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).
Correct answer: A. ATP has three attached phosphate groups. The one
that is closest to the ribose is the alpha phosphate group, the second closest one is
the beta phosphate, and the farthest phosphate group is the gamma phosphate. The
question asks to propose a reason why ATP levels would decrease. Each one of the
answer choices indicates that ATP is being converted into another substance. Answer
choice D suggests that ATP loses one phosphate group and forms AMP, which is
incorrect. AMP has only one phosphate group, and ATP has three, so in order to form
AMP from ATP, there would have to be a loss of two phosphate groups. Therefore, answer
choice D is incorrect. Since there needs to be a loss of one phosphate group to convert
ATP to ADP, the phosphate group farthest away from the ribose would be lost first. The
phosphate group that is lost first would be the gamma phosphate group. The gamma
phosphate has the highest energy (most unstable) bond and is best used to couple with
energetically unfavorable processes. This gamma phosphate group is attached to the
beta phosphate group in the second position, and thus, option choice A is correct since
the gamma phosphate group is being detached from the beta phosphate group.
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