Characterization of the mitochondrial porin from Drosophila melanogaster.
Mitochondrial porin was isolated from the fruit fly Drosophila melanogaster at different developmental stages, starting from whole mitochondria. The porin from adults' mitochondria was fully characterized. The protein had a molecular mass of 31 kDa as judged from sodium dodecylsulfate electrophoretograms. It was very resistive against digestion with V8 proteinase of Staphylococcus aureus and a larger number of fragments were only obtained after digestion with papain. Drosophila porin showed little interaction with antibodies raised against mitochondrial porins from mammalia and Neurospora crassa, but a strong reactivity with antibodies raised against yeast porin. Reconstitution experiments with planar lipid bilayer membranes showed that the protein was able to form ion-permeable pores with a single-channel conductance of 0.41 nS in 0.1 M KCl. At low transmembrane voltages Drosophila porin had the properties of a general diffusion pore with an estimated effective diameter of about 1.7 nm and a small selectivity for anions over cations. Voltages larger than 20 to 30 mV resulted in a closure of the pore. The closed states of the pore were found to be cation-selective. The addition of a synthetic polyanion to the aqueous phase on one side of the membrane resulted in an asymmetric shift of the voltage dependence and the pore became already closed at very small voltages negative at the cis-side (the side of the addition of the polyanion).
De Pinto
V
V
Dipartimento Farmaco-Biologico, Università di Bari, Italy.
Benz
R
R
Caggese
C
C
Palmieri
F
F
D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D004331:Drosophila melanogaster / Q000032:analysis* / Q000254:growth & development; D004553:Electric Conductivity; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007473:Ion Channels / Q000502:physiology / Q000648:ultrastructure; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism; D008564:Membrane Potentials; D008928:Mitochondria / Q000032:analysis*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D018272:Porins; D012697:Serine Endopeptidases / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D007473:Ion Channels; D008051:Lipid Bilayers; D010446:Peptide Fragments; D018272:Porins; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase