Effects of human insulin on glucose metabolism in the yeast Saccharomyces cerevisiae were studied in this report. Under two conditions of growth limitation (glucose-grown cells during transition to stationary phase or spheroplasts during incubation in synthetic glucose medium), human insulin (10 and 1 microM, respectively) enhanced glycogen accumulation and glycogen synthase activity by 40-60% compared to control cells. Glycogen phosphorylase activity was also increased under the same conditions, but this stimulation was diminished by 35-45% in insulin-treated compared to control cells. Thus, under growth limitation, insulin causes glycogen phosphorylase and glycogen synthase to become more sensitive to inactivation and activation, respectively. In glucose-induced spheroplasts, insulin (1 microM), in addition to glycogen accumulation, led to about 2-fold increases of the rates of ethanol production and glucose oxidation compared to control cells, and the maximal concentration of hexose 6-phosphate was increased by 30-40%. In contrast, glucose transport as well as the levels of the allosteric regulators, fructose 2,6-bisphosphate and cAMP, were not altered at all. Snf1 kinase is assumed to be involved in the regulation of glycogen metabolism in yeast, although it does not seem to be modulated directly by the glucose concentration. Snf1 kinase activity was elevated 5-10-fold in response to insulin both during glucose induction of yeast spheroplasts and during transition to stationary phase of glucose-grown cells. We conclude that Saccharomyces cerevisiae and insulin-sensitive mammalian cells share some parts of the signaling cascades regulating oxidative and nonoxidative glucose metabolism in response to glucose and insulin.
A low affinity insulin-binding protein in the plasma membrane of Saccharomyces cerevisiae has been identified recently (Müller, G., Rouveyre, N., Upshon, C., Gross, E., and Bandlow, W., preceding paper in this issue). Since the mammalian insulin receptor functions as a tyrosine kinase with autophosphorylation capacity, kinase studies were performed with the partially purified insulin-binding protein preparation. Incubation with [gamma-32P]ATP in vitro led to phosphorylation of the 53-kDa insulin-binding protein on serine but not on tyrosine residues. In addition, a 70-kDa polypeptide, copurified with the insulin-binding protein preparation, was tyrosine-phosphorylated under the same conditions. Phosphorylation of both proteins was enhanced by human insulin. These results obtained by immunoprecipitation and immunoblotting using specific anti-phosphoserine/threonine/tyrosine antibodies were confirmed by phosphoamino acid analysis of the individual immunoprecipitated and gel-purified 32P-labeled phosphoproteins. During gel filtration, the 53-kDa protein coeluted as a 300-kDa complex together with the 70-kDa phosphotyrosine-containing protein and was coimmunoprecipitated with the latter using an anti-phosphotyrosine antibody, strongly arguing for complex formation between the two proteins. The data presented raise the possibility that stimulation of glycogen synthesis by insulin in yeast is mediated by a 53-kDa insulin-binding protein and a 70-kDa phosphotyrosine-containing protein which are organized in a large plasma membrane-bound signaling complex. Elucidation of the function and molecular mode of interaction of these components in yeast may help to understand metabolic insulin signaling in mammalian cells.
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.
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