Verlag des Forschungszentrums Jülich
JUEL-3546
Schroers, Andreas
Funktionelle und strukturelle Eigenschaften des mitochondrialen Phospharcarriers aus Accharomyces cerevisiae
83 S., 1998
Uptake of inorganic phosphate into mitochondria is essential for energy metabolism. The
import of phosphate into the mitochondrial matrix is mediated by the phosphate carrier (PIC),
a 32 kDa protein which catalyzes the electroneutral import of phosphate (H2PO4)
compensated by the antiport of a hydroxyl ion. We have expressed wild type and mutant PIC from
yeast mitochondria in Escherichia coli inclusion bodies. PIC was solubilized from inclusion
bodies, purified and reconstituted into proteoliposomes in a functionally active form.
The mitochondrial phosphate carrier (PIC) belongs to the mitochondrial carrier family
(MCF). Members of this family comprise six transmembrane segments and have been
shown to exist as dimers in the solubilized state. Beneath the strictly coupled transport-
functions of the PIC there is an uncoupled transport mode which can be observed after
modification of the carrier with HgCl2. After the analysis of mutants in which the three cysteines
of the wt-PIC were exchanged with serine, the modification of the cystein residue in position
28 turned out to be the trigger for uncoupling. By the introduction of amino acids different in
size and in charge in position of cystein 28 a positive charge was found to be responsible for
the induction of the uncoupled efflux mode.
In order to investigate a functional necessity of the dimerization and the molecular
mechanism of coupling, we have developed a method to isolate tagged proteins as monomers,
to construct defined dimers from these monomers, and to isolate specific dimeric forms. By
functional analysis of heterodimers consisting of functionally different monomers the
following conclusions can be drawn: (i) Dimerization is essential for functionality, (ii) formed
dimers are stable, (iii) there is functional crosstalk between the subunits of the dimer, (iv)
modification (mutation) of one monomer is sufficient for uncoupling. Furthermore the functional
analysis of heterodimers with different amino acids in position 28 provided evidence for
one single pathway within the functional active dimer.
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