Insulin signaling in the yeast Saccharomyces cerevisiae. 2. Interaction of human insulin with a putative binding protein.
A putative insulin-binding protein (Kd = 0.5 +/- 0.2 microM for human insulin) was partially purified from solubilized plasma membranes of Saccharomyces cerevisiaeby wheat germ agglutinin and insulin affinity chromatographies. The binding affinities of various mutant insulin analogues correlated well with their capacities to activate glycogen synthase and SNF1 kinase in glucose-induced yeast spheroplasts, the ranking of their relative efficacies in yeast and in isolated rat adipocytes being similar. Using a bifunctional cross-linker and two different experimental protocols, a 53-kDa polypeptide contained in the insulin-binding protein preparation was specifically affinity cross-linked to [125I]monoiodo[B26]insulin. The relative rankings of the insulin analogues with respect to inhibition of cross-linking and binding to the partially purified insulin-binding protein were identical. Incubation of intact yeast spheroplasts with [125I]monoiodo[AI4]insulin led to specific and time-dependent association of the radiolabeled insulin with the cell surface followed by its internalization and degradation. These processes were considerably delayed by low temperature and energy depletion of the spheroplasts, suggesting involvement of the ATP-dependent endosomal apparatus. These data provide evidence for the existence of a low-affinity insulin-binding protein in the plasma membrane of Saccharomyces cerevisiae.
Müller
G
G
Hoechst Marion Roussel Deutschland GmbH, D-65926 Frankfurt am Main, Germany. Guenter.Mueller@hmrag.com
Rouveyre
N
N
Upshon
C
C
Grobeta
E
E
Bandlow
W
W
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D006801:Humans; D007328:Insulin / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D009154:Mutation; D011485:Protein Binding; D051381:Rats; D011972:Receptor, Insulin / Q000302:isolation & purification* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
D007328:Insulin; D011972:Receptor, Insulin