The Role of the N and C Termini of Recombinant Neurospora Mitochondrial Porin in Channel Formation and Voltage-dependent Gating

Abstract

To investigate the role of the N and C termini in channel function and voltage-dependent gating of mitochondrial porin, we expressed wild-type and mutant porins from Neurospora crassa as His-tag fusion products in Escherichia coli. Large quantities of the proteins were purified by chromatography across a nickle-nitrilotriacetic acid-agarose column under denaturing conditions. The purified His-tagged wild-type protein could be functionally reconstituted in the presence of detergent and sterol and behaved in black lipid bilayer membranes indistinguishably from native porin isolated from Neurospora crassa mitochondria. Mutants of porin lacking part of the N terminus (DeltaN2-12porin, DeltaN3-20porin), part of the C terminus (DeltaC269-283porin), or both (DeltaN2-12/DeltaC269-283porin) also showed channel forming activity. The mutant porin lacking the C terminus had a smaller single channel conductance than the wild-type protein, but its other biophysical properties were identical. DeltaN2-12porin and DeltaN3-20porin formed noisy channels with decreased channel stability. These channels were still voltage-dependent. DeltaN2-12/DeltaC269-283porin lost channel stability and had altered gating characteristics. These results are discussed with respect to different models that have been proposed in the literature for the structure of mitochondrial porin channels.

Role of the N‐ and C‐termini of porin in import into the outer membrane of Neurospora mitochondria

“…Rather, the truncated proteins seem to adopt a conformation in the membrane that renders them more sensitive to proteolytic attack. In support of this hypothesis, we have observed that porin molecules lacking the N-terminus form unstable channels which rapidly fluctuate between numerous open substates [20].…”

“…The porin mutants used in this study were constructed as described previously [20] and cloned into the vector pGEM4 (Promega). Briefly, restriction fragments of the Neurospora porin cDNA clone [18] were replaced by PCR products lacking either amino acid residues 2-12 or 3-20, to create A2-12porin and A3-20porin, respectively (Fig.…”

“…The aminoterminal residues of porin have the potential to form an amphipathic c~-helix [17,18], and may lie at one surface of the membrane [19]. This region of the protein is not essential for either pore formation or voltage-dependent gating [20]. However, a potential role for this sequence as an import signal was suggested on the basis of its similarity to mitochondrial presequences and to the amino-terminus of Tom70 [18,21].…”

Swapping of the N‐terminus of VDAC1 with VDAC3 restores full activity of the channel and confers anti‐aging features to the cell

“…This structure represents an addition to the b-barrel pore structure and was proposed to have functionally relevant properties [20][21][22][23][24][25][26]35]. It has been recently proposed that the final N-terminal sequence (i.e.…”

Probing the orientation of yeast VDAC1 in vivo

“…The FLAG1, which is 11-20 residues from the N-terminus, will most likely have a trans exposure (see Fig. 1); especially if the Nterminus is as dynamic as proposed [8,13,[22][23][24][25]. Residues 1-10 continue through the lumen towards exposure of the extreme Nterminal residue to the cis side.…”