Stable plasma membrane expression of the soluble domain of the human insulin receptor in yeast

Abstract

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. ß

The post-genomic era of interactive proteomics: Facts and perspectives

“…In the SRS, the translocation of hSos is dependent on a protein-protein interaction: the bait X is fused to C-terminally truncated hSOS, which is active but unable to target the plasma membrane. The bait is co-expressed with a prey Y, which can be either an integral membrane protein or a soluble protein that is anchored to the membrane by means of a myristoylation signal [38]. If X and Y interact, the hSos fusion is recruited to the plasma membrane and substitutes for the defective cdc25-2 allele (Fig.…”

“…The integral membrane protein anchors the cassette in the lipid bilayer and prevents its diffusion to the nucleus. If a cytoplasmic or a nuclear protein is selected as bait, a sequence motif that confers fatty acid modification can be used to attach the bait to the membrane [38,65]. The interacting partner Y, which can be either an integral membrane protein or a cytoplasmic protein (Fig.…”

Insulin or insulin-like studies on unicellular organisms: a review

“…Aspects of inter-and intracellular signaling systems in cell survival, proliferation, differentiation, chemosensory behaviour, and programmed cell death in free-living unicellular organisms have been observed in S. cerevisiae and Tetrahymena by Christensen et al (1998a). Angermayr et al (2000) suggested a failure of the active insulin receptor kinase domain in coupling to yeast (glucose) signaling cascades. Intracellular systems present many common features in uniand multicellular organisms, like the physiological responses obtained in studies using the yeast Schizosaccharomyces pombe that provide evidence for a insulin signaling pathway (Buckley et al, 2002).…”

“…They asserted that this protein could be the "receptor" that mediates insulin-induced downstream metabolic effects. Angermayr et al (2000) described the stable expression of the complete cytoplasmic protein fragment of hInsR including the juxtamembrane domain in yeast cells, with an apparent molecular mass of 48 kDa, and autophosphorylation capacity. They found that this autophosphorylation occured in tyrosine residues and that the tyrosine kinase activity did not interfere with glucose signalling in yeast.…”

Screening for Insulin-Like/Mimetic Drugs Using Lower Eukaryotes

“…Alternatively, the assays for human receptor activation may be based on signal transduction pathways activated by receptor tyrosine kinase activity: I. The soluble domain of the human insulin receptor can be stably expressed at the plasma membranes of Saccharomyces cerevisiae [42]. II.Tyrosine phosphorylation of some yeast proteins is increased upon incubation of intact cells or spheroblasts with human insulin [43].…”