Journal
BIOCHEMICAL JOURNAL
Volume 364, Issue -, Pages 669-677Publisher
PORTLAND PRESS
DOI: 10.1042/BJ20011836
Keywords
dynein; embryo implantation; microtubule-associated protein; trophinin
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Funding
- NICHD NIH HHS [R01 HD34108] Funding Source: Medline
- NIGMS NIH HHS [R01 GM55147] Funding Source: Medline
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Tastin was originally identified as an accessory protein for trophinin, a cell adhesion molecule that potentially mediates the initial attachment of the human embryo to the uterine epithelium. However, no information regarding tastin's function is available to date. The present study is aimed at understanding the role of tastin in mammalian cells. Hence, we examined the intracellular localization of tastin in human cell lines transfected with an expression vector encoding influenza virus haemagglutinin (HA)tagged tastin. Ectopically expressed HA-tastin was seen as a pattern resembling the fibres that overlap the microtubular cytoskeleton. When HA-tastin-expressing cells were cultured with nocodazole to disrupt microtubule (MT) polymerization, tastin was dispersed to the entire cytoplasm and an MT sedimentation assay showed tastin in the supernatant; however, tastin was sedimented with polymeric MTs in cell lysates not treated with nocodazole. Sedimentation assays using HA-tastin mutants deleted at the N- or C-terminus revealed MT-binding activity associated with the N-terminal basic region of tastin. A yeast two-hybrid screen for tastin-interacting proteins identified Tctex-1, one of the light chains of cytoplasmic dynein, as a tastin-binding protein. Immunoprecipitation and Western-blot analysis confirmed binding of HA-tagged tastin and FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp- Lys epitope)-tagged Tctex-1 in human cells. Furthermore. in vitro assays have demonstrated the binding between a fusion protein. glutathione S-transferase-Tctex-1, and in vitro translated S-35-labelled tastin. As Tctex-1 is a component of a MT-based molecular motor, these results suggest that tastin plays an important role in mammalian cells by associating with the microtubular cytoskeleton.
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