| Abstract |
Almost 500 Escherichia coli proteins are secreted post-translationally through the
bacterial translocase (SecA-SecYEG). Secretory proteins are comprised by the signal
peptide and the mature domain. The two parts have targeting signals and bind on
distinct sites on the translocase. The subject of the thesis is to resolve the nature of the
targeting signals on the mature domain. Using several secretory proteins we found
that ~100 amino acids are sufficient for targeting. Lowering the amino acids length,
the affinity is reducing, implying that ~100 amino acids are necessary for targeting.
The reduction coincides with the removal of the hydrophobic patches of the sequence.
Mutagenizing the hydrophobic amino acids of the patches and making the sequence
neutral the affinity is lowering. In the absence of a signal peptide the affinity is
abolished. We conclude that for mature domain targeting, the substrate must have at
least one hydrophobic patch. It is known that a protein is stable in a soluble
environment when it buries its hydrophobic core by folding. A prerequisite for
targeting is the protein to be unfolded. How do secretory proteins manage to expose
their hydrophobic patches in a soluble environment? Testing the behavior of the
secreted proteins in soluble environment we discovered that 50% remain soluble,
targeting and translocation competent. A condition that we name natively unfolded.
The other 50% precipitates mainly because of their signal sequence. The cell deals
with the aggregating proteins by having several different chaperones (SecA, SecB,
Trigger factor) that prevent their aggregation. The chaperones apart from preventing
the aggregation they maintain the proteins into a translocation competent state. Next
we tested if the cytoplasmic proteins become targeted. Cytoplasmic proteins have
hydrophobic patches similar to the secreted and they become targeted to the
translocase. The period of time they remain targeting competent is less compared to
the secreted. Concluding the mature domain targeting signals are 1. The hydrophobic
segments and 2. The non-native code. The secretory proteins have been evolved to
stay unfolded and become targeted. The expense of staying unfolded –in some casesis
to aggregate, a cost that the cell takes in order the proteins to remain non-native.
Next we searched for the targeting signals on SecA. SecA has hydrophobic patches
on its cytoplasmic side. We immobilized the c-tail on a hydrophobic patch the affinity
was reduced 10 times. We identified a hydrophobic patch that participates in targeting although it is not unique. We conclude that targeting is mediated through hydrophobic
interactions.
To test if the signal peptide affects targeting, we exchanged the signal peptide of
alkaline phosphatase with 23 other E.coli signal peptides. The targeting changed
slightly but the secretion and the activation energy of the holoenzyme was highly
affected.
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