Purification, functional characterization, and cDNA sequencing of mitochondrial porin from Dictyostelium discoideum.
Porin of Dictyostelium discoideum was extracted from mitochondria with Genapol X-80 and was purified by hydroxyapatite and CM-cellulose chromatography. The purified protein displayed a single band of 30 kDa in SDS-polyacrylamide gel electrophoresis. The formation of channels in artificial lipid bilayer membranes defined its function as a channel-forming component. Its average single-channel conductance was 3.9 nanosiemens in 1 M KCl, which suggested that the effective diameter of the channel is approximately 1.7 nm at small transmembrane potentials. The channel displayed a characteristic voltage dependence for potentials higher than 20 mV. It switched to substates of smaller conductance and a selectivity different to that of the open state. The closed state was stabilized at low ionic strength. The cDNA sequence of mitochondrial porin from D. discoideum was determined. It showed little sequence similarities to other known mitochondrial porins. The functional similarity, however, was striking. Localization of the porin in the mitochondrial outer membrane was confirmed by immunogold labeling of cryosections of fixed cells.
Troll
H
H
Fakultät für Biologie, Universität Konstanz, Federal Republic of Germany.
Malchow
D
D
Müller-Taubenberger
A
A
Humbel
B
B
Lottspeich
F
F
Ecke
M
M
Gerisch
G
G
Schmid
A
A
Benz
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000502:physiology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004023:Dictyostelium / Q000235:genetics*; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D015723:Gene Library; D006801:Humans; D006882:Hydroxyapatites; D008051:Lipid Bilayers; D008564:Membrane Potentials; D008928:Mitochondria / Q000378:metabolism / Q000648:ultrastructure; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000235:genetics; D018272:Porins; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D001425:Bacterial Outer Membrane Proteins; D006882:Hydroxyapatites; D008051:Lipid Bilayers; D018272:Porins; D004247:DNA; D017886:Durapatite