Charge and structural components of mitochondrial leader sequences

Thomas Sweeney Heard, Purdue University

Abstract

Mitochondrial leader sequences have been found to be statistically enriched for positively charged residues, with only a few known leader sequences possessing negatively charged residues. Mutational studies that have introduced negatively charged residues into various leader sequences have shown a general, but not absolute, trend toward reduced import. The leader sequence of rat liver aldehyde dehydrogenase (ALDH) has been previously determined by NMR to form a helix-linker-helix structure. In in vitro experiments, a negative charge introduced into this leader did not prevent import provided that a net positive charge remained in the N-helical segment. When the net charge of the N-terminal helical segment was reduced to zero, import could be recovered by removing the linker, which resulted in a longer, more stable leader. This structural recovery of import was effective enough to compensate for a net charge of zero within the first ten residues, even when a glutamate is the first charged side chain in the sequence. α-Isopropylmalate synthase (Leu 4p) has a leader sequence that also forms a continuous helical structure and contains two glutamates at the N-terminus. This sequence did not import ALDH well. The continuous helical structure of the IPMS4 leader was not as important as a region on the C-terminal side of the glutamates that exits in the native IPMS protein. This region is more rich in positive charge, demonstrating that the targeting signal of a leader does not have to be very close to the N-terminus. In vivo experiments with the native and mutant leaders of ALDH, using the green fluorescent protein (GFP) as the passenger protein, revealed that the native leader was capable of accepting negatively charged resides as demonstrated in the in vitro assays, but linker-deleted leader was found neither to import as well as the native sequence, nor to compensate for loss in positive charge. The folding properties of GFP also revealed that this protein could not be imported by a post-translational mechanism challenging the generally held hypothesis that mitochondrial import is exclusively post-translational.

Degree

Ph.D.

Advisors

Weiner, Purdue University.

Subject Area

Cellular biology|Molecular biology

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