In the past, I have provided multiple lines of evidence to establish the plausibility of front-loading evolution.However, we have focused primarily on the transition from a unicellular to a multicellular eukaryotic state.Let’s now take one step back and begin pondering whether eukaryotic cells were themselves front-loaded to appear. For without the eukaryotic cell design, it is unlikely that the planet would possess anything analogous to an animal or plant.
While we still don’t really understand the origin of the eukaryotic cell, there is a strong consensus about the origin of the mitochondria, a very important organelle of eukaryotes.According to the endosymbiotic theory, mitochondria are the descendents of bacteria. The theory postulates that a phagocytic cell engulfed some aerobic bacteria and rather than digest them, a symbiotic relationship was established, where each partner benefited from the new relationship. This relationship then set the stage for the ultimate stream-lining of the bacteria, such that they were transformed into mitochondria through the transfer of much of their gadgetry to the host nucleus.
In a nutshell, the essence of the argument for the endosymbiotic origin of mitochondria is that mitochondria look like they share a common ancestor with bacteria. The argument is quite convincing, as there are numerous mitochondrial genes whose sequences are much more similar to bacterial sequence than that which exists in the nucleus of the same cell. In fact, this is an example where no one piece of evidence carries the day, but instead it’s the cumulative power of multiple lines of evidence.
Since mitochondria were once bacteria, might this transition have been front-loaded to happen?
The main obstacle for this transition would occur long after the bacteria had established a symbiotic union with its host.At some point, the bacteria transfer their genes to the host’s nucleus, which would mean the host’s ribosomes in the cytoplasm would have to synthesis the bacterial proteins.And here is where the problem comes in – how do you specifically transfer the bacterial proteins in the cytoplasm, now mixed with the host’s own proteins, back into the proto-mitochondria?
If we think about this transition through the lens of PICERAS, the mitochondria are intimately tied with the pillar of energy, as they regenerate the ATP needed to fuel the cell’s molecular machines. They excel at this function because of the pillar of compartmentalization, where these metabolic reactions are optimized by the localized conditions within the mitochodria brought about by the composition of mitochondrial proteins themselves.So how do we get the mitochondrial proteins that are synthesized by ribosomes in the cytoplasm across the mitochondrial membranes?
Eukaryotic cells use the pillar of seclusion, where mitochondrial proteins have a small set of amino acids attached to the front end of the protein that act as a signal to set them apart from non-mitochondrial proteins.This signal is known as the mitochondrial targeting sequence and contains around thirty amino acids that form an alpha helix that is mostly basic and hydrophobic.Thus, any protein that has this mitochondrial targeting sequence attached to its front end will be transported into the mitochondiria.To go a long way in solving the transport problem posed by the endosymbiotic transition, we need only account for the existence of this mitochondrial targeting sequence, as standard processes of recombination or transposition can paste it onto the front end of any protein.
So where did the mitochondrial targeting sequence come from?Answer – they have always existed in bacteria. One recent study  used two different computer programs to determine that at least one out of every twenty proteins from E. coli possess a mitochondrial targeting sequence. The researchers then took a candidate E. coli protein from the list (YhaR) and expressed it in baker’s yeast.When this was done, the E coli protein was transported to the mitochondria! Not only is this all further evidence of the endosymbiotic theory, the researchers reached the following conclusion:
Multiple sequence alignments of the bacterial versions of these proteins show ragged amino-termini, with YhaR from E. coli having one of the longer amino-terminal extensions. Ectopic expression of bacterial YhaR results in targeting of the protein to yeast mitochondria, suggesting that in some cases, during the course of evolution, this preadaptation meant little or no mutagenesis of upstream regions in bacterial genes to render the proteins they encode competent for import into ‘‘protomitochondria.’’
The gene products translated in the cytosol then needed to be recognized for translocation into protomitochondria. Although seemingly problematic, it is now clear that physicochemical properties of a sizeable number of bacterial proteins like YhaR, present in diverse phyla of extant bacteria and therefore likely to have been inherent in proteins of the ancestral proteobacter, were available as a preadaptation to be used as the basis to specify mitochondrial targeting.
A key phase in the evolution of mitochondria was made possible because of preadaptation.We thus have our first plausible example of front-loading the evolution of the modern, eukaryotic cell.
