Over 90% of the ~3000 different proteins present in mature chloroplasts are encoded on nuclear DNA and translated in the cytosol [cell fluid outside the nucleus]. These proteins are synthesized in precursor form – each bearing an amino-terminal targeting signal called a transit peptide – and are imported into the organelle by an active, post-translational targeting process (Figure 1). This process is mediated by molecular machines in the outer and inner envelope membranes, referred to as ¡®translocon at the outer envelope membrane of chloroplasts¡¯ (Toc) and ¡®translocon at the inner envelope membrane of chloroplasts¡¯ (Tic), respectively. Upon arrival in the stroma [chloroplast interior], the transit peptide is removed and the protein either takes on its final conformation or is sorted to one of several internal compartments in a separate targeting process. (Emphasis added in all quotes.)

Chloroplasts are complex organelles comprising six distinct suborganellar compartments: they have three different membranes (the two envelope membranes and the internal thylakoid membrane), and three discrete aqueous compartments (the intermembrane space of the envelope, the stroma and the thylakoid lumen). One of the consequences of this structural intricacy is that the internal routing of chloroplast proteins is a surprisingly complex process. While envelope proteins may employ variations of the Toc/Tic import pathway to arrive at their final destination, proteins destined for the thylakoid membrane or lumen employ one of four distinct targeting pathways (Figure 1). Thylakoid membrane proteins are targeted by the signal recognition particle (SRP)-dependent and spontaneous insertion pathways, whereas lumenal proteins are targeted by the Sec and Tat pathways....

1. The transit peptide needs to fit the receptor on the membrane, and another protein has to be ready to cleave it (remove it).
2. The transit peptides have to be precise to avoid having the protein arrive at the wrong organelle, like the endoplasmic reticulum, mitochondrion or peroxisome – organelles which also accept polypeptides with shipping labels.
3. Transit peptides are varied. ¡°One might therefore expect chloroplast transit peptides to share well-defined primary or secondary structural motifs,¡± they say. ¡°On the contrary, transit peptides are remarkable in their heterogeneity. They vary in length from 20 to >100 residues, and have no extended blocks of sequence conservation.¡±
4. The transit proteins ¡°do not seem to form secondary structure in aqueous solution¡± but once they arrive at their target membrane, they seem to take on a characteristic structure.
5. The polypeptides (precursor proteins) are threaded through the needle of specialized gates in the membrane. There, additional molecular machines (chaperones) make sure they do not fold prematurely.
6. To get a polypeptide through a membrane involves three steps: contact, docking, and translocation, when the transit peptide is cleaved. This requires energy: a high concentration of ATP must be present for the operation.
7. The Toc and Tic squads, like a delivery organization with a variety of employees skilled in particular tasks but working on common goals, is made up of multiple proteins, each with their own task to perform, all working in coordination.
8. Once inside the outer membrane, the polypeptide has to get past the inner membrane. Another set of specialized proteins are available for that task.
9. A third import apparatus has to complete the task of getting the polypeptide to its final destination. Many go to the thylakoid membrane, rich with light-harvesting structures and ATP synthase.
10. Those polypeptides bound for the thylakoid membrane have a secondary shipping label (transit peptide). In addition, they may have a ¡°stop-transfer¡± signal to indicate their destination.
11. Removal of the secondary transit peptide can occur by ¡°one of two very different pathways,¡± called Sec and Tat. Sec transports proteins in an unfolded state, but Tat can transport them in a folded state. Each pathway involves multiple proteins working together.
12. In the Tat pathway, ¡°There is even evidence that some proteins are exported in an oligomeric form¡± [i.e., several proteins bound together in a complex], ¡°which points to a remarkable translocation mechanism,¡± they remark. Is this like squeezing a completed sweater through the eye of a needle? ¡°...we currently know very little about this mechanism,¡± they say. ¡°Somehow, this system must transport a wide variety of globular proteins – some over 100 kDa [kilodaltons] – while preserving the proton motive force and avoiding loss of ions and metabolites.¡± Their surprise at this indicates it is quite a feat.
13. The translocation process can expend 30,000 protons, ¡°a substantial cost by any standard.¡± According to current theory, a pH difference between inner and outer membrane provides the proton flow, but that pH balance must be carefully monitored and regulated.
14. Another pathway named SRP inserts proteins into the lumen. The authors claim this pathway was ¡°clearly inherited from the cyanobacterial progenitor of the chloroplast,¡± but admit that there are differences in the insertion pathways and events at the thylakoid membrane in chloroplasts. ¡°...it is fair to state that, while the major players in this pathway have been identified, their modes of action remain unclear and we do not understand how such highly hydrophobic proteins are bound by soluble factors, shuttled to the membrane and then handed over to membrane apparatus and inserted.¡±
15. Evolutionists who expected the SRP pathway from E. coli bacteria to act the same in chloroplasts, where homologous proteins were detected, learned otherwise: ¡°Surprisingly, this is not the case. In vitro assays for the insertion of a range of membrane proteins have shown that the vast majority of such proteins do not rely on any of the known protein transport machinery, including SRP, FtsY, Alb3 or the Sec/Tat apparatus, for insertion.¡± Nor do they rely on nucleoside triphosphates or proton flow.
16. Speaking of the apparent spontaneous insertion of the thylakoid proteins, they comment, ¡°This unusual pathway for membrane protein insertion appears to be unique to chloroplasts.¡± Though the typical insertion components are not involved, they believe it would be ¡°overly simplistic¡± to assume that this pathway requires no ¡°complex insertion apparatus.¡±
17. Other pathways than those described above are used for other proteins to get inside the chloroplast. Some are encoded by the chloroplast DNA, translated in the interior, then transported to their destinations.
18. Chloroplasts have to transport not only the essential light-harvesting proteins, but also ¡°housekeeping¡± proteins for structural maintenance. These must be imported at their own separate rates depending on the stage of development or the environmental conditions, and have their own specific transit peptides.

