IMPORT, PROCESSING, AND 2-DIMENSIONAL NMR STRUCTURE OF A LINKER-DELETED SIGNAL PEPTIDE OF RAT-LIVER MITOCHONDRIAL ALDEHYDE DEHYDROGENASE

Previous NMR studies (Karslake, C., Piotto, M. E., Pak, Y. M., Weiner, H., and Gorenstein, D. G. (1990) Biochemistry 29, 9872-9878) had show n that a 22-amino acid signal peptide of rat liver aldehyde dehydrogen ase (ALDH) when bound to a micelle had two amphiphilic alpha-helices, one located at the N terminus and the other at the C terminus. It was shown that deletion of either helix caused the precursor protein not t o be imported (Wang, Y., and Weiner, H., (1993) J. Biol. Chem. 268, 47 59-4765). The two helices are separated by a Arg-Gly-Pro flexible ''li nker'' region, and to test the role of this linker region in the impor t and processing of the precursor protein, we deleted it from the ALDH signal peptide and precursor protein. The 19-amino acid signal peptid e of ALDH, to which has been added 3 residues at the C terminus and fr om which has been deleted the 3-residue flexible linker region, has be en studied by two-dimensional NMR in a dodecylphosphocholine micelle. In this membrane-like environment the peptide contains a single alpha- helical segment that extends almost the entire length of the peptide. NH exchange experiments show residues on the hydrophobic face of the p eptide to exchange much more slowly than those of the hydrophilic face . Combined with the previous study, these results suggest that precurs or protein import simply requires a sufficiently long amphiphilic heli x (or helices) to bind stably to the membrane. The N and C helices of native ALDH are only about 6-8 residues long; this represents only abo ut two turns of a helix, and either helix on its own does not provide enough stabilization to ensure folding and binding to the membrane. Th e linker-deleted ALDH peptide contains a single helix of 12-14 residue s that is long enough to provide a hydrophobic surface that can stably interact with the hydrophobic interior of the membrane. The function of the C helix in the native signal peptide is therefore to enhance th e stability and binding of the N-terminal signal to the membrane. Sign ificantly, unlike native ALDH precursor protein, the linker-deleted si gnal peptide precursor protein could no longer be processed after impo rt into mitochondria. As explained by modeling of the alpha-helix and the NH exchange rate data, the precursor protein requires that the fir st several residues of the mature protein be part of the hydrophobic m embrane associated face of the helix. By removing 3 residues in the ce nter of the helix, a phase shift of the amphiphilic helix has occurred , moving the first several residues of the mature protein toward the h ydrophilic face. The proteolytic signal processing protein therefore a ppears unable to recognize these residues.