Lysine Residues at Positions 234 and 236 in Yeast Porin Are Involved in Its Assembly into the Mitochondrial Outer Membrane

Abstract

Various point mutations of lysyl residues in yeast mitochondrial porin (283 residues) were tested for their ability to assemble in vitro into the outer membranes of intact yeast mitochondria. Assembly was evaluated by protection from proteinases. The extent of assembly of two of the mutants, K234E and K236E porins, was much less than for wild-type in either post-translational or co-translational assembly assays. Lysine to glutamate mutants at other positions and K234R porin assembled as well as wild-type, but K234Q porin was poorly inserted. When both Lys-234 and Lys-236 were mutated, K234R/K236R porin was inserted better than K234Q/ K236Q porin, which was inserted better than K234E/ K236E; however, none of these mutants assembled as well as wild-type porin. It was concluded that optimal assembly of yeast porin depended on the presence of positively charged residues at both positions 234 and 236 and a lysine at one of these positions. After undergoing the assembly reaction, mutants that were vulnerable to proteinase K (i.e. K234E, K234Q, and K236E porins) seemed to be incompletely digested and were, to varying degrees, resistant to extraction by Na 2 CO 3 (pH 11.5). These experiments suggested that these mutants were incompletely inserted into the outer membrane. Both Lys-234 and Lys-236 are included in an internal pentapeptide, VKAKV, that is conserved in porins from protists, plants, and animals, and it is possible that, at least, the lysines in this tract are one of the signals for the membrane assembly of these proteins.Mitochondrial porin (i.e. the 283-residue mitochondrial outer membrane voltage dependent anion channel) forms large holes in the mitochondrial outer membrane that permit the passage of metabolites necessary for reactions that occur in the interior compartments of mitochondria (for review, see Ref. 1). It is likely that each channel is formed by a single M r 31,000 porin polypeptide (2). Although the tertiary structure of eukaryotic porin is still unknown, it is thought to be largely comprised of amphipathic ␤-strands in the form of a ␤-barrel. This conclusion is based in part on observations that fungal porin does not contain sufficiently long hydrophobic tracts to serve as membrane anchors (3) and in part, in analogy with the known structures of bacterial porins (4, 5). The distribution of amino acid residues that contribute to the channel properties of porin has been interpreted to mean that yeast porin has 13 membrane spanning tracts: a single amphipathic ␣-helix and 12 amphipathic ␤-strands (6). According to this model, charged amino acids in the membrane spanning tracts line the aqueous pore.Mitochondrial porins are encoded by nuclear genes and synthesized by cytosolic ribosomes. Porin has been shown to assemble in vitro by both post-translational (7-9) and co-translational processes (10). Porin assembly is independent of the mitochondrial membrane potential but it seems to depend on energy in the form of ATP (3, 9, 11). As is the case for other outer membrane proteins, the mature (i.e. ...

Characterization of Oyster Voltage-Dependent Anion Channel 2 (VDAC2) Suggests Its Involvement in Apoptosis and Host Defense

“…The GLK motif, a specific amino-acid triplet in VDACs, plays a vital role in ATP nucleotide binding and nucleotide-dependent channel gating [ 41 ] and is conserved in VDACs though mutations of the lysine residue, has only limited effect on the functionality of the domain [ 37 ]. The VKAKV-like sequence is present in all the available plant and animal VDAC sequences [ 42 ] and is involved in the insertion of the protein into MOM. BLAST analysis of CgVDAC2 showed a porin3 domain, which is a typical characteristic of pore-forming proteins [ 43 ].…”

Deletion Variants of Neurospora Mitochondrial Porin: Electrophysiological and Spectroscopic Analysis

“…P-229 is also absent in 228porin, which may alter the topology of the loop that contains it, perhaps interrupting interactions responsible for gating. K-234 and K-236, which are required for the stable assembly of yeast VDAC1 into the mitochondrial outer membrane (47), are also within this proposed loop.…”

Isolation of mitochondria from Saccharomyces cerevisiae using magnetic bead affinity purification

“…To characterize the integrity of mitochondria prepared by the two methods, isolated mitochondria were treated with proteinase K. If isolated organelles are intact, then protease treatment should degrade Tom70, a protease-sensitive outer membrane protein, but should not affect the intermembrane space protein cytochrome b2 (Cyb2). Since the outer membrane protein porin is protease-resistant [ 38 ], the level of porin served as a control for protein load in these studies. We find that proteinase K treatment decreased Tom70 levels but not Cyb2 levels in crude and magnetic bead-purified mitochondria ( Fig 5A and 5B ).…”