The Tat pathway manages the remarkable feat of transporting large, folded proteins without collapsing the delta-pH, and we currently know very little about this mechanism. Most membrane proteins use a possibly ¡®spontaneous¡¯ insertion mechanism that just does not make sense at the moment – why do these proteins need so little assistance from translocation apparatus, when membrane proteins in other organelles and organisms need so much? And how do these thylakoid proteins avoid inserting into the wrong membrane? We have gone some way toward understanding the rationale for the existence of all these pathways, but the thylakoid may still have surprises in store.

If you survived this long and mind-numbing description of chloroplast protein transport, you have gasped at the complexity of it all. On a general level, getting a protein from one place to another sounds simple. But look how many players are involved, how many checks and balances, how many protection mechanisms and signals are required to get the packages delivered accurately. And this is all just to get the chloroplast to start to get ready to begin to commence doing its job: harvesting light for photosynthesis (and that¡¯s another story: if this one was over your head, run for cover).
You saw these authors refer to the Darwinian just-so story that once upon a time, a bacterium engulfed a cyanobacterium that had learned how to harvest light, and the two shared their technologies, forming a glorious partnership that led to plants. For that to be true, all these new protein transporting mechanisms had to be invented to get the genes moved into the nucleus, and then to get their translated proteins back into the chloroplasts. Why didn¡¯t the chloroplast DNA stay put? Did they offer any plausible way an unpredictable series of accidents led to all this complexity? Assuredly not. Considering the difficulty in getting just one protein right by chance, it strains credibility, light-years beyond the breaking point, to think that these complexes of complicated proteins – all working like a company – arose by chance. Nor would an unbiased person presume that time would suffice for thousands of beneficial mutations to occur for even one of the pathways to emerge, even assuming natural selection – that magic wand of the Darwinists – preserved them. But one component is useless if not part of a functioning system. That is the power of the argument from irreducible complexity. We¡¯re talking about three thousand proteins needing special delivery to make a chloroplast work, and each one requires dozens if not hundreds of amino acids in the correct sequence. Would one of the Toc proteins have any selective value if the other components were not present to help it get the polypeptide through the outer membrane? Clearly not. Even if all the Toc proteins emerged somehow, and managed to squeeze the polypeptide through the outer membrane, the polypeptide would just sit there uselessly without the Tic proteins to get it through the second membrane. If it got past the inner membrane, it would be useless unless it folded correctly with the aid of chaperones, and then made it to the exact destination in the chloroplast, where, working together with other proteins, it could perform its spectacular feat: converting light energy to chemical energy. But all the players in the system need that energy to do their jobs!
It may be tedious to wade through some of these articles about cellular mechanisms, but take the time once in awhile, because the power of the the message – intelligent design – is in the details. Hold this evidence up in the face of the Darwin Party and ask them some hard-hitting questions: how could such coordinated complexity arise by unguided, mindless, purposeless processes? Did Fed Ex or UPS emerge from a tornado in a junkyard? It¡¯s details like this that convinced Antony Flew, the prominent (former) atheist, that the case for intelligent design was compelling, and over many years, convinced him to become a theist. With some hard heads and hard hearts, overkill and persistence is necessary.