But this story gets more interesting.To appreciate the twist that comes next, let’s step back to make sure we can visualize the process of transporting mitochondrial proteins into the mitochondria.
First, like gram negative bacteria, the mitochondria have two membranes:
We want to focus on the outer membrane, as this is where the mitochondrial proteins, synthesized by the ribosomes in the cell’s cytoplasm, must be targeted for entry into the mitochondria.
Mitochondrial proteins are sent to something called the TOM complex that is found in the outer membrane.The figure below shows a simplified version of this complex that is composed of a receptor, that binds the mitochondrial targeting sequence on mitochondrial proteins, and shuttles it to the channel for entry into the mitochondria (TIM is the inner membrane transporter).
The figure below provides more detail:
You can see the mitochondrial protein on the top with its exposed mitochondrial targeting sequence (the + signs near the N).The mitochondrial targeting sequence binds to the receptor (R) which passes the protein to the TOM channel (shown as GIP in this picture).The protein is then threaded through both TOM and TIM, using the same proton gradient that mitochondria generate for their ATP synthases.
Now, take a look at a more detailed figure:
As you can see, TOM is composed of multiple proteins.Tom7, Tom22, and Tom40 are the core elements of this complex, where Tom40 form the pore itself and is likely to be the descendent of bacterial porins (protein pores found in the outer membranes of gram negative bacteria).
It turns out that the mitochondrial targeting sequence interacts with multiple components of this system:
The intracellular sorting of newly synthesized precursor proteins (preproteins) to mitochondria depends on the “mitochondria-targeting sequence” (MTS), which is located at the amino termini of the preproteins. MTS is required, however, not only for targeting newly synthesized preproteins to mitochondria, but also for all the following steps along the mitochondrial protein import pathway. MTS of nascent preproteins is first recognized by a cytoplasmic molecular chaperone, MSF, and then by Tom70 and Tom20 of the mitochondrial outer membrane receptor complex, Tom5 and Tom40 of the outer membrane protein translocation machinery, Tim23 of the inner membrane protein translocation machinery, and finally the processing peptidase, MPP, in the matrix. MTS is a multi-role sorting sequence which specifically interacts with various components along the mitochondrial protein import pathway. Recognition of MTS at multiple steps during the import of preproteins may contribute to the strict sorting of proteins destined for mitochondria. 
The player I want to focus on is Tom20.Why?Because Tom20 is the receptor protein that snags the mitochondria targeting sequence.Tom20 is not essential, as yeast strains have been constructed where Tom20 was removed and the yeast remain viable.This means that the receptor simply enhances the efficiency of the transport process.
Tom20 has been studied in fungal and mammalian systems and shown to have the a domain structure as shown in Figure 1.
Figure 1.Domain organization of Tom20 in animals and fungi (modified from Fig 5 from ). N = amino terminus (the front-end of the protein) and C = carboxyl terminus (the back end of the protein).
The gray boxes represent regions that are disordered, the blue box represents a TPR domain that binds the MTS, and the black box represents the region that spans the outer membrane of the mitochondria.The line above represents the regions exposed to the cytoplasm.
So the black box region sits in the membrane and the blue box, with TPR domains, snags the MTS.
When scientists used the sequence of this protein to query databases for homologs in plants and protozoa, nothing was found.Could it be that plants and fungi don’t have Tom20?
Biochemical techniques were used is isolate a protein from mitochndria that was the same size as Tom20 and interacted with the membrane pore.Antibodies raised against this protein worked to block protein transport into the mitochondria.The gene for this protein was isolated and the protein’s structure was recently determined .The results are shown in Figure 2.
Figure 2.Domain organization of Tom20 in plants and algae (modified from Fig 5 from ). N = amino terminus (the front-end of the protein) and C = carboxyl terminus (the back end of the protein).
If you compare this plant Tom20 to animal/fungi Tom20, you’ll notice the same pattern of domains in reverse.This becomes even more clear if we try to visualize these proteins in the membrane (Figure 3).
Figure 3. Tom20 from animals/fungi (A.) compared to Tom20 from plants (B.)Yellow box represents membrane, with cytoplasm on top.
Notice that the pattern is the same, except that for animals, it is the back end of the protein that is anchored in the membrane while for plants and algae, it is the front-end of the protein that is anchored.
