Growth, Differentiation and Sexuality

194 H.D. Osiewacz and A. Hamann
ever, a lowered ROS scavenging system can be tolerated
in this mutant because respiration switches
to being dependant on the alternative oxidase AOX,
generating lower ROS than is the case for standard
respiration (Borghouts et al. 2002b). This switch is
a retrograde response induced by impairments of
the assembly of cytochrome c oxidase due to copper
depletion.
Cytochrome c oxidase binds three copper
molecules per complex. One copper is bound to
subunit I, and two atoms in subunit II (Capaldi
1990; Yoshikawa et al. 1998; Richter and Ludwig
2003). Depletion of copper was demonstrated to
substantially impair COX assembly (Glerum et al.
1996; Nobrega et al. 2002; Barros et al. 2004).
Consequently, copper deficiency results in
a failure to assemble complex IV of the respiratory
chain, and therefore leads to a deficiency in
standard respiration. In principle, such a situation
should be lethal for a strict aerobe like P. anserina.
However,inthegriseamutant,duetothefactthat
copper deficiency is not complete because low
levels of copper are transported into the cell via
a low-affinity copper uptake system, low standard
respiration is observed. Moreover, P. anserina
possesses a copper-independent, iron-containing
alternative terminal oxidase, AOX, which can replace
the COX in the respiration chain (Borghouts
et al. 2001). AOX is located upstream of complex
III of the respiratory chain, and therefore the
formation of electron motive force is restricted
to complex I. The OXPHOS complexes, organized
in large supramolecular structures termed
respirasomes in this mutant, clearly differ from
that of the wild-type strains (Krause et al. 2004),
and the generation of ATP through this kind of
respiration chain is reduced (Fig. 10.1A). Most
interestingly, the mutant’s life span is increased
about 60% compared to the wild-type strain. This
life span increase can be explained by different
factors: (1) a reduction of mtDNA reorganizations
due to copper deficiency (see above) and (2)
a reduced production of ROS. In comparison
to the standard COX-dependent respiration, the
alternative respiration results in the generation of
a lower membrane potential (Wagner and Moore
1997). This lower membrane potential and the
bypass of the superoxide anion generation at
complex III result in a lower production of ROS
through the alternative pathway. Which one of the
two factors given above contributes most to the
increase in life span remains unclear. However, it
is interesting to note that, in contrast to the ex1
mutant of P. anserina, the grisea mutant is not
immortal,althoughinbothmutantsthealternative
respiratory pathway is induced. Part of the answer
appears to be that ex1, due to the deletion of large
parts of the CoxI gene, respires exclusively via
the alternative pathway whereas grisea uses both
pathways.
In the long-lived grisea mutant, copper
metabolism is affected in the whole cell. Consequently,
not only mitochondrial functions are
impaired. Importantly, the copper-zinc-dependent
superoxide dismutase (Cu/Zn SOD, SOD1),
a scavenging enzyme in the cytoplasm and the
mitochondrial intermembrane space, is severely
impaired, resulting in a failure to cope with
accumulating oxidative stress in aging cultures
(Borghouts et al. 2002b). The inability of the
mutant to detoxify cytosolic ROS, and a partial
respiration via COX resulting in lower ROS generation
seem to be the key determinants of life span
increase in this mutant.
In order to raise more specific data, transgenic
strains in which more specific targets are affected
have been constructed and analysed. One example
is transgenic strain Cox5:ble in which the nuclearencoded
fifth subunit of cytochrome c oxidase
(Cox5) has been replaced by a selection marker
(Dufour et al. 2000). This mutant respires exclusivelyviaAOXandhasalifespanwhichisincreased
at least tenfold. In another example, Stumpferl et al.
(2004) replaced the endogenous PaCox17 gene by
a selection marker. The PaCox17 gene encodes
a copper transporter which is involved in the
delivery of copper to complex IV of the respiratory
chain (Fig. 10.3). The knockout strain respires via
the alternative pathway and is characterized by
a 17-fold increase in mean life span. However, apart
fromtheswitchinrespiration,thistypeofmitochondrial
copper depletion was found to lead to
a stabilization of the mtDNA and to a changed profile
of the two superoxide dismutases. In contrast
to the wild-type strain, in which juvenile cultures
express high amounts of mitochondrial superoxides
dismutase 2 (SOD2) and low activity of SOD1
but senescent cultures high SOD1 and low SOD2,
the transgenic strain constitutively expresses
high levels of SOD1. Thus, although a specific
target gene has been addressed, the outcome of
modification is multiple once again (Fig. 10.4).
Induction of PaAox is also observed in a strain
carrying a thermosensitive mutation of the gene
oxa1 (Sellem et al. 2005). The OXA1 protein is
involved in the assembly and insertion of differ-