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Genes Cells ; 24(11): 731-745, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31554018


Cluap1/IFT38 is a ciliary protein that belongs to the IFT-B complex and is required for ciliogenesis. In this study, we have examined the behaviors of Cluap1 protein in nonciliated and ciliated cells. In proliferating cells, Cluap1 is located at the distal appendage of the mother centriole. When cells are induced to form cilia, Cluap1 is found in a novel noncentriolar compartment, the cytoplasmic IFT spot, which mainly exists once in a cell. Other IFT-B proteins such as IFT46 and IFT88 are colocalized in this spot. The cytoplasmic IFT spot is present in mouse embryonic fibroblasts (MEFs) but is absent in ciliogenesis-defective MEFs lacking Cluap1, Kif3a or Odf2. The cytoplasmic IFT spot is also found in mouse embryos but is absent in the Cluap1 mutant embryo. When MEFs are induced to form cilia, the cytoplasmic IFT spot appears at an early step of ciliogenesis but starts to disappear when ciliogenesis is mostly completed. These results suggest that IFT-B proteins such as Cluap1 accumulate in a previously undescribed cytoplasmic compartment during ciliogenesis.

Cílios/metabolismo , Citoplasma/metabolismo , Proteínas do Citoesqueleto/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Animais , Cílios/ultraestrutura , Citoplasma/ultraestrutura , Fibroblastos , Proteínas de Choque Térmico , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cinesina , Camundongos , Camundongos Knockout , Proteínas Supressoras de Tumor
FEBS Lett ; 589(15): 1778-86, 2015 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-25979175


C-Glucosyltransferase is an enzyme that mediates carbon-carbon bond formation to generate C-glucoside metabolites. Although it has been identified in several plant species, the catalytic amino acid residues required for C-glucosylation activity remain obscure. Here, we identified a 2-hydroxyflavanone C-glucosyltransferase (UGT708D1) in soybean. We found that three residues, His20, Asp85, and Arg292, of UGT708D1 were located at the predicted active site and evolutionarily conserved. The substitution of Asp85 or Arg292 with alanine destroyed C-glucosyltransferase activity, whereas the substitution of His20 with alanine abolished C-glucosyltransferase activity but enabled O-glucosyltransferase activity. The catalytic mechanism is discussed on the basis of the findings.

Glucosiltransferases/metabolismo , Soja/enzimologia , Sequência de Aminoácidos , Catálise , Domínio Catalítico , Cromatografia Líquida de Alta Pressão , Glucosiltransferases/química , Glucosiltransferases/classificação , Espectrometria de Massas , Dados de Sequência Molecular , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos
J Cell Biol ; 204(2): 203-13, 2014 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-24421334


Axonemal dynein complexes are preassembled in the cytoplasm before their transport to cilia, but the mechanism of this process remains unclear. We now show that mice lacking Pih1d3, a PIH1 domain-containing protein, develop normally but manifest male sterility. Pih1d3(-/-) sperm were immotile and fragile, with the axoneme of the flagellum lacking outer dynein arms (ODAs) and inner dynein arms (IDAs) and showing a disturbed 9+2 microtubule organization. Pih1d3 was expressed specifically in spermatogenic cells, with the mRNA being most abundant in pachytene spermatocytes. Pih1d3 localized to the cytoplasm of spermatogenic cells but was not detected in spermatids or mature sperm. The levels of ODA and IDA proteins were reduced in the mutant testis and sperm, and Pih1d3 was found to interact with an intermediate chain of ODA as well as with Hsp70 and Hsp90. Our results suggest that Pih1d3 contributes to cytoplasmic preassembly of dynein complexes in spermatogenic cells by stabilizing and promoting complex formation by ODA and IDA proteins.

Proteínas Reguladoras de Apoptose/fisiologia , Dineínas do Axonema/metabolismo , Espermatozoides/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Axonema/metabolismo , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Fertilidade/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , RNA Mensageiro/metabolismo , Espermatozoides/ultraestrutura , Testículo/metabolismo