The Mitochondrial Free Radical Theory of Aging - Supernova: Pliki

Transgenic Copies of mtDNA: Techniques and Hurdles
amino-acid sequence. Import involves the unfolding of this structure, 13 so that a protein
which has just been imported is in fact completely useless until it has resumed its proper
configuration. The refolding process seems to differ greatly from one protein to another:
some seem to refold all on their own, while others need help from “chaperone” molecules.
Mitochondrially encoded proteins must also, somehow, fold into the correct configuration
as they come into existence during translation, but we have no idea whether the machinery
(if any) that does this will work on a protein that is emerging from the membrane rather
than from a ribosome. We’ll just have to try it.
15.8. Fallacious “Existence Proofs” That This Cannot Work
As if all the obstacles discussed in this chapter were not enough, the plausibility of
this proposed treatment has also been challenged on the basis that we have no idea what
other, as yet unknown vital function mtDNA may have, that has caused its retention during
evolution. It is certainly fair to ask why mtDNA still exists, and if no satisfactory answer
can be found then one would be justified in worrying that its replacement by nuclear
transgenes might have unknown deleterious effects. That is why I laid out in so much
detail, in Section 10.2, the best guess as to why mtDNA still exists. Given such a robust
explanation, I claim that there is no reason to anticipate such problems. If they arise, of
course, we will have to tackle them; but we can cross that bridge when we come to it. Two
possibilities should be mentioned, however, since they may be testable.
One suggestion 14 was based on a similarity between one mt-coded gene, ATPase subunit
8, and a bacterial toxin called hok. Why is this potentially relevant? Well, the role of a protein
in the cell comprises not only what it does, but also what it does not do; many mutants in
model organisms cause their phenotype by expressing a perfectly correct protein in an
incorrect place, where it happens to be toxic. We have no information* regarding how the
various mitochondrially-encoded proteins might interact with cytosolic ones, as they would
be in danger of doing if they were constructed by cytosolic ribosomes. It would be highly
valuable to explore this possibility at an early stage in the attempt to develop the proposed
transgenes; this can of course be done before import has been perfected, since it is the
behaviour of the protein prior to import which is of interest. If any toxicity is discovered,
techniques to circumvent it may include use of a presequence or chaperone that prevents
the offending protein from adopting its usual three-dimensional configuration (and hence
activity) until after import, as will be discussed in another context in Section 15.10. 15
Another proposed reason “why mitochondria need a genome” 16a is that the mt-coded
proteins have sequence characteristics that are preferentially recognised as targets for export
from the cell, so that if they were nuclear-coded they would end up on the outside of the cell
before they could be targeted to mitochondria. This is an intriguing idea, but is based only
on sequence similarities, not on observation of such export. And again, even if it turns out
to be true it is something that we might hope to subvert by suitable presequences and/or
chaperones.
Finally, as noted in Section 15.4, there has been one failed attempt at allotopic expression—with
ATPase subunit 6. (Actually there have surely been many other such failures, but
this one has actually been reported—albeit only in a meeting abstract.) 16b Not only did the
transgenic copy fail to rescue a mutation in the endogenous gene: it actually impaired
OXPHOS in mitochondrially wild-type cells. This was interpreted by some as implying
cytosolic toxicity. However, the study involved expressing the protein at very high levels. If
its import was failing, due for example to its hydrophobicity (see Section 15.9), then the
* However, this is another possible explanation for the non-function of a transgenic ATPase subunit 6 (see
Section 15.4)
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