This reverse similarity is not restricted to domain organization, but also extends into the sequence of the two proteins:
Comparison of the plant and animal Tom20 transmembrane domains and the proximal cytosolic regions suggests striking structural similarities, but features within the sequences occur in reverse order. First, there are conserved glycine and aromatic residues within the region predicted to be in the bilayer of the outer mitochondrial membrane. Secondly, an aspartate residue is found at the cytosolic membrane interface. Thirdly, this aspartate is followed by a region of 13 residues in which charged residues (five basic, one acidic) predominate. 
The researchers summarize the significance of all this:
The transmembrane domain and its flanking regions of the plant and animal Tom20s only show clear sequence and structural similarities when viewed in reverse, and genetic mechanisms underlying protein evolution, such as duplication, cyclic permutations, and limited insertions and deletions, could not easily result in the sequence reversal that we observe in these two proteins. We therefore hypothesize that two distinct TPR protein ancestors existed prior to the split of the lineage giving rise to plants and protozoans from that giving rise to animals and fungi. These distinct ancestral TPR proteins independently gave rise to the Tom20 in plants and the Tom20 in animals and fungi. 
In other words, this is an example of convergent evolution at the molecular level.Two membrane proteins independently evolved to carry out the same function. The two proteins have the same pattern of protein domains, but in reverse.Remarkable sequence similarity also exists, but again, seen only in reverse.
So how did this occur?We can think of this example of convergent evolution as the unfolding of the preadapted state.This original preadapted state channeled the blind watchmaker, much as a seeing eye dog can lead a blind man.And if it wasn’t for the lucky fact that the proteins are in reverse, an example of convergent evolution would be scored by everyone as an example of common descent.
So what was the preadapted state?Two key parts appear to be the original bacterial porin (which evolved to become Tom40) and the MTS-like sequences that exist on one out of every twenty bacterial proteins.Since the MTS sequence interacts with Tom40, the bacterial design was already poised to facilitate the endosymbiotic union.This interaction would set up a selection pressure that would be guided by the architecture and composition of the bacterial porin and the MTS.In essence, the combined demands of the porin and the MTS would function as bait to fish Tom20 out of the bacterial tool box of protein domains.Inside this toolbox was the TPR domain which seemed to fit the MTS nicely.From there, we simply attach a membrane spanning region, which could be picked up from many other bacterial proteins through duplication and recombination. In this case, natural selection did not stumble upon some solution, any solution, that just happened to work. No, no. It hit the same target – twice.And it did so roughly about the same time: after plants and animals/fungi split apart, but before animals and fungi split apart, and before green algae and plants split apart.
This design logic was laid out and explained in The Design Matrix.After surveying test tube experiments where scientists generated ATP-binding proteins from random peptides, I observed:
The whole experiment is an intelligent use of chance. First, you fish out the proteins that weakly express a function you are trying to find, which is easily accomplished by using the function itself as bait in the pool, cleansing away all the other sequences that do not meet your criteria. Once the candidates are isolated, you start over with them, but this time, the bait is more complex, as it is not merely the function, but also includes the sequence of the binder. The mutation steps that followed were built around a strategy that kept most of the identified sequence constant while tweaking on its periphery. The result was the isolation of a protein with improved function.
Life’s designer may have also made intelligent use of chance. Only in this case, the “bait” was not a simple molecule like ATP, nor a single complex of ATP and a protein. Instead, the bait could have been the entire cell, or set of heterogeneous cells. What the blind watchmaker could subsequently find was then constrained by the carefully chosen initial conditions. Just as the researchers, as artificial selectors, set up their in vitro selection experiment such that it was rigged to find ATP-binding proteins, so too may life’s initial conditions have been rigged by the design of the cell’s architecture and the choice of which components to employ. In such a case, this chosen state would then act as the surrogate for the artificial selector.
Tom20 represents a decent candidate for such design.
1.Rebecca Lucattini, Vladimir A. Likic´, and Trevor Lithgow. 2004. Bacterial Proteins Predisposed for Targeting to Mitochondria.Mol. Biol. Evol. 21:652–658.
2. Omura, T. 1998. Mitochondria-Targeting Sequence, a Multi-Role Sorting Sequence Recognized at All Steps of Protein Import into MitochondriaJ. Biochem 123: 1010-1016.
1. , , , , Convergent evolution of receptors for protein import into mitochondria (2006) Current Biology, 16 (3), pp. 221-229.