact with RNA to regulate genes

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Three eukaryote-like Orc1/Cdc6 proteins functionally interact and mutually regulate their activities of binding to the replication origin in the hyperthermophilic archaeon Sulfolobus solfataricus P2

The crenarchaeon Sulfolobus solfataricus has the potential to be a powerful model system to understand the central mechanism of eukaryotic DNA replication because it contains three active origins of replication and three eukaryote-like Orc1/Cdc6 proteins. However, it is not known whether these SsoCdc6 proteins can functionally interact and collectively contribute to DNA replication initiation. In the current work, we found that SsoCdc6-1 stimulates DNA-binding activities of SsoCdc6-3. In contrast, SsoCdc6-3 inhibits those of both SsoCdc6-1 and SsoCdc6-2. These regulatory functions are differentially affected by the C-terminal domains of these SsoCdc6 proteins. These data, in conjunction with studies on physical interactions between these replication initiators by bacterial two-hybrid and pull-down/Western blot assays, lead us to propose the possibility that multiple SsoCdc6 proteins might coordinately regulate DNA replication in the archaeon species. This is the first report on the functional interaction among the archaeal multiple Cdc6 proteins to regulate DNA replication.     

Tint maps to mouse chromosome 6 and may interact with a notochordal enhancer of Brachyury

At the proximal part of mouse chromosome 17 there are three well-defined genes affecting the axis of the embryo and consequently tail length: Brachyury, Brachyury the second, and the t-complex tail interaction (T1, T2, and tct). The existence of T1 and tct in fact defines the classical "t-complex" that occupies approximately 40 cM of mouse chromosome 17. Their relationship to each other and various unlinked interacting genes has been enigmatic. The tint gene was the first of the latter to be identified. We report here its genetic mapping using a microsatellite scan together with outcrosses to Mus spretus and M. castaneous followed by a subsequent testcross to T, T1, and T2 mutants. Surprisingly, tint interacts with T2 but not with T1. The implications of our data suggest that T2 may be part of the T1 regulatory region through direct or indirect participation of tint.     

Phosphorylation of histone H3 is required for proper chromosome condensation and segregation

Phosphorylation of histone H3 at serine 10 occurs during mitosis in diverse eukaryotes and correlates closely with mitotic and meiotic chromosome condensation. To better understand the function of H3 phosphorylation in vivo, we created strains of Tetrahymena in which a mutant H3 gene (S10A) was the only gene encoding the major H3 protein. Although both micronuclei and macronuclei contain H3 in typical nucleosomal structures, defects in nuclear divisions were restricted to mitotically dividing micronuclei; macronuclei, which are amitotic, showed no defects. Strains lacking phosphorylated H3 showed abnormal chromosome segregation, resulting in extensive chromosome loss during mitosis. During meiosis, micronuclei underwent abnormal chromosome condensation and failed to faithfully transmit chromosomes. These results demonstrate that H3 serine 10 phosphorylation is causally linked to chromosome condensation and segregation in vivo and is required for proper chromosome dynamics.     

Cdc13 cooperates with the yeast Ku proteins and Stn1 to regulate telomerase recruitment

The Saccharomyces cerevisiae CDC13 protein binds single-strand telomeric DNA. Here we report the isolation of new mutant alleles of CDC13 that confer either abnormal telomere lengthening or telomere shortening. This deregulation not only depended on telomerase (Est2/TLC1) and Est1, a direct regulator of telomerase, but also on the yeast Ku proteins, yKu70/Hdf1 and yKu80/Hdf2, that have been previously implicated in DNA repair and telomere maintenance. Expression of a Cdc13-yKu70 fusion protein resulted in telomere elongation, similar to that produced by a Cdc13-Est1 fusion, thus suggesting that yKu70 might promote Cdc13-mediated telomerase recruitment. We also demonstrate that Stn1 is an inhibitor of telomerase recruitment by Cdc13, based both on STN1 overexpression and Cdc13-Stn1 fusion experiments. We propose that accurate regulation of telomerase recruitment by Cdc13 results from a coordinated balance between positive control by yKu70 and negative control by Stn1. Our results represent the first evidence of a direct control of the telomerase-loading function of Cdc13 by a double-strand telomeric DNA-binding complex.     

The human Mis12 complex is required for kinetochore assembly and proper chromosome segregation

During cell division, kinetochores form the primary chromosomal attachment sites for spindle microtubules. We previously identified a network of 10 interacting kinetochore proteins conserved between Caenorhabditis elegans and humans. In this study, we investigate three proteins in the human network (hDsn1Q9H410, hNnf1PMF1, and hNsl1DC31). Using coexpression in bacteria and fractionation of mitotic extracts, we demonstrate that these proteins form a stable complex with the conserved kinetochore component hMis12. Human or chicken cells depleted of Mis12 complex subunits are delayed in mitosis with misaligned chromosomes and defects in chromosome biorientation. Aligned chromosomes exhibited reduced centromere stretch and diminished kinetochore microtubule bundles. Consistent with this, localization of the outer plate constituent Ndc80HEC1 was severely reduced. The checkpoint protein BubR1, the fibrous corona component centromere protein (CENP) E, and the inner kinetochore proteins CENP-A and CENP-H also failed to accumulate to wild-type levels in depleted cells. These results indicate that a four-subunit Mis12 complex plays an essential role in chromosome segregation in vertebrates and contributes to mitotic kinetochore assembly.