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RESEARCH
Our
research focuses on two broad aspects of mitochondrial function
i) How does mitochondrial porin fold and function in the outer membrane?
ii) How is mitochondrial DNA maintained?
i) How
is mitochondrial porin arranged across the outer membrane?
Mitochondrial porin resides in the outer membrane, and allows movement of
small metabolites across the lipid bilayer. It acts as a
voltage-dependent, anion-selective channel, leading to its other name,
VDAC. In addition to allowing the exchange of substrates and products for
mitochondrial metabolism, porin has also been implicated as a key
regulator in processes such as apoptosis. The protein is predicted to
span the mitochondrial outer membrane as a series of beta-strands, as
does its bacterial namesake. Our phylogenetic analysis of almost 300
mitochondrial porin sequences indicated a common arrangement of
beta-strands in porins from plants, animals and fungi (Young et al.
2007). Our initial efforts towards determining porin structure
concentrated on expression of mitochondrial porin in Escherichia coli, and refolding in detergent. We identified a
mixture of ionic and non-ionic detergents (SDS and DDM) that support
folding of porin at high concentrations (Bay et al.
2008a). The next stage was to assess the effects of the native
fungal sterol, ergosterol, and its human counterpart, cholesterol on the
folded state of detergent-solubilized porin. Subtle changes in folding
were detected when ergosterol was added to LDAO-solubilized porin, while
only cholesterol influenced the folding of porin in SDS/DDM (Bay et al.
2008b).
Since
this work, three other labs have published structures of human VDAC
obtained through NMR or x-ray crystallography (Ujwal et al. 2008;
Bayrhuber et al. 2008; Hiller et al. 2008). Our focus has
shifted to in vivo studies of
the Neurospora porin, namely
i) is it
essential in Neurospora?
ii) do
variants that form pores in artificial membranes also function in vivo,
and if so, is their function altered from that of wild-type?
iii)
which portions of the protein are essential for interactions with other
cellular components?
ii) Mitochondrial DNA replication
Function of the C-terminal extension of the
mitochondrial DNA polymerase of Saccharomyces
Mitochondrial
DNA is replicated by a dedicated, nuclear-encoded mitochondrial DNA
(mtDNA) polymerase. The mtDNA polymerase of fungi bears an enigmatic
carboxyl-terminal extension (CTE) of up to 300 amino acids. CTE are
limited to several groups of fungi, but vary extensively in length and
amino acid sequence. Using yeast, we have demonstrated that only the
polymerase-proximal portion of the CTE is required for mtDNA maintenance,
and that certain variant mtDNA polymerases that lack more than half of
the CTE are either temperature sensitive for mtDNA maintenance (Young, et al.
2006). Current work focuses on the following questions:
i) What role does the CTE play in the enzymatic activites
of fungal mitochondrial DNA polymerases?
ii) What are the crucial parts of the CTE, and do they
interact with other components of the mitochondrial DNA replication
machinery?
Other contributors
to mtDNA maintenance
A variety of laboratory strains of Saccharomyces are in common use for mitochondrial studies.
However, known marker alleles such as ade2
and his3D-200 and have negative effects on
fidelity of mtDNA replication, and mtDNA maintenance, respectively. Other
“hidden” traits, such as the hap1
mutation in the S288c-based strains, and the version of the mitochondrial
DNA polymerase present, also affect mitochondrial function (Young and
Court, in press). The link between mitochondrial translation and mtDNA
maintenance remains a topic of interest.
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