Interaction of non‐classical detergents with the mitochondrial porin

Abstract

The effect of different families of detergents on the solubilization and purification of the pore-forming protein (porin) of the mitochondrial outer membrane of bovine heart was investigated in detail. With Tritons, dimethylamine oxides and zwittergents, porin solubilization with respect to total mitochondrial membrane protein was more efficient with the more hydrophobic members of each series. With most detergents the protein eluted as protein-detergent micelles in the void volume of hydroxyapatite/cehte columns. In contrast, the protein was bound to the column material and was eluted after the addition of salt to the elution buffer when the detergents octylglucoside, zwittergent 2-314 and lauryl (dimethyl)-amine oxide were used. The protein purified in the presence of the latter detergent had a higher pore-forming activity in lipid bilayer membranes compared to porin isolated in the presence of Triton X-100. The binding of porin to the hydroxyapatite/celite column was used to study the lipid content of the active pore-forming complex. The analysis revealed that the complex contained no phospholipid but rather five molecules of cholesterol/polypeptide chain.The mitochondria1 outer membrane contains general diffusion pores which are responsible for the free permeability of this membrane towards small hydrophilic solutes [l]. The active component of this molecular sieve is a protein, called mitochondrial porin or voltage-dependent anion channel [2 -41. Mitochondria1 porins were isolated from a variety of eukaryotic cells and their pore-forming properties were studied in reconstitution experiments with planar lipid bilayers and liposomes [2 -51. According to these investigations the mitochondrial pore has a diameter of about 2 nm in the open state and is slightly anion-selective at low transmembrane potentials. Voltages larger than 20 mV result in a shift of the pore into closed states, which have a reduced permeability towards hydrophilic solutes and a completely different selectivity from the open state.The role of the mitochondrial outer membrane in the physiology and metabolism of mitochondria was underestimated in the past. It has been described as a simple barrier that prevented the destruction of the mitochondrial inner membrane during osmotic swelling. More recent papers gave some insight into the function of the mitochondrial outer Abbreviations. (cHxN)zC, dicyclohexylcarbodiimide; CaE3, octyl trioxyethylene; CsE4, octyl tetraoxyethylene; CsE5, octyl pentaoxyethylene; C8(HE)S0, octyl (hydroxyethyl) sulfoxide; Cs(DHP)SO, octyl (dihydroxypropane) sulfoxide; DDAO, decyl (dimethyl)-amine oxide; LDAO, lauryl (dimethy1)-amine oxide; IysoPtdCho, lysophosphatidylcholine; MEGA 6, exanoyl-N-methylglucamide; MEGA 7, heptanoyl-N-methylglucamide; MEGA 8, octanoyl-N-methylglucamide; MEGA 9, nonaoyl-N-methylglucamide; MEGA 10, decanoyl-N-methylglucamide; MEGA 1 1 , undecanoyl-N-methylglucamide; MEGA 12, dodecanoyl-N-methylglucamide; NDAO, nonaoyl (dimethyl)-amine oxide; octyl-POE, octyl-polydisperse-oligo(oxyethy1-ene);...

Porin Is Present in the Plasma Membrane Where It Is Concentrated in Caveolae and Caveolae-related Domains

“…Porin might be present in caveolae and caveolae-like domains simply because these are stations on the way to its final destination(s). Porin binds cholesterol (28,41,42), and caveolae are especially rich in cholesterol, playing an important role in its traffic (43). Thus an alternative working hypothesis may be that porin mRNA(s) are translated in the cytosol, and some molecules are transported to the caveolar region by means of caveolin-cholesterol complexes.…”

“…It might have been due to technical reasons (i.e. labeling conditions) or to the protein transmembrane disposition; it is well known that porin is deeply embedded in the phospholipid bilayer (28), and thus poorly accessible to bulky hydrophilic molecules. For our purposes the matter is not highly relevant, because our goal was to isolate only NHS-SS-biotinylated porin molecules, certainly originating from caveolar domains of the plasma membrane.…”

Positive residues involved in the voltage‐gating of the mitochondrial porin‐channel are localized in the external moiety of the pore

“…The purification of bovine heart porin in LDAO was performed as has been described in [9]. The purified protein was ultradialysed against 2070 LDAO, 10 mM Tris-HCl (pH 7.0) and 1 mM EDTA (solubilization buffer) and concentrated in an Amicon apparatus (PM 10 membrane).…”

“…One method uses the detergent Triton X-100 [8] and the other the detergent lauryl (dimethyl)-amine oxide (LDAO) [9]. Triton X-100 has a very long hydrophilic tail which shields the surface-exposed hydrophilic domains of the protein.…”

“…LDAO, on the other hand, has a very small polar head group which leaves part of the hydrophilic domains, including some positively charged groups of the protein, exposed to the surrounding water [7]. These charged groups are either totally positively charged or predominantly positively charged at neutral pH, since the LDAO-purified protein binds only to cation-and not to anion-exchangers [7,9]. The interaction between the LDAO-porin and the cation exchanger column is thus a suitable system to investigate the positively charged residues located on the external surface of the functionally active pore.…”