Date on Master's Thesis/Doctoral Dissertation

8-2009

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Biochemistry and Molecular Biology

Committee Chair

Wattenberg, Brian

Author's Keywords

Hydrophobic pockets; Mitochondrial targeting; Carboxy-terminal tail

Subject

Proteins

Abstract

Here, I address the function of the carboxy-terminal hydrophobic helix of the pro-apoptotic protein Bax. There has been considerable controversy as to whether this sequence is required for the targeting and insertion of Bax into the mitochondrial outer membrane. The Bax carboxy-terminal tail is tucked into a hydrophobic pocket within the closed/inactive conformation of Bax. Apoptotic stimulation results in an opening of the Bax conformation, exposing a mitochondrial-targeting signal and subsequent insertion of Bax into the mitochondrial outer membrane. Here, I confirm that the Bax tail alone can specifically target and anchor a cytosolic passenger protein to the mitochondria. Surprisingly, however, I find that the carboxy-terminal tail is not responsible for the specific targeting of Bax to the mitochondria rather than other cellular membranes. Specifically, replacing the Bax tail with an ER-targeting tail-anchor had no effect on Bax mitochondrial targeting, in the context of full-length Bax. This contrasts to the targeting function of tail-anchor signals in other tail-anchored proteins. In addition, I demonstrated that the Bax tail has a negative regulatory effect on Bax activation. Mutations that disrupt the interaction of the Bax tail with the hydrophobic pocket resulted in an open/active conformation of Bax and constitutive mitochondrial targeting. Deletion of the Bax tail also resulted in an open/active conformation of Bax, however the anchor-deleted form of Bax was not associated with mitochondria. This indicates a requirement of the Bax tail for mitochondrial translocation. By introducing charged residues into the tail sequence to block insertion of the sequence into the hydrophobic bilayer, I show that insertion of the Bax tail is required for Bax mitochondrial targeting. My data support a model whereby the Bax tail must be released from its hydrophobic pocket to initiate the change into an open/active conformation. The tail then functions as an anchor to stabilize Bax at the mitochondrion after the initial addressing step.

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