Passage 3: ATP
Regulation of cellular ATP level is critical for diverse biological processes and may be
defective in diseases such as cancer and mitochondrial disorders. While mitochondria
play critical roles in ATP level regulation, we still lack a systematic and quantitative
picture of how individual mitochondrial-related genes contribute to cellular ATP levels
and how dysregulated ATP levels may affect downstream cellular processes. Advances
in genetically encoded ATP biosensors have provided new opportunities for addressing
these issues. ATP biosensors allow researchers to quantify the changes of ATP levels in
real-time at the single-cell level and characterize corresponding effects at the cellular,
tissue, and organismal level.
Mitochondria are best known as the powerhouses of the cell that produce ATP via
oxidative phosphorylation (OXPHOS). Mitochondrial ATP synthesis involves tricarboxylic
acid (TCA) cycle enzymes, electron transport chain complexes, and ATP synthase, in
which acetyl-coenzyme A (CoA) derived from food molecules is oxidized to produce
ATP. During bioenergetic reactions, mitochondria also produce other physiologically
important molecules such as reactive oxygen species.
Two studies presented a new generation of ATP biosensors. In the first, a circularly
permuted green fluorescent protein (cpGFP) was combined with a bacterial ATPbinding protein to generate a reporter that quantitatively reports cellular ATP to
adenosine diphosphate (ADP) ratio. Because of differential binding affinities to ATP
and ADP, the reporter exhibits different degrees of conformational change in cpGFP
and, thus, different fluorescent signal changes in response to ATP or ADP binding.
In the second study, Imamura and colleagues developed a series of highly sensitive
and selective Förster resonance energy transfer (FRET)-based reporters for cellular
ATP concentrations. FRET-based reporters contain a subunit of a bacterial ATP
synthase fused between cyan fluorescent protein (CFP) and yellow fluorescent
protein (YFP). The reversible binding of ATP leads to conformational changes of
the synthase subunit, resulting in altered spatial proximities between fluorescent
proteins and thus changes in the FRET signal.
Dissecting the regulation and function of ATP at the single-cell level. Adapted from
Zhang et al. (2018).
CpGFP creates a blue stain when bound to ATP and a red stain when bound to
ADP, and researchers discovered a new toxic agent that blocked ATP synthase
but increased the amount of glycolysis in the mitochondria. What would be
most likely the resulting effect?
A) mostly blue staining
B) mostly red staining
C) mostly purple staining
D) not enough information
Correct answer: B. Since the question stem informs us that the colors
correspond with what the detector binds to, a higher intracellular concentration
of a particular species would correspond to the respective color. This means that
red would result from high ADP concentration, blue would result from high ATP
concentration, and purple would be the result of roughly equal concentrations of
the two species. Therefore, we should investigate the concentration of both species
in the cell. The question stem also states that there is a new toxin that inhibits
ATP synthase but stimulates glycolysis. Glycolysis produces two molecules of ATP
for every molecule of glucose. While this can be used for energy for the body, the
body and cells primarily rely on oxidative phosphorylation, producing high-energy
electron carriers (during glycolysis, the TCA cycle, and pyruvate dehydrogenase) to
fuel the ATP synthase via the electron transport chain (ETC). The ETC makes about
30-32 ATP per glucose molecule, and this is the primary source of ATP for the cell.
Since the toxin outlined in the question increases glycolysis (which does produce
some ATP), it still inhibits ATP synthase, so the net effect on ATP production is
still negative. This large reduction in ATP will cause there to be a large buildup of
ADP in the cells and thus be a very unbalanced concentration of ADP in the cell
because cellular processes would consume more ATP than is being produced,
whose consumption necessarily entails producing ADP. This unbalanced high
concentration of ADP will then yield mostly red staining, making answer choice B
correct.
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