Co-culturing experiments reveal the uptake of myo-inositol phosphate synthase (EC 5.5.1.4) in an inositol auxotroph of Saccharomyces cerevisiae

Microbial Cell Factories

Hana D. Alebous¹  Macy Vickers²  Margaret D. Johnson²  Mary E. Harris²  Erika Steele³ 

[1] Department of Biological Sciences, School of Science, The University of Jordan, PO Box 11942, Amman-Jordan, Jordan;Department of Biological Sciences, The University of Alabama, PO Box 870344, 35487, Tuscaloosa, AL, USA;The University of Alabama, The Institute of Social Science Research, PO Box 8702161, 35487, Tuscaloosa, AL, USA;

关键词: Inositol auxotroph;    Saccharomyces cerevisiae;    Myo-inositol Phosphate Synthase (MIP);    Protein secretion;    Cellular supernatant;    Extracellular vesicle;    Co-culturing;   

DOI  :  10.1186/s12934-021-01610-6

来源: Springer

【 摘 要 】

BackgroundMyo-Inositol Phosphate Synthase (MIP) catalyzes the conversion of glucose 6- phosphate into inositol phosphate, an essential nutrient and cell signaling molecule. Data obtained, first in bovine brain and later in plants, established MIP expression in organelles and in extracellular environments. A physiological role for secreted MIP has remained elusive since its first detection in intercellular space. To provide further insight into the role of MIP in intercellular milieus, we tested the hypothesis that MIP may function as a growth factor, synthesizing inositol phosphate in intercellular locations requiring, but lacking ability to produce or transport adequate quantities of the cell–cell communicator. This idea was experimentally challenged, utilizing a Saccharomyces cerevisiae inositol auxotroph with no MIP enzyme, permeable membranes with a 0.4 µm pore size, and cellular supernatants as external sources of inositol isolated from S. cerevisiae cells containing either wild-type enzyme (Wt-MIP), no MIP enzyme, auxotroph (Aux), or a green fluorescent protein (GFP) tagged reporter enzyme (MIP- GFP) in co- culturing experiments.ResultsResulting cell densities and microscopic studies with corroborating biochemical and molecular analyses, documented sustained growth of Aux cells in cellular supernatant, concomitant with the uptakeof MIP, detected as MIP-GFP reporter enzyme. These findings revealed previously unknown functions, suggesting that the enzyme can: (1) move into and out of intercellular space, (2) traverse cell walls, and (3) act as a growth factor to promote cellular proliferation of an inositol requiring cell.ConclusionsCo-culturing experiments, designed to test a probable function for MIP secreted in extracellular vesicles, uncovered previously unknown functions for the enzyme and advanced current knowledge concerning spatial control of inositol phosphate biosynthesis. Most importantly, resulting data identified an extracellular vesicle (a non-viral vector) that is capable of synthesizing and transporting inositol phosphate, a biological activity that can be used to enhance specificity of current inositol phosphate therapeutics.

【 授权许可】

CC BY