Interaction of peptides corresponding to mitochondrial presequences with membranes.

D.W Hoyt,D.M Cyr,L.M Gierasch,M.G Douglas

doi:10.1016/s0021-9258(18)54692-5

D.W Hoyt, D.M Cyr + Show 2 more

Open Access

https://doi.org/10.1016/s0021-9258(18)54692-5

Copy DOI

Abstract

The transport of the F1-ATPase beta-subunit precursor into mitochondria is dependent upon a presequence at its amino terminus. Within the mitochondrial membrane translocation site the potential amphiphilic character of the presequence region may be necessary to stabilize binding to the mitochondrial inner membrane. To better understand its role in protein import, the interaction of the F1 beta-presequence with lipid membranes was measured using circular dichroism and surface tensiometry. These studies reveal that a 20-residue peptide containing the F1 beta-presequence binds to phospholipid vesicles (Kd = 4.5-6.0 x 10(-8)M and adopts a predominantly alpha-helical structure. Although the presequence peptide binds avidly to lipids, it does not appear to penetrate deeply into the bilayer to perturb a reporter probe in the membrane interior. Compared with the effect of the peptides with demonstrated membrane insertion and lytic properties, the F1-beta-presequence appears to displace phospholipid head groups but not insert deeply into the bilayer. High concentrations (greater than 50 microM) of presequence peptides are required to noticibly perturb import of the full length F1 alpha- or F1 beta-subunit precursors. Thus, the F1 beta-presequence alone is not sufficient to efficiently compete for import but may require a protein context or a minimal length to assist insertion into the transport site. These observations are discussed in light of the different requirements for import of various presequence containing precursors into mitochondria.

Highlights

Into mitochondria is dependent upon a presequence at Insight into the mechanism of presequence selectivity and its amino terminus
Structure of Presequence Peptides-The F1-ATPase @-subunit presequence is required for the import of the precursor protein into mitochondria (Vassarotti et al, 1987; Bedwellet al., 1987;Walker et al, 1990). the @-subunit precursor contains redundant import information (Bedwell et al, 1987), as little as the first 15 residues alone is sufficient to promote import of this precursor or other passenger proteins w into mitochondria both in vivo and in vitro (Vassarotti et al, 1987; Walker et al, 1990)
Mitochondria were incubated for 5 min with the indicated concentration of peptide prior to theinitiation of import a t

Summary

RESULTS

Structure of Presequence Peptides-The F1-ATPase @-subunit presequence is required for the import of the precursor protein into mitochondria (Vassarotti et al, 1987; Bedwellet al., 1987;Walker et al, 1990). the @-subunit precursor contains redundant import information (Bedwell et al, 1987), as little as the first 15 residues alone is sufficient to promote import of this precursor or other passenger proteins w. Mitochondrial DeDtide conformationfrom CD SDectra and SDS. These conditions have been shown in earlier studies to influence the conformation properties of peptides under. The observed CD spectra (Fig. 2) were fit to combinations of a-helix, p-structure,and random coil standards (Greenfield andFasman, 1969). Both the D and L sequence peptides and the control

Presequenceand Signal peptides

Kd nM

DISCUSSION