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The Signal Recognition Particle (SRP)-dependent membrane trafficking pathway plays a role in the regulatory control of the heat-shock transcription factor, sigma32


In E. coli, the heat shock transcription factor, s32, senses and responds to perturbations in protein homeostasis. The output of the heat-shock response (HSR) is finely tuned to the amount of stress present; and fine-tuning requires several s32 regulon members (molecular chaperones and proteases) whose ability to impart regulatory control on s32 depends on the protein homeostatic state of the cell. While regulation of s32 has been well studied, several lines of evidence indicate a missing player or mechanistic step involved in s32 regulation. Specifically, several s32 mutants were found to no longer be sensitive to the regulatory controls of chaperones and proteases. While these mutants are no longer inhibited by chaperones or degraded by proteases in vivo, they are sensitive to both chaperone-mediated inhibition and protease-dependent degradation in vitro, strongly indicating a missing component in s32 regulation.

In this work, I identified a new player to be the Signal Recognition Particle (SRP), a component of the co-translational trafficking machinery involved in transporting proteins to the inner membrane. I identified an interaction between s32 and the co-translational trafficking machinery in vivo; and determined that s32 interacted directly with the SRP through several in vitro studies. Additionally, I identified a membrane-localized s32 population and that localization is dependent on the SRP-dependent trafficking pathway. I also found that a severely-defective s32 regulatory mutant, I54Ns32, is additionally defective in SRP-binding; and that this defect in binding led to a significant decrease in membrane fractionation of the mutant.

To understand the biological significance of SRP-binding and membrane localization, I tethered s32 to the membrane in vivo, and found that membrane-tethered s32 is more sensitive to the regulatory controls of both proteases and chaperones. Most importantly, membrane-tethering I54Ns32 restored its sensitivity to chaperones and proteases, indicating that the regulatory defects observed in I54Ns2 are due to its defect in membrane localization. These observations have led me to propose that SRP and subsequent transport of s32 to the membrane is an additional regulatory step for s32 control, allowing s32 to sense flux of proteins to the membrane in addition to protein folding homeostasis.

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