Crossing Guard Found in Mitochondrial Membranes

by Ross Anderson, Ph.D.

I’m sure that most readers have heard of mitochondria, and how important they are to all eukaryotic cells. They are commonly referred to as the cell’s “powerhouse” as they are responsible for synthesizing the bulk of ATP required to power many reactions in a cell.

A characteristic of all eukaryotic cells is that they have membrane-bound organelles within them. Some, like the nucleus and mitochondria, are bounded by two membranes, but most are bounded by a single membrane. Regardless of the number of membranes, all must have transport mechanism in place to get proteins into them in order to function properly. Each organelle utilizes different designs to transport proteins into them, or out of them.

Two organelles, the endoplasmic reticulum (ER) and the mitochondria, have different complexes; however, there is one complex that is common to both. It is called Ubx2. A recent paper in Nature by Christoph U. Martensson et al., with a ho-hum title “Mitochondrial Protein Translocation-Associated Degradation,” is actually about quality control inside the cells of our bodies. The researchers found some exciting news about this protein complex Ubx2: it acts like a crossing guard, ferrying passengers safely through the membrane. But this is no ordinary crossing guard. It also acts as a bouncer or cop when bad proteins try to clog up the channel. And it has access to the whole search-and-rescue team. Without this entire system knowing what to do in advance of trouble, the whole cell could die.

Making the Ingredients

Making ATP in the mitochondria requires the use of many different proteins that work as parts of large complexes. Some of these proteins are located in the matrix region of the mitochondria (the inner parts), and many are located between membranes, either the inner membrane or the outer membrane. A mitochondrion has a small genome of its own, but a large majority of its proteins are encoded by genes located in the nucleus—not in the mitochondrial DNA. Consequently, these genes must be transcribed in the nucleus, then translated into proteins in the cytosol of the cell (i.e., outside the mitochondria).

Transporting the Ingredients

After the precursor proteins have been transcribed in the cytosol, they must then be transported into the mitochondria and folded properly before they are able to function as proteins or enzymes. Because we live in a fallen world, not everything proceeds perfectly or is made perfectly; mutations occur. As such, sometimes proteins do not have the correct amino acid sequence, or even if they do, they simply don’t fold properly. Proteins destined for the mitochondria are maintained in an unfolded state until they are transported into the mitochondria where they are folded by special helper enzymes called chaperonins. This is accomplished by the binding of certain proteins in the cytosol known as heat-shock proteins (Hsp’s). Hsp’s are also called chaperones, because they bind to unfolded regions of proteins and prevent them from engaging in improper interactions that might lead to an improperly folded protein.

Meet TOM

If mitochondrial protein precursors are not transported into the mitochondria properly, they will accumulate on the outer surface of the mitochondria. This leads to a mitochondrial-induced stress response by the cell whereby a number of cellular activities are affected. As a consequence of improper transport by the transport channel, TOM (Translocon Outer Membrane), becomes clogged and proteins can no longer be transported into the mitochondria. This can lead to down-regulation of mitochondrial proteins which will compromise the synthesis of ATP.

The TOM itself is actually a complex of proteins. The various members, numbered like Tom20 and Tom70, work together for the efficient transport of mitochondrial proteins through the outer membrane. Each protein of the complex is encoded by its own gene, and the various genes are carefully regulated so that the correct number of components are ready for assembly of the TOM. As the name implies, TOM is located in the outer membrane and forms the channel through which proteins traverse the outer membrane. The TOM joins another protein complex, TIM (Translocon Inner Membrane), located in the inner membrane. TIM forms a channel through the inner membrane. Together these two complexes of proteins form a complete channel through both membranes. The TOM core complex is made up of Tom40, Tom22, and various small Tom proteins. This complex associates with Toms 20 and 70.

The Quality Control Team

Together, the proteins Ubx2 and Cdc48 promote clearance of stalled precursor proteins from the general entry gate of mitochondria, the TOM complex, for proteasomal degradation. Credit: Christoph Mårtensson, University of Freiburg

The authors found that Ubx2 associates with Tom40 and some of the other proteins of the complex, except for Tom70. Under conditions where a cell is stressed, another protein, Cis1 binds to Tom70 and recruits Msp1 which aids in the removal of non-imported precursor proteins. However, under ‘normal’ non-stressed conditions, Ubx2 binds. In the ER (endoplasmic reticulum), Ubx2 functions with two proteins, DOA10 and HRD1, which, together, recruit an ATPase (an ATP-powered enzyme), Cdc48, and function in the removal and degradation of proteins from channels in the ER membrane if they failed to make it through. So in the ER, Ubx2 associates with DOA10 and HRD1, but at the mitochondria, it has a different role. There, Ubx2 associates with Tom40, 22 and 20. This suggested to the authors that there must be two populations of Ubx2; one for the ER and one for mitochondria.

