Bilayer‐penetrating properties enable apocytochrome c to follow a special import pathway into mitochondria

Abstract

In this study, we have investigated the protein/lipid interactions of two mitochondrial precursor proteins, apocytochrome c and pCOX IV‐DHFR, which exhibit mitochondrial import pathways with different characteristics. In‐vitro‐synthesized apocytochrome c was found to bind efficiently and specifically to liposomes composed of negatively charged phospholipids and showed a (at least partial) translocation across a lipid bilayer, as reported previously for the chemically prepared precursor protein [Rietveld, A. & de Kruijff, B. (1984) J. Biol. Chem. 259, 6704–6707; Dumont, M. E. & Richards, F. M. (1984) J. Biol. Chem. 259, 4147–4156]. Negatively charged liposomes were shown to efficiently compete with mitochondria for import of in‐vitro‐synthesized apocytochrome c into the organelle, suggesting an important role for negatively charged phospholipids in the initial binding of apocytochrome c to mitochondria. In contrast, the purified and in‐vitro‐synthesized precursor fusion protein pCOX IV‐DHFR, consisting of the presequence of yeast cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase was unable to translocate across a pure lipid bilayer. The data indicate that the ability of apocytochrome c to spontaneously translocate across the bilayer is not shared by all mitochondrial precursor proteins. The implications of the special protein/lipid interaction of apocytochrome c for import into mitochondria will be discussed.

The mature form of imported mitochondrial proteins undergoes conformational changes upon binding to isolated mitochondria

“…6B). Furthermore, mAspAT bound to liposomes consisting of synthetic phospholipids that mimicked the outer mitochondrial membrane (for their lipid composition, see Endo et al, 1989;Jordi et al, 1992), thus supporting the notion that mitochondrial proteins bind to the lipid component of the membrane. However, the bound enzyme did not show increased proteinase sensitivity.…”

The Toxoplasma gondii protein ROP2 mediates host organelle association with the parasitophorous vacuole membrane

“…By what alternative mechanism is ROP2hc translocated across the MOM? ROP2hc import, presumably exposing its NH 2 terminus to the intermembrane space (IMS), has features reminiscent of apocytochrome c import (Stuart Neupert, 1990;Jordi et al, 1992;Dumont, 1996). ROP2hc shares some potentially important physical features with the IMS-localized cytochrome c and its receptor cytochrome c heme lyase (CCHL) (Lill et al, 1992).…”

Mmicular mechanisms of cytochrome c biogenesis: three distinct systems

“…Besides direct binding to CCHL, another factor shifting the equilibrium of the import reaction involves folding of apocytochrome c in the intermembrane space. Subsequent to synthesis on cytoplasmic ribosomes, the apoprotein has a protease‐sensitive unfolded conformation that may be important for penetration of the outer membrane (Jordi et al ., 1992). After the binding of apocytochrome c to CCHL within the intermembrane space (Nicholson et al ., 1988), a protease‐resistant, native form appears after covalent attachment of haem.…”