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Meet the Souped-up Version of ATP Synthase

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A vacuole. No big deal. A bubble inside a bigger bubble, the cell. A storage place for fats, perhaps. One could be forgiven in Victorian days for being so dismissive. Now we know better. Who ever would have dreamt that turbo-charged rotary engines line the membranes of vacuoles, setting the acidity inside to tightly regulated specifications?

Our popular video on ATP synthase illustrates one variety of rotary motor in the cell: the F-type ATPase. These molecular marvels, a tenth the size of the bacterial flagellum but clocked at up to 42,000 rpm, take in protons and churn out ATP molecules. Another type of rotary motor, the vacuolar ATPase (V-ATPase) works much the same way, but in reverse. It spends ATP “currency” to pump protons into vacuoles against the concentration gradient, regulating their acidity. Essential organelles in all three kingdoms of life, V-ATPases are involved in many cellular functions. A 2013 paper in BioArchitecture introduces them:

Vacuolar or V-type ATPases are more distantly related rotary motors that work in reverse to ATP synthases. Using the energy from ATP hydrolysis they pump cations across membranes in a manner similar to man-made rotary pumps. V-type ATPases are important for the acidification of intracellular compartments and generate trans-membrane electrochemical potential gradients that provide the energy for sym- or antiporters to actively transport ions and molecules through membranes. They have been reported to maintain up to 100-fold differences in proton concentration between the cytosol and intracellular compartments. Again there are three versions; the eukaryotic V-type ATPase, and two simpler ones, the bacterial V-type ATPase and the archaeal A-type ATPase, which are structurally indistinguishable and are therefore often summed up as “A-type ATPases/synthases.” [Emphasis added.]

Your life depends on these motors. Wikipedia lists a few of the roles they play in your body:

V-ATPases are found within the membranes of many organelles, such as endosomes, lysosomes, and secretory vesicles, where they play a variety of roles crucial for the function of these organelles. For example, the proton gradient across the yeast vacuolar membrane generated by V-ATPases drives calcium uptake into the vacuole through an H+/Ca2+ antiporter system (Ohya, 1991). In synaptic transmission in neuronal cells, V-ATPase acidifies synaptic vesicles. Norepinephrine enters vesicles in exc by V-ATPase.

V-ATPases are also found in the plasma membranes of a wide variety of cells such as intercalated cells of the kidney, osteoclasts (bone resorbing cells), macrophages, neutrophils, sperm, midgut cells of insects, and certain tumor cells. Plasma membrane V-ATPases are involved in processes such as pH homeostasis, coupled transport, and tumor metastasis. V-ATPases in the acrosomal membrane of sperm acidify the acrosome. This acidification activates proteases required to drill through the plasma membrane of the egg. V-ATPases in the osteoclast plasma membrane pump protons onto the bone surface, which is necessary for bone resorption. In the intercalated cells of the kidney, V-ATPases pump protons into the urine, allowing for bicarbonate reabsorption into the blood.

(We won’t consider tumor metastasis a “function” but rather a dysfunction.) But think of it; you would never have been born without a V-ATPase enabling a protease to drill through the egg cell’s tough membrane. Your kidneys need V-ATPases. Your bones need them. Your immune system and nervous system need them. Are you beginning to appreciate these motors?

A new paper in Nature took the closest-ever look at the V-ATPase in yeast cells. Using cryo-electron microscopy, three researchers from Toronto were able to see in unprecedented detail the components of these molecular engines, and for the first time, deduce how they move and work together during operation. Here’s how they compared V-ATPase to the more well-known F-type ATP synthase:

The eukaryotic V-ATPase is the most complex rotary ATPase: it has three peripheral stalks, a hetero-oligomeric proton-conducting proteolipid ring, several subunits not found in other rotary ATPases, and is regulated by reversible dissociation of its catalytic and proton-conducting regions.

V-ATPase has two extra stators, looking something like flying buttresses holding the whole machine in place while it whirs along. The carousel-like V0 subunit, with its ten ring segments, “functions as a ten-step motor,” they say. And because it must force protons into the organelle against a strong concentration gradient, the machine is hermetically sealed against leakage.

The illustrations of V-ATPase in the paper are beautiful. The authors also included three video clips: one of the actual motors in situ, and two animations showing how the parts interact. Notice the machine metaphors in this quote:

The E- and G-subunits of the peripheral stalks undergo a bending motion along their elongated coiled-coil region, reminiscent of the action of a cantilever spring (Fig. 4c, Extended Data Fig. 6g-i and Supplementary Videos 2 and 3). The N-terminal domain of the a-subunit swings parallel to the membrane, moving to and away from the rotation axis of the rotor like the arm of a record player.

It’s long been a mystery why the top and bottom portions of the engine don’t have an integer ratio. The outer V1 part, for instance, digests three ATP per cycle. The inner V0 part, which pumps protons, has ten units. This creates a non-integer 3:10 ratio. Is this wasteful? Does it create slippage in the machinery? It appears now that there is a reason for it.

The resulting series of structures shows ten proteolipid subunits in the c-ring, setting the ATP:H+ ratio for proton pumping by the V-ATPase at 3:10, and reveals long and highly tilted transmembrane ?-helices in the a-subunit that interact with the c-ring. The three different maps reveal the conformational changes that occur to couple rotation in the symmetry-mismatched soluble catalytic region to the membrane-bound proton-translocating region. Almost all of the subunits of the enzyme undergo conformational changes during the transitions between these three rotational states. The structures of these states provide direct evidence that deformation during rotation enables the smooth transmission of power through rotary ATPases.

Although this is still under investigation, something about the mismatch contributes to the efficiency, perhaps by keeping the motor spinning. The conformational changes are coordinated for that function:

Overall, the structures presented here show that in the V-ATPase both the rotor and stator parts of the engine undergo coordinated conformational changes. This combination of flexibility and rigidity is probably a key attribute for the function of these highly efficient macromolecular machines.

And now, the Darwinese. This is all they have to say about evolution:

The existence of these conformational changes is not obvious from inspection of crystal structures of the individual subunits. However, when visualized as a movie, each subunit appears to have evolved to perform these motions.

That should keep them out of trouble with censors who might wonder if they are secret advocates for intelligent design.

The cognitive dissonance between the design evidence and the evolutionary belief is even starker in the BioArchitecture piece referred to above. There, four Australian researchers go out of their way to point out the similarities between the rotary ATPases and human-engineered motors. They talk about the different types of “fuel” that the motors use, and how lipids act like “motor oil” for “lubrication.” They compare the angle of placement of multiple ATPases to the angle in a V8 engine. They speak of turbines, stepping motors, gear ratios, superchargers, crankshafts, brakes, ratchets, scaffolds, torsion bars, pushrods, and more! It’s wonderful. The stage is all set for a design inference! Then comes the conclusion:

Molecular machines, like the rotary ATPases described here, seem to have much in common with man-made machines. However, the analogies hold only to a certain point and are in large parts not fully understood. What is evident is that several billion years of evolution have resulted in biological motors that are unsurpassed in efficiency, fine-tuning to their environment and sustainability.

To borrow Charlie Brown’s most-used word, “AAAAAGGGGHHH!”