Interestingly, Ubx2 itself, must be transported into the mitochondria in order to function. Once in the mitochondria, Ubx2 can associate with the mature Tom40 complex only in the presence of Tom22 and Tom70. Once associated with Tom40 the complex of Toms20, 22, 40 and 70 can recruit Cdc48 to the TOM complex. However, in order for Cdc48 to associate with the TOM there must be yet another two proteins, Ufd1 and Np14. If you can keep track of all this complexity, Cdc48 also has associated proteins, Vms1 and Msp1, required for mitochondrial-associated degradation of proteins!

Putting it All Together

A mitochondrion’s main function is to run its powerhouse, a sequence of molecular machines called the electron transport chain. This chain produces ATP from the food that we eat. The scientists in the Nature paper reported that, without Ubx2 and Vms1 or Msp1, the stability of the electron transport chain complexes (respiratory chain) in the inner membrane was compromised. As a result, proteins would get tagged with ubiquitin (the “kill-me” tag) for destruction and, as a result, would accumulate at the TOM. Thus, the TOM complex recruits Ubx2 which, in turn, recruits Cdc48 and its partner proteins, Udf1 and Np14. The Cdc48 complex then extracts arrested proteins from the TOM channel. The extracted proteins are then degraded by a proteasome (another complex of proteins that degrades faulty proteins and recycles the amino acids). The authors dubbed this quality control system ‘mitoTAD’ for mitochondrial translocation-associated degradation. Thus the “mitoTAD maintains the full functionality of the TOM complex for correct import of precursor proteins into the mitochondria.” Without it, aberrant precursor proteins would accumulate and clog the TOM. That, obviously, could lead to the detriment of the cell and the entire organism.

Sticking to the Science

Most likely, all the authors believe that evolution is the explanation for how this came to be. To their credit, though, they did not engage in evolutionary storytelling to try and prop up an evolutionary ideology. They clearly answered the three questions an objective scientist should ask:

• What is it?
• What does it do?
• How does it do it?

It was refreshing, therefore, to see a paper stick to the science. That said, anyone reading this paper with an open mind would be amazed at the complex quality control involved in the seeming “simple” task of ushering proteins through a membrane. The transport systems usually work well, but it is evident that foresight was involved in knowing how to handle emergencies, like when protein precursors get stuck in the channel. Does this not all show clearly the hallmarks of intelligent design? Look how many proteins are required. Each of these must be able to bind to others, so that they function as a whole.

Irreducible Complexity Squared

Mutation studies by the authors showed that when one or more of the needed proteins are absent, the function of the whole is lost. Assembly of one complex of proteins is required before the proper assembling and functioning of another complex; e.g., assembly of the TOM complex sets the stage for recruitment of the Ubx2 complex which, in turn, sets the stage for recruitment of the Cdc48 complex. Thus, we see irreducible complexity at two points: an irreducibly complex collection of parts arranged in an irreducibly complex sequence over time.

This is very much like a Rube Goldberg machine. (If you don’t know what that is, see this entertaining modern example on YouTube.) Clearly, one malfunction anywhere in the sequence stops everything downstream that depends on it. Nobody would think the device in the video would arise by chance. Let’s be consistent, therefore, and apply the same reasoning in this case. The inference to the best explanation therefore—arising from the scientific facts themselves—is that intelligence with foresight played a fundamental role in the creation of this system. Bible-believers like me joyfully attribute that mind to the wonder-working hand of the Creator God of Genesis.

Ross Anderson (PhD biochemistry) is professor of biochemistry at The Master’s University in southern California. Dr Anderson’s expertise is in the area of biochemistry and molecular biology. He has taught Biochemistry and helped to direct research projects of graduate and medical students at Baylor College of Medicine, Houston, TX. Dr. Anderson was a post-doctoral researcher in the Molecular Genetics Division of the Department of Ophthalmology at the Houston Neurosensory Center.

Dr Anderson was a member of both the undergraduate and graduate faculty at Lamar University, Beaumont, TX. There he taught and directed the research activities of undergraduates and Masters of Science degree candidates in Biology. Currently he is professor of biochemistry at The Master’s University in southern California.

Dr Anderson’s research interests include structure-function studies of DNA polymerizing enzymes and the synthesis and expression of synthetic human genes in bacterial hosts. He has authored or co-authored several publications in major, peer-reviewed journals. He is a member of the American Chemical Society and Sigma Xi Research Society.

Recommended Resource: Foresight, a new book by Brazilian scientist Marcos Eberlin, gives numerous examples in nature of processes and systems that required planning and foresight to work. This pro-ID book has been endorsed by 3 Nobel laureates.