Verlag des Forschungszentrums Jülich
JUEL-4110
Köberling, Oliver
Mechanismen der Qualitätskontrolle bei der Sec-abhängigen Proteintranslokation in Gram-positiven Bakterien
121 S., 2004
Translocation of proteins across the bacterial cytoplasmic membrane is catalyzed by the Sectranslocase consisting of the SecA ATPase and the transmembrane proteins SecY, SecE and SecG. SecA mediates the stepwise translocation of the export protein by ATP dependent cycles of insertion and deinsertion into the SecYEG channel. Initiation of translocation requires the formation of functional interactions between at least SecA, SecY and the precursor protein. This proofreading mechanism ensures that among the homologous proteins only those designated for export are translocated. During the secretion of heterologous proteins this quality control step can cause a severe bottleneck.
To investigate the molecular mechanisms underlying the quality control in vivo a Bacillus subtilis SecA replacement mutant (RMA) containing a clean chromosomal replacement of the B. subtilis secA gene by the secA gene of Staphylococcus carnosus was used as a genetic tool. This gene replacement results in an artificially increased proofreading activity of the hybrid translocase resulting in a conditional growth defect at 25 °C due to pleiotropic export defects at low temperature. In contrast, the heterologous outer membrane protein A (OmpA) of Escherichia coli is selectively excluded from export even at the permissive temperature (37 °C) . Furthermore the RMA shows defects in sporulation and development of genetic competence.
In this work spontaneous mutants of the RMA were selected for suppression of the cold sensitive growth and the sporulation defect, respectively . Interestingly, all suppressor mutants now also showed an improved OmpA export suggesting a reduced quality control activity compared to the RMA. The identification of the mutations of seven mutants showed that the suppressors contained single amino acid exchanges in the SecA of S. carnosus. Suprisingly, the positions of four mutations correspond exactly to the positions of known mutations in the SecA of E. coli resulting in a pre-activation of the SecA protein by elevation of the basal ATPase activity ("superactive"
SecA). Therefore it is very likely that the respective mutations of the suppressor mutants
restore the OmpA export by super-activation of the S. carnosus SecA protein. This strongly
suggests that in the RMA a non optimal activation of the SecA ATPase activity by the
heterologous OmpA protein is the limiting step during translocation. Another mutation was located in a region of the S. carnosus SecA which, as identified in the SecA of E. coli, interacts with SecY. This strongly suggests that in this mutant the activation of the SecA ATPase by the OmpA is restored by optimized interactions between the SecA of S. carnosus and the SecY of B. subtilis.
Since the RMA is an artificial genetic system, five single mutations were transferred
independendly into the corresponding positions of the SecA of B. subtilis. Expression of three
mutated SecA proteins in the homologous background indeed resulted in a more than 3-fold improved export of a variant on the B. subtilis alkaline phosphatase PhoB which is only poorly exported in the B. subtilis wildtype. This suggests a conserved function of the respective amino acid residues in the regulation of the ATPase activity in the B. subtilis and S. carnosus SecA. The results obtained in this study show for the first time that the efficiency of translocation of a heterologous protein is dependent on its ability to activate the SecA ATPase. A non optimal activation can be bypassed either by optimizing interactions between SecA, SecY and the export
protein or by pre-activating SecA.
Since the SecY protein is another important component of the translocation initiation complex several mutations which result in a reduction of the proofreading activity when present in the transmembrane segment 10 of the E. coli SecY were introduced into the corresponding positions of the B. subtilis SecY. Indeed, in two cases the export of the PhoB variant was significantly improved, clearly showing that SecA and SecY are essential components of the quality control system of the B. subtilis translocase and variations in either of the subunits can improve the translocation of proteins which are only poorly exported in a B. subtilis wildtype strain.
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