Post-Translational Targeting of a Tail-Anchored Green Fluorescent Protein to the Endolpasmic Reticulum

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  • Masaki Ryuichi
    Department of Physiology, and Division of Cell Biology, Liver Research Center, Kansai Medical University
  • Kameyama Keiichi
    Department of Biomolecular Science, Faculty of Engineering, Gifu University
  • Yamamoto Akitsugu
    Department of Physiology, and Division of Cell Biology, Liver Research Center, Kansai Medical University Department of Bio-Science, Nagahama Institute of Bio-Science and Technology

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Abstract

Microsomal aldehyde dehydrogenase (msALDH) is a tail-anchored protein localized to the cytoplasmic face of the endoplasmic reticulum (ER). The carboxyl-terminal 35 amino acids of msALDH possess ER-targeting sequences in addition to a hydrophobic membrane-spanning domain. To study the mechanism for ER targeting of this protein in vivo, we took advantage of a green fluorescent protein-msALDH fusion protein containing the last 35 amino acids of msALDH [GFPALDH(35)]. When expressed from cDNA in COS-7 cells, the fusion protein was localized to the ER. We then prepared a recombinant fusion protein and injected it into the cytoplasm of COS-7 cells. The injected protein was correctly localized to the ER after a 30-min incubation at 37°C. However, a recombinant fusion protein that contained only the transmembrane domain of msALDH failed to be targeted to the ER. When the assay was carried out at 4°C, the recombinant GFPALDH (35) remained in the cytoplasm. Moreover, incubation of COS-7 cells under conditions of ATP depletion resulted in the cytoplasmic distribution of the injected protein. These results indicate that GFPALDH (35) is targeted to the ER post-translationally via an ATP-dependent pathway. This microinjection system worked effectively in different mammalian cell types, suggesting a common mechanism for ER targeting of the tail-anchored protein.

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