Direct Membrane Insertion of Voltage-dependent Anion-selective Channel Protein Catalyzed by Mitochondrial Tom20

Abstract

Insertion of newly synthesized proteins into or across the mitochondrial outer membrane is initiated by import receptors at the surface of the organelle. Typically, this interaction directs the precursor protein into a preprotein translocation pore, comprised of Tom40. Here, we show that a prominent β-barrel channel protein spanning the outer membrane, human voltage- dependent anion-selective channel (VDAC), bypasses the requirement for the Tom40 translocation pore during biogenesis. Insertion of VDAC into the outer membrane is unaffected by plugging the translocation pore with a partially translocated matrix preprotein, and mitochondria containing a temperature-sensitive mutant of Tom40 insert VDAC at the nonpermissive temperature. Synthetic liposomes harboring the cytosolic domain of the human import receptor Tom20 efficiently insert newly synthesized VDAC, resulting in transbilayer transport of ATP. Therefore, Tom20 transforms newly synthesized cytosolic VDAC into a transmembrane channel that is fully integrated into the lipid bilayer.

Membrane insertion of the chloroplast outer envelope protein, Toc34:constrains for insertion and topology

“…Both proteins were found to associate with the membrane (Fig. 3B, lane 2); however, after competition for nonspecific binding using N-liposomes (Schleiff et al, 1999;, only a small amount of Tic40 remained bound to the liposomes (Fig. 3B, lane 3), and this was rapidly degraded by the addition of thermolysin (Fig.…”

“…The multilamellar vesicles were extruded 21 times through a 100 nm pore polycarbonate filter mounted in the mini-extruder (Liposofast, Armatis, Mannheim, Germany) to give unilamellar liposomes (MacDonald et al, 1991). The insertion of Toc34 and mutants into the liposomes was carried out as described (Schleiff et al, 1999;). …”

The outer membrane form of the mitochondrial protein Mcr1 follows a TOM‐independent membrane insertion pathway

“…We further tested the insertion capacity of mitochondria harbouring the tom40-3 temperature-sensitive allele of Tom40. Mitochondria containing this Tom40 variant show an impaired import of presequence-containing precursor proteins [9,17]. Mitochondria isolated from this mutated strain could insert the 34 kDa form with efficiency similar to that of mitochondria isolated from the corresponding parental strain (Fig.…”

A GTP-driven motor moves proteins across the outer envelope of chloroplasts

“…The labeled protein was incubated with empty or proteoliposomes in 20 mM Hepes (pH 7.6)͞125 mM NaCl͞0.5 mM MgCl 2 , and indicated amounts of nucleotide for 5 min at 25°C, followed by competition with N-liposomes [10 mM final lipid concentration, extruded in 20 mM Hepes (pH 7.6)͞125 mM NaCl; ref. 25] for 10 min at 25°C. Both liposome species were separated by centrifugation through a cushion containing 200 mM sucrose.…”