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  • 1
    Electronic Resource
    Electronic Resource
    Oxford BSL : Blackwell Science Ltd, UK
    Molecular microbiology 29 (1998), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Profilin-deficient Saccharomyces cerevisiae cells show abnormal growth, actin localization, chitin deposition, bud formation and cytokinesis. Previous studies have also revealed a synthetic lethality between pfy1 and late secretory mutants, suggesting a role for profilin in intracellular transport. In this work, we document further the secretion defect associated with the pfy1Δ mutant. Electron microscopic observations reveal an accumulation of glycoproteins in the bud and in the mother cell. The MATa, pfy1Δ cells mate as well as wild-type cells, while the mating efficiency of MATα, pfy1Δ cells is reduced. Pulse-chase experiments demonstrate an accumulation of the 19 kDa α-factor precursor and delayed secretion of the mature α-factor. The TGN protein Kex2p is the principal enzyme responsible for the endoproteolytic cleavage of the α-factor precursor. An immunofluorescence detection of Kex2p shows an altered localization in pfy1Δ cells. Instead of a discrete punctate distribution, the enzyme is dispersed throughout the cytoplasm. A high-copy-number plasmid containing MID2, which encodes a potential transmembrane protein involved in cell cycle control, suppresses the abnormal growth, actin distribution, α-factor maturation and the accumulation of intracellular membranous structures in pfy1Δ cells.
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  • 2
    ISSN: 1615-6102
    Keywords: Actin ; Cytoskeleton ; Physarum polycephalum ; Profilin ; Yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Profilin is a ubiquitous actin-monomer-binding protein. The protistPhysarum polycephalum contains two profilins, ProA and ProP, present in amoebae and plasmodia, respectively. We have used mutantSaccharomyces cerevisiae cells in an attempt to observe distinct functions for the two profilins. Profilin-deficient yeast cells (Δpfy1) have delocalized actin cortical patches, do not contain visible actin cables, have reduced mating efficiency and do not grow at 37 °C or in the presence of caffeine. Deletion of theSRV2 gene (Δsrv2), coding for the adenylyl cyclase-associated protein, also results in an altered actin distribution and an inability to survive on rich medium. We found that the Δpfy1 and Δsrv2 mutant phenotypes were corrected equally well by the overexpression of Physarum ProA or yeast Pfy1p profilins. The Δpfy1 cells overexpressing ProP have improved mating efficiency and a normal distribution of actin cortical patches. These cells, however, have barely detectable actin cables, do not grow at 37 °C, and are sensitive to caffeine. Also, the expression of ProP does not correct the growth defect of the Δsrv2 cells. These results suggest that the two Physarum proteins are not functionally equivalent in yeast cells. No difference was detected in the affinity of ProA and ProP for poly-L-proline, while ProA has a slightly greater affinity than ProP for phosphatidylinositol 4,5-biphosphate.
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  • 3
    Publication Date: 2008-11-16
    Description: Congenital dyserythropoietic anemia (CDA) type I is an inherited autosomal recessive macrocytic anemia associated with ineffective erythropoiesis and the development of secondary hemochromatosis. Distinct erythroid precursors with inter-nuclear chromatin bridges and spongy heterochromatin are pathognomonic for the disease. The mutated gene (CDAN1) encodes a ubiquitously expressed protein of unknown function, codanin-1. Based on the morphological features of CDA type I erythroblasts and the preliminary data on the Drosophila homolog, dlt, which was found to be required for cell survival and cell cycle progression, we investigated the location and the behavior of codanin-1 during the cell cycle. Using immunofluorescence and immune electron microscopy, we localized codanin-1 to the heterochromatin in interphase cells. During the cell cycle, high levels of codanin-1 were observed in S phase. At mitosis, codanin-1 underwent phosphorylation, which coincided with exclusion from condensed chromosomes. The proximal CDAN1 gene promoter region, never before characterized, was found to contain 5 putative E2F1 binding sites. E2F transcription factors are the main regulators of G1/S transition. Cotransfection of an E2F1 expression plasmid increased luciferase activity, confirming that E2F1 activates the transcription of CDAN1, and chromatin immunoprecipitation identified the codanin-1 promoter as a direct target of E2F1. Taken together, these data suggest that codanin-1 is a cell cycle-regulated protein active in S-phase. Based on the localization of codanin-1 to the heterochromatin and the spongy appearance of heterochromatin in CDA I, we suggest that codanin-1 may be involved in heterochromatin organization during DNA replication. This represents the first work towards understanding the function of the proteins involved in CDAs.
    Print ISSN: 0006-4971
    Electronic ISSN: 1528-0020
    Topics: Biology , Medicine
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  • 4
    Publication Date: 2010-11-19
    Description: Abstract 1004 Congenital dyserythropoietic anemia type I (CDA1) is an inherited recessive macrocytic anemia associated with ineffective erythropoiesis. The disorder is characterized by the accumulation of erythroid precursors containing spongy heterochromatin and internuclear chromatin bridges. The mutated gene (CDAN1) encodes an ubiquitously expressed protein (codanin-1) of unknown function. We have previously shown that codanin-1 is a direct transcriptional target of the E2F1 transcription factor and that the levels of codanin-1 increase during S-phase and decrease during mitosis. In an attempt to further define the role of codanin-1, we conducted a yeast two-hybrid screen using a human bone marrow library and found that codanin-1 binds to Asf1a. Asf1 (anti silencing function) is a H3/H4 histone chaperone involved in the chromatin structure dynamics by its role in nucleosome assembly and disassembly. Using coimmunoprecipitation experiments we confirmed that histone chaperone Asf1a is a direct binding partner of codanin-1. Minimal 100 amino acids domain of codanin-1, involved in binding Asf1, was identified and defined as the Asf1-binding domain. We found that codanin-1 binds to the conserved N-terminal core of Asf1a, where histones and other histone chaperones also bind. FLAG-tagged codanin-1 or its Asf1-binding domain immunoprecipitated from transfected Hela cells and subsequently coimmunoprecipitated histone H3 and Asf1a simultaneously. A pull-down assay of purified Asf1-binding domain in the presence of core histones showed, however, no direct binding of this domain of codanin-1 to H3/H4 histones. By using the replication-independent nucleosome formation assay we noticed that the nucleosome assembly activity of GST-Asf1a was severely decreased by the addition of the purified Asf1-binding domain of codanin-1, a phenotype similar to the one observed in Asf1 depletion. One possible explanation is that binding of codanin-1 inhibits dissociation of histones from Asf1a, which therefore cannot be deposited onto DNA. It will be of interest to determine if codanin-1 is involved in modulating Asf1a activity in vivo and also in response to DNA replication or damage and to determine its role in erythroid heterochromatin formation. Disclosures: No relevant conflicts of interest to declare.
    Print ISSN: 0006-4971
    Electronic ISSN: 1528-0020
    Topics: Biology , Medicine
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  • 5
    Publication Date: 2005-11-01
    Print ISSN: 0898-6568
    Electronic ISSN: 1873-3913
    Topics: Biology , Medicine
    Published by Elsevier
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