Biochemical, molecular, and functional characterization of porin isoforms from potato mitochondria.
The mitochondrial outer membrane of eukaryotic cells contains a voltage-dependent anion channel termed porin. In the organisms studied so far only one type of porin has been identified at the protein level. Here we present a biochemical and molecular genetic analysis of two different porin polypeptides of M(r) 34,000 and 36,000 from the outer membranes of potato mitochondria (termed POM 34 and POM 36, respectively). N-terminal sequencing and the use of labeled oligonucleotide mixtures derived from these amino acid sequences allowed the isolation of cDNA clones encoding the 34- and 36-kDa proteins. They have similar steady state protein levels and share about 75% identical amino acids suggesting that they represent isoforms. In addition, a third cDNA clone coding for a slightly different isoform of the 36-kDa protein was characterized. The polypeptides encoded by the three cDNA clones share the highest degree of sequence identity with mitochondrial porins from fungi and mammals. Tentative models of the secondary structure of the 34- and 36-kDa proteins suggest the occurrence of a 16-stranded beta-barrel typical for bacterial and mitochondrial porins. Purification of the 34-kDa protein by hydroxyapatite chromatography allowed conductance measurements in artificial bilayers. The 34-kDa protein is a voltage-dependent, channel-forming component with single channel conductances of 3.5 and 2.0 nanosiemens in 1 M KCl. In spite of the striking functional similarities to mitochondrial porins from other organisms neither the 34- nor the 36-kDa proteins are able to complement the respiratory defect of a yeast por- mutant.
Heins
L
L
Institut für Genbiologische Forschung Berlin GmbH, Federal Republic of Germany.
Mentzel
H
H
Schmid
A
A
Benz
R
R
Schmitz
U K
UK
D000595:Amino Acid Sequence; D001483:Base Sequence; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D008928:Mitochondria / Q000737:chemistry*; D008969:Molecular Sequence Data; D018272:Porins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D011198:Solanum tuberosum / Q000737:chemistry*
D018076:DNA, Complementary; D018272:Porins