Structural and functional studies on the signal peptide of rat liver mitochondrial aldehyde dehydrogenase

Yi Wang, Purdue University

Abstract

It was previously shown that the signal peptide of rat liver mitochondrial aldehyde dehydrogenase in a micelle environment contained a N-helix, a C-helix and a flexible linker between the two helices. It was now determined that both the N-helix and the C-helix the signal peptide are necessary for it to function in the import of ALDH into mitochondria. Switching the positions of the two helices did not affect the import. Replacing the C-helix of ALDH signal peptide with the N-terminal helix of the cytochrome c oxidase subunit IV signal peptide also did not affect import. However, the import efficiency was remarkably decreased by replacing the C-helix with the C-terminal random coil of the cytochrome c oxidase subunit IV signal peptide. Circular dichroism studies showed that the stability of the N-helix of ALDH signal peptide was much weaker than that of the COX IV's N-helix. A helical conformation at the C-terminal portion would increase the stability of the N-helix of the ALDH signal peptide. Deletion of the linker caused the two short N-helix and C-helix to merge into a long continuous one. This linker deleted signal peptide had a similar import efficiency as did the native ALDH signal peptide. However, the linker deleted signal peptide could not be processed after import. It is suggested that a short, possibly 8-12 residue, amphiphilic helix at the N-terminal portion of presequence is necessary and sufficient to direct protein import into mitochondria. The interaction of ANCN (ALDH signal peptide with its C-helix replaced by the N-terminal helix of COX IV), ANCC (ALDH signal peptide with its C-helix replaced by the C-random coil of COX IV) and the linker deleted ALDH signal peptides with phospholipid bilayers was investigated by various techniques. Results showed that ANCC, which displayed a weak import capability, had a very low affinity for binding to the negatively charged membranes. Both ANCN and -linker showed a relatively high affinity. It appears that -linker possesses a stronger hydrophobic effect with membranes while ANCN had a higher electrostatic interaction. Based on these studies, a model was proposed to describe the interaction of mitochondrial signal peptides with negatively charged phospholipid membranes involving electrostatic interaction for initial binding and hydrophobic interaction for insertion.

Degree

Ph.D.

Advisors

Weiner, Purdue University.

Subject Area

Biochemistry|Molecular biology

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