KDEL tagging: a method for generating dominant-negative inhibitors of the secretion of TGF-beta superfamily proteins.
Most endoplasmic reticulum (ER)-retained proteins contain a carboxy-terminal signal sequence called the ER retention signal motif such as the Lys-Asp-Glu-Leu (KDEL) motif. Using this molecular mechanism, we developed a new dominant-negative assay, designated the KDEL-tag trap assay, to negatively regulate secretion of disulfide bond-dependent protein dimers, as typified by TGF-beta superfamily proteins. First, we tested this method on the Nodal protein Xnr5, which is a well-studied mesoderm inducer in vertebrates. Tagging of Xnr5 protein with KDEL at the carboxy-terminus effectively blocked the secretion of Xnr5, resulting in complete inhibition of mesoderm induction in Xenopus embryogenesis. Second, we examined the usefulness of the KDEL-tag trap assay on BMPs, which are well-known negative regulators of neural induction and ventralizing factors during early development, and demonstrated that the functions of the BMP family proteins BMP4 and ADMP were blocked by the KDEL-tag trap assay. Moreover, the technical feasibility of the KDEL-tag trap assay was confirmed in a cell culture system using mouse osteoblasts. Taken together, these results suggest that the KDEL-tag trap assay can be adapted to inhibit a variety of plasma membrane or secreted proteins of a multimeric nature.
Department of Education (Sciences), Shizuoka University, Suruga-ku, Shizuoka, Japan.
Shinya
S
Matsukawa
Yuki
Y
Moriyama
Tadayoshi
T
Hayata
Haruka
H
Sasaki
Yuzuru
Y
Ito
Makoto
M
Asashima
Hiroki
H
Kuroda
D000818:Animals; D001692:Biological Transport; D002478:Cells, Cultured; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005799:Genes, Dominant; D051379:Mice; D018832:Molecular Chaperones; D009154:Mutation / Q000235:genetics; D055457:Nodal Protein / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism*; D010957:Plasmids; D021382:Protein Sorting Signals; D055411:TGF-beta Superfamily Proteins / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis / Q000378:metabolism
D018832:Molecular Chaperones; D055457:Nodal Protein; D009842:Oligopeptides; D021382:Protein Sorting Signals; D055411:TGF-beta Superfamily Proteins; C073762:lysyl-aspartyl-glutamyl-leucine