Post-translational targeting of a tail-anchored green fluorescent protein to the endolpasmic reticulum.
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 degrees 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 degrees 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.
Masaki
Ryuichi
R
Department of Physiology, and Division of Cell Biology, Liver Research Center, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi, Osaka 570-8506. masaki@takii.kmu.ac.jp
Kameyama
Keiichi
K
Yamamoto
Akitsugu
A
D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008845:Microinjections; D016253:Microscopy, Immunoelectron; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013696:Temperature
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate