Download Study of a point mutation in the mitochondrially

A66
Biochemical Society Transactions (200 I ) Volume 29, Part 3
Chlamydomonas reinhardtii mitochondrial mutants lacking
complex I activity: characterization of the mutations and
assembly of the multimeric complex I
P.Cardol, R.F.Matagne, C.Rernacle
Genetics of microorganisms, B22, University of Liege, B-4000
Liege, Belgium
3
In Chlamydomonas reinhardtii, only five subunits (ND1, ND2,
ND4, ND5, ND6) of the mitochondrial NADHubiquinone oxidoreductase (complex I) are encoded in the mitochondrial
genome. Four mitochondrial mutations leading to complex I inactivation have been identified: dumj is a frameshift mutation in the
3’ non coding sequence of the ndj gene, resulting in a low
amount of the corresponding transcript; durn17 and durn20 are
frameshift mutations located in nd6 and ndl , respectively; durn25
is a deletion eliminating two amino acids of ND1. The four
mutants are currently investigated for complex I assembly by
BN-PAGE analyses and detection of complex I by staining and
immunological reactions. Frameshift mutations in ndl or nd6
prevent a correct assembly of complex I, although a peripheral
arm seems to be present. In contrast, the capacity to assemble a
complete complex I is preserved in dumj and durn21, although,
for the former mutation, with an apparent decreased efficiency or
a reduced stability of the complex.
Supported by grants from the Belgian FNRS (1.5.21 1.99 and
2.4552.01). P.C. and C.R. are Research Fellow and Research
Associate from FNRS, respectively.
5 FUNCTION A N D REGULATION O F FUNGAL
ALTERNATIVE OXIDASE
T. loseph-Horne, C. Reeve, A. Hall’ and P. M. Wood
Department of Biochemistry, School of Medical Sciences,
University of Bristol, University Walk, Bristol, BS8 1 TO, UK. ‘$
Syngenta, Jealott 5 Hill, International Research Centre, Bracknell,
Berkshire, RG42 6EI; UK.
Most fungi utilise a branched respiratory chain involving several
N A D H dehydrogenases and at least two terminal oxidases
(cytochrome c oxidase and alternative oxidase (AOX)). Whilst
little is known concerning the function and regulation of the
N A D H dehydrogenases, significant advances have recently been
made in understanding the roles of the terminal oxidases. This
present study will concentrate on AOX from the phytopathogen,
Botrytis cinerea. As observed in other fungi, B. cinerea developmentally expresses a functional AOX. Experiments with specific
inhibitors showed that both core and alternative pathways were
necessary to maintain electron flux rates. Inhibition of AOX led
to a partial collapse of mitochondrial membrane potential. This
implies that electron flow from N A D H to AOX leads to proton
translocation, almost certainly mediated by Complex I. As
further differences from the plant AOX, activity was up regulated
by high matrix ADPATP ratios, whilst there was no evidence for
pyruvate regulation. In addition to AOX’s involvement in pmf
generation, AOX provides a protective mechanism against ROS
generation after inhibition of the core pathway, which may have
particular relevance during pathogenesis.
Biogenesis of the eukaryotic photosystem I complex
6 K.AII
and S.Purton
4 Study of a point mutation in the mitochondrially-encoded
subunit 111 of cytochrome oxidase affecting the complex
assembly in yeast
A. Kapazoglou and B. Meunier
Wolfson Institute for Biomedical Research, University College
London, Cower Street, London WC1E 6AE
Mitochondria1 cytochrome oxidase is a complex multimeric
enzyme with up to 13 subunits, which has both nuclear and mitochondrial genetic origin. Three subunits (subunits I, I1 and 111)
are mitochondrially-encoded and form the catalytic core of the
enzyme. Subunits I and I1 bind the haems and copper redox
centres. Subunit 111 has no redox centre and is likely to be
involved in the assembly or stability of the complex.
In yeast, the mutation S203L in subunit 111abolishes the respiratory growth and alters the optical signal of cytochrome oxidase.
We have isolated second-site reversions which partially restore the
respiratory function: G104S in subunit 111, and Q137K and
A224V in subunit I. interestingly, disease-associated mutations
located in these regions have been reported. We are currently
studying the effects of the primary mutation and of the reversions
on the enzyme activity and assembly.
0 2001 Biochemical Society
Department of Biology, University College London, Darwin
Building, Cower Street, London WC1E 6BI; U.K.
Photosystem I (PSI) is a membrane protein complex composed of
a large number of polypeptide subunits, designated PsaA to
PsaN. In eukaryotes, the genes for these subunits are distributed
between the nuclear and chloroplast genomes. The function of
many of the small nuclear-encoded subunits is not known, and
mutational studies using cyanobacteria have not proved useful
because of fundamental differences between the prokaryotic and
eukaryotic complexes (for example, PsaG, PsaH and PsaN are all
absent from cyanobacterial PSI). We are investigating the role of
three nuclear-encoded subunits: PsaI PsaL and PsaN, which may
be involved in the assembly of PSI. We have cloned the genes for
these subunits in Chlamydomonas. The sequencing of psaN has
been completed and the sequencing of psaI andpsaL is in
progress. These genes and the rest of the PSI nuclear-encoded
genes will be used as probes to screen a large collection of PSI
nuclear mutants. Isolation of mutants lacking the genes will
provide an insight into their function and a valuable null
background for structure-function studies, in which site-directed
variants of the gene are re-introduced into the nuclear genome.
Progress to date will be reported.