Stable plasma membrane expression of the soluble domain of the human insulin receptor in yeast.
The soluble cytoplasmic kinase domain of the human insulin receptor was N-terminally equipped with either an N-acetylation or a dual-acylation motif (MGC box, to allow myristoylation/palmitoylation) and expressed in yeast cells under the control of the inducible CUP1 promoter. Although the cellular concentration was about the same in both instances (reflecting similar stability against proteolysis), only the myristoylated protein was capable of autophosphorylation to a significant extent and was active to phosphorylate endogenous yeast proteins at tyrosine residues in vivo. Cellular subfractionation showed that the insulin receptor was associated with plasma membranes, from where it was not extractable with high salt or alkali, but a significant fraction was also localized in the nuclear fraction. The myristoylated protein is absent from the cytoplasm. No effect of expression of either the acetylated or the myristoylated version on growth and respiration on various carbon sources was detected, suggesting a failure of the active insulin receptor kinase domain to couple to yeast (glucose) signalling cascades.
Angermayr
M
M
Institut für Genetik und Mikrobiologie, Maria-Ward-Strasse 1a, Munich, Germany.
Strobel
G
G
Müller
G
G
Bandlow
W
W
D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D006801:Humans; D008668:Metallothionein / Q000235:genetics; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D017434:Protein Structure, Tertiary; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011972:Receptor, Insulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D015003:Yeasts / Q000235:genetics*
C007121:copper thionein; D019000:Phosphotyrosine; D008668:Metallothionein; D011972:Receptor, Insulin; D017027:Protein Tyrosine Phosphatases