Lost in Transition: Start-Up of
Glycolysis Yields Subpopulations
of Nongrowing Cells
Johan H. van Heerden, Meike T. Wortel, Frank J. Bruggeman, Joseph J. Heijnen, Yves J. M. Bollen,
Robert Planqué, Josephus Hulshof, Tom G. O’Toole, S. Aljoscha Wahl, Bas Teusink*
Introduction: Cells use multilayered regulatory systems to respond adequately to changing environments or perturbations. Failure in regulation underlies cellular malfunctioning, loss of fitness, or
disease. How molecular components dynamically interact to give rise to robust and adaptive responses
is not well understood. Here, we studied how the model eukaryote Saccharomyces cerevisiae can
cope with transition to high glucose levels, a failure of which results in metabolic malfunctioning and
Methods: We combined experimental and modeling approaches to unravel the mechanisms used by
yeast to cope with sudden glucose availability. We studied growth characteristics and metabolic state
at population and single-cell levels (through flow cytometry and colony plating) of the wild type and of
mutants unable to transit properly to excess glucose; such mutants are defective in trehalose synthesis,
a disaccharide associated with stress resistance. Dynamic 13C tracer enrichment was used to estimate
dynamic intracellular fluxes immediately after glucose addition. Mathematical modeling was used to
interpret and generalize results and to suggest subsequent experiments.
Results: The failure to cope with glucose is caused by imbalanced reactions in glycolysis, the essential
pathway in energy metabolism in most organisms. In the failure mode, the first steps of glycolysis carry
more flux than the downstream steps, resulting in accumulating intermediates at constant low levels of
adenosine triphosphate (ATP) and inorganic phosphate. We found that cells with such an unbalanced
glycolysis coexist with vital cells with normal glycolytic function. Spontaneous, nongenetic metabolic
variability among individual cells determines which state is reached and consequently which cells
survive. In mutants of trehalose metabolism, only 0.01% of the cells started to grow on glucose; in
the wild type, the success rate was still only 93% (i.e., 7% of wild-type yeast did not properly start up
glycolysis). Mathematical models predicted that the dynamics of inorganic phosphate is a key determinant in successful transition to glucose, and that phosphate release through ATP hydrolysis reduces the
probability of reaching an imbalanced state. 13C-labeling experiments confirmed the hypothesis that
trehalose metabolism constitutes a futile cycle that would provide proper phosphate balance: Upon a
glucose pulse, almost 30% of the glucose is transiently shuttled into trehalose metabolism.
Discussion: Our work reveals how cell fate can be determined by glycolytic dynamics combined
with cell heterogeneity purely at the metabolic level. Specific regulatory mechanisms are required to
initiate the glycolytic pathway; in yeast, trehalose cycling pushes glycolysis transiently into the right
direction, after which cycling stops. The coexistence
of two modes of glycolysis—an imbalanced state
and the normal functional state—arises from the
fundamental design of glycolysis. This makes the
imbalanced state a generic risk for humans as well,
extending our fundamental knowledge of this central pathway that is dysfunctional in diseases such
as diabetes and cancer.
FIGURES IN THE FULL ARTICLE
Fig. 1. The coexistence of two glycolytic
states underlies glucose-tolerant tps1∆
Fig. 2. pHi reveals distinct metabolic
Fig. 3. Metabolic subpopulations are caused
by small variation in metabolic variables,
and their sizes can be manipulated.
Fig. 4. Generalized core model of glycolysis
can reach two stable, coexisting states.
Fig. 5. 13C tracer enrichment reveals highly
dynamic flux distributions through the
Figs. S1 to S18
Tables S1 to S8
Initiation of glycolysis can have two outcomes. Upon
glucose availability, glycolysis can end up in either a functional steady state or an unviable imbalanced state with
imbalanced fluxes between ATP-consuming (Vupper) and ATP-producing steps (Vlower). In wild-type yeast, the transient
activation of trehalose cycling pushes glycolysis toward the
viable steady state. Failure to do so results in metabolic
malfunctioning, as observed in mutants in trehalose biosynthesis (tps1∆).
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Cite this article as J. H. van Heerden et al.,
Science 343, 1245114 (2014).
The list of author affiliations is available in the full article online.
*Corresponding author. E-mail: firstname.lastname@example.org
RESEARCH ARTICLE SUMMARY
www.sciencemag.org SCIENCE VOL 343 28 FEBRUARY 2014 987