The role of the N and C termini of recombinant Neurospora mitochondrial porin in channel formation and voltage-dependent gating.
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.
Popp
B
B
Lehrstuhl für Biotechnologie, Theodor-Boveri-Institut (Biozentrum) der Universität Würzburg, Am Hubland, D-97074 Würzburg, Federal Republic of Germany.
Court
D A
DA
Benz
R
R
Neupert
W
W
Lill
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D004875:Ergosterol / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015640:Ion Channel Gating; D007473:Ion Channels / Q000378:metabolism*; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009492:Neurospora crassa / Q000235:genetics / Q000378:metabolism*; D018272:Porins; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D050994:Voltage-Dependent Anion Channels
D017931:DNA Primers; D004271:DNA, Fungal; D005656:Fungal Proteins; D007473:Ion Channels; D008565:Membrane Proteins; D018272:Porins; D011993:Recombinant Fusion Proteins; D050994:Voltage-Dependent Anion Channels; D004875:Ergosterol