Fig 1.
Decreased intracellular inositol levels in mck1Δ cells.
(A) Cells were grown in SC medium to the early stationary phase. Intracellular inositol levels were determined as described in “Experimental procedures” (3 independent experiments with 3 replicates each). Values shown are mean ± SEM. (B) Inositol restored the growth of mck1Δ cells at elevated temperature. WT and mck1Δ cells were diluted and plated on SC plates in the presence or absence of 75 M inositol and incubated at indicated temperatures for 2 days.
Fig 2.
Rate of synthesis of inositol-3-phosphate and inositol is decreased in mck1Δ cells.
Cells were cultured in SC medium supplemented with 75 M inositol to the mid log phase and transferred to SC inositol free medium for 3 h. [13C6]glucose was added at a final concentration of 0.2% and cells were incubated for 15 min. Levels of 13C labeled inositol-3-phosphate (A) and inositol (B) in cell extracts were determined by LC-MS (6 independent experiments). Values shown are mean ± SEM.
Fig 3.
MIPS activity is decreased in mck1Δ cells.
(A) Strains expressing His-Xpress tagged MIPS were constructed by knocking out the INO1 gene with the KanMX gene, which is then replaced with the His-Xpress INO1 fusion gene. (B) Cells expressing the His-Xpress tagged MIPS were grown in SC medium to the early stationary phase. Enzymatic activity of MIPS protein purified from cell extracts was determined as described in “Experimental procedures” (2 independent experiments with 3 replicates each). Values shown are mean ± SEM. (C) MIPS protein levels are not changed in gsk3 mutant cells. 30 g protein from total cell extract were resolved with 10% SDS-PAGE and analyzed by Western blot.
Fig 4.
VPA does not decrease MIPS activity in mck1Δ cells.
Cells expressing His-Xpress tagged MIPS were grown in SC medium to the mid log phase and treated with 1 mM VPA for 3 h. MIPS activities were determined as described in “Experimental procedures” (3 independent experiments with 3 replicates each) (*p<0.05). Values shown are mean ± SEM. Statistical significance was determined by unpaired t test.
Fig 5.
Model of VPA-induced inositol depletion via inhibition of Mck1.
MIPS is the rate-limiting enzyme of inositol synthesis. Mck1 is a positive regulator of MIPS that is required for optimal de novo synthesis of inositol. Positive regulation of MIPS by Mck1 is inhibited by VPA, thus accounting for VPA-induced inhibition of MIPS and concomitant decrease in inositol synthesis. Inositol depletion causes a plethora of cellular consequences, including those that contribute to mood-stabilization.