The ADP-ATP antiporter in the mitochondrial inner membrane

25 Jun The ADP-ATP antiporter in the mitochondrial inner membrane

Question
Problem Set 3

1. The ADP-ATP antiporter in the mitochondrial inner membrane can exchange ATP for ATP, ADP for ADP, and ATP for ADP. Even though mitochondria can transport both ADP and ATP, there is a strong bias in favor of exchange of external ADP for internal ATP in actively respiring mitochondria. You suspect this bias is due to the conversion of ADP into ATP inside the mitochondrion. ATP synthesis would continually reduce the internal concentration of ADP and thereby create a favorable concentration gradient for import of ADP. The same process would increase the internal concentration of ATP, thereby creating favorable conditions for export of ATP.

To test your hypothesis, you conduct experiments on isolated mitochondria. In the absence of substrate (when the mitochondria are not respiring and membrane is uncharged), you find that ADP and ATP are taken up at the same rate. When you add substrate, the mitochondria begin to respire, and ADP enters mitochondria at a much faster rate than ATP. As you expected, when you add an uncoupler (dinitrophenol, which collapses the pH gradient) along with the substrate, ADP and ATP again enter the matrix at the same rate. When you add an inhibitor of ATP synthase (oligomycin) along with the substrate, ADP is taken up much faster than ATP. Your results are summarized in Table 1 below. You are puzzled by the results with oligomycin, since your hypothesis predicted that the rates of uptake would be equal.

Table 1: Entry of ADP and ATP into isolated mitochondria

Experiment

Substrate

Inhibitor

Relative Rates of

Entry into Matrix

1

absent

none

ADP = ATP

2

present

none

ADP > ATP

3

present

dinitrophenol

ADP = ATP

4

present

oligomycin

ADP > ATP

When you show your results to your advisor, she compliments you on your fine experiments and agrees that they disprove the hypothesis. She suggests that you examine the structures of ATP and ADP if you wish to understand the behavior of the antiporter.

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Figure 1. Structures of ATP and ADP.

What is the correct explanation for the biased exchange by the ADP-ATP antiporter under some of the experimental conditions and an unbiased exchange under others?

2. The uncoupler dinitrophenol was once prescribed as a diet drug to aid in weight loss. How would an uncoupler of oxidative phosphorylation promote weight loss? Why do you suppose that dinitrophenol is no longer prescribed?

3. The yeast two-hybrid system depends on the modular nature of many transcription factors, which have one domain that binds to DNA and another domain that activates transcription. Domains can be interchanged by recombinant DNA methods, allowing hybrid transcription factors to be constructed. Thus, the DNA-binding domain of the E. coli LexA repressor can be combined with the powerful VP16 activation domain from herpesvirus to activate transcription of genes downstream of a LexA DNA binding site (Figure 3).

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Figure 3. Activation of transcription by a hybrid transcription factor (LexA DBD joined to VP16 AD)

If the two domains of the transcription factor can be brought into proximity by protein-protein interactions, they will activate transcription. This is the key feature of the two-hybrid system. Thus, if one member of an interacting pair of proteins is fused to the DNA-binding domain of LexA (to form the “bait”) and the other is fused to the VP16 activation domain (to form the “prey”), transcription will be activated when the two hybrid proteins interact inside a yeast cell. It is possible to design powerful screens for protein-protein interactions if the gene whose transcription is activated is essential for growth or can give rise to a colored product.

To check out the ability of the system to find proteins with which the protein Ras interacts, hybrid genes were constructed that contained the LexA DNA-binding domain fused to Ras (LexA-Ras) and the other fused to nuclear lamin (LexA-lamin). A second pair of constructs contained the VP16 activation domain alone (VP16) or fused to the adenylyl cyclase gene (VP16-CYR). Adenylyl cyclase is known to interact with Ras and serves as a positive control. Nuclear lamins do not interact with adenylyl cyclase and so serve as a negative control. These plasmid constructs were introduced into a strain of yeast containing copies of the His3 gene and the LacZ gene, both with LexA-binding sites positioned immediately upstream. Individual transformed colonies were tested for the ability to grow on a plate lacking histidine, which requires expression of the His3 gene. In addition, colonies were tested for tested for the ability to grow as blue colonies (as compared to the normal white colonies) when grown in the presence of an appropriate substrate (XGAL) for beta-galactosidase. The set-up for the experiment is outlined in Table 3.

