The Mitochondrial Free Radical Theory of Aging - Supernova: Pliki

Transgenic Copies of mtDNA: Techniques and Hurdles
Chlamydomonas reinhardtii, offers even more opportunity: its mtDNA encodes only seven
of the 13. 21 But, currently, there is no information regarding the missing six. Much further
work is clearly needed in identifying and experimenting with nuclear-coded homologues of
our mt-coded genes.
A second highly relevant aspect of import is chaperones. The (nuclear-coded) beta
subunit of the yeast ATPase has an anomalously long presequence; it was recently shown 15
that this sequence acts as a cis-chaperone, keeping the business end of the protein in a
semi-unfolded state while it is still in the cytosol, so that import is easier than if it had folded
up into its final configuration. An even more encouraging finding of this study 15 was that
this property of the presequence was not specific to that one protein: it also accelerated the
import of other proteins to which it was attached. It has not yet been tried on the mt-coded
proteins; the results will be of great interest.
15.11. Do We Really Need Complex I?
I mentioned in Section 2.4.4 that the Saccharomyces cerevisiae (yeast) mtDNA encodes
only six of the 13 proteins that ours does, but that this was not helpful in the same way as the
plants discussed above, because the seven others are not present in the S. cerevisiae nucleus
either. The huge, 40-odd-subunit Complex I that accepts electrons from intramitochondrial
NADH and passes them to ubiquinone has been completely discarded, and in its place there
is an enzyme which is composed of only one (nuclear-coded) polypeptide. This enzyme
does exactly the same job ... except for one thing: it does no proton-pumping.
This led most people, myself included, to give no further thought to the relevance of
yeast to development of this technology. Seo et al 22 were not so unimaginative. A number of
diseases are known which stem from mutations in Complex I subunits, so it is clear that we
need it; but, they thought, do we actually need its proton-pumping? Could the toxicity of
these mutations be primarily due, instead, to the production of more LECs, or to the impaired
recycling of NADH back to NAD + ? To test this possibility, they introduced a copy of
the yeast gene into mammalian cells: this was easy, because (being nuclear-coded) it already
uses the same genetic code, and furthermore the mammalian mitochondrial processing
peptidase (the protein which removes the presequence of imported proteins after they are
imported into mitochondria) recognises the same cleavage sequence as the yeast one, so no
re-engineering whatsoever was needed. At the time of writing only very preliminary results
are available, but cells mutant in Complex I but expressing this transgenic yeast gene appear
to grow faster than controls, and also to use less glucose, indicating that they are performing
OXPHOS with the yeast protein. A further indication of OXPHOS function is that these
cells can grow well on galactose, which is a much poorer substrate for glycolysis than glucose.
23 Furthermore, they appear to generate fewer LECs: this may be because Complex I is
probably the major site of LEC production in the respiratory chain, whereas the yeast enzyme
may work by direct two-electron transfer from NADH to ubiquinone, i.e., not generate
the risky ubisemiquinone intermediate. (Recall that this is thought to be how the mammalian
FAD-dependent electron transporters, such as Complex II, avoid LEC
production—see Section 11.2.1.) If this idea really works, the impact on development of
protein import would be dramatic, since, of the six mt-coded proteins which have so far not
been found nuclear-coded in any species, four are subunits of Complex I.
15.12. Cotranslational Import
The idea that a protein’s hydrophobicity hinders its importability into the mitochondrion
is based on the assumption that the complete protein exists in the cytosol at some point.
This is not necessarily so. Many extracellular proteins are secreted from the cell while they
are being built, so that only a very short stretch of the protein is ever in the cytoplasm. There
183