Table 3: Experiments to test the two-hybrid system

Plasmid Constructs

Growth on Plates

Color on Plates

Bait

Prey

Lacking Histidine

With XGAL

LexA-Ras

LexA-lamin

VP16

VP16-CYR

LexA-Ras

VP16

LexA-Ras

VP16-CYR

LexA-lamin

VP16

LexA-lamin

VP16-CYR

A. Fill in Table 3 (or make a new one in Excel and paste that into your solutions) with your expectations. Use a plus sign to indicate growth on plates lacking histidine and a minus sign to indicate no growth. Write “blue” or “white” to indicate the color of colonies grown in the presence of XGAL.

B. For any entries in the table that you expect to grow in the absence of histidine or to form blue colonies with XGAL, describe (or sketch) the structure of the active transcription factor on the lacZgene.

C. If you want two proteins to be expressed in a single polypeptide chain, what must you be careful to do when you fuse the two genes together?

4. You are baffled. You are studying the control of cyclic AMP levels in brain slices and have confirmed that signal molecules such as isoproterenol that act through

β-adrenergic receptors cause a modest increase in cyclic AMP, as expected from G-protein-mediated coupling between the receptor and adenylyl cyclase. You find a puzzling synergy, however, between isoproterenol and a number of pharmacological agents that by themselves have no effect on cyclic AMP levels. What is the basis for this paradoxical augmentation of cyclic AMP levels?

A biochemist friend of yours has suggested a possible explanation: she found in in vitro experiments that βγ subunits from inhibitory trimeric G proteins stimulate type II adenylyl cyclase, which is expressed in the brain. To test this idea in cells, you plan to express the cDNAs encoding the component proteins in human kidney cells, which lack the receptors found in brain. In this way you hope to reconstruct the effects you observed in brain slices, but in a much simpler background.

You transfect the kidney cells with various combinations of cDNAs encoding type II adenylyl cyclase, the dopamine receptor (which interacts with an inhibitory G protein), and a mutated (constitutively active) α s* subunit. You measure the levels of cyclic AMP in the resulting cell lines in the absence or presence of quinpirole, which activates the dopamine receptor (see figure 1). You also measure the effects of pertussis toxin, which blocks the signal from Gi-coupled receptors by modifying the α i subunit in such a way that it can no longer bind GTP or dissociate from its βγ subunit (see figure 4)

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Figure 4. Measurements of cyclic AMP levels in cells transfected with cDNAs for the dopamine receptor, type II adenylyl cyclase, and αs*

A. Did you succeed in reproducing the paradoxical result you observed in brain slices in the transfected kidney cells? Explain.

B. Explain the effects of pertussis toxin in your experiments.

C. What do your experiments indicate is required for maximal activation of type II adenylyl cyclase? Propose a molecular explanation for the augmented activation of type II adenylyl cyclase.

D. Predict the results of expressing the cDNA for the α subunit of transducin, which does not bind to adenylyl cyclase but binds tightly to free βγ subunits.

5. Consider a signaling pathway that proceeds through three protein kinases that are sequentially activated by phosphorylation. In one case the kinases are held in a signaling complex by a scaffolding protein; in the other, the kinases are freely diffusing (See Figure 5 below). Discuss the properties of these two types of organization in terms of signal amplification, speed, and potential for cross talk between signaling pathways.

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Figure 5. A protein kinase cascade organized by a scaffolding protein or composed of freely diffusing components.

6. Phosphorylase kinase integrates signals from cyclic AMP-dependent and Ca2+-dependent signaling pathways that control glycogen breakdown in liver and muscle cells (See Figure 6). Phosphorylase kinase is composed of four subunits. One is the protein kinase that catalyzes addition of phosphate to glycogen phosphorylase to activate it for glycogen breakdown. The other three subunits are regulatory proteins that control the activity of the catalytic subunit. Two contain sites for phosphorylation by PKA, which is activated by cyclic AMP. The remaining subunit is calmodulin, which binds Ca2+ when its cytosolic concentration rises. The regulatory subunits control the equilibrium between the active and inactive conformations of the catalytic subunit. How does this arrangement allow phosphorylase kinase to serve its role as an integrator protein for the multiple pathways that stimulate glycogen breakdown?

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Figure 6. Integration of cyclic AMP-dependent and Ca2+-dependent signaling pathways by phosphorylase kinase in liver and muscle cells. G1P is glucose 1-phosphate, the product of the cleavage of a glucose from glycogen using a