Functional characterization of the conserved "GLK" motif in mitochondrial porin from Neurospora crassa.
Mitochondrial porin facilitates the diffusion of small hydrophilic molecules across the mitochondrial outer membrane. Despite low sequence similarity among porins from different species, a "glycine-leucine-lysine" (GLK) motif is conserved in mitochondrial and Neisseria porins. To investigate the possible roles of these conserved residues, including their hypothesized participation in ATP binding by the protein, we replaced the lysine residue of the GLK motif of Neurospora crassa porin with glutamic acid through site-directed mutagenesis of the corresponding gene. Although the pores formed by this protein have size and gating characteristics similar to those of the wild-type protein, the channels formed by GLEporin are less anion selective than the wild-type pores. The GLEporin retains the ability to be cross linked to [alpha-(32)P]ATP, indicating that the GLK sequence is not essential for ATP binding. Furthermore, the pores formed by both GLEporin and the wild-type protein become more cation selective in the presence of ATP. Taken together, these results support structural models that place the GLK motif in a part of the ion-selective beta-barrel that is not directly involved in ATP binding.
Runke
G
G
Department of Microbiology, University of Manitoba, Winnipeg, MB R3T 2N2, Canada.
Maier
E
E
O'Neil
J D
JD
Benz
R
R
Court
D A
DA
D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002942:Circular Dichroism; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D015640:Ion Channel Gating; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009492:Neurospora crassa / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050994:Voltage-Dependent Anion Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D005656:Fungal Proteins; D011994:Recombinant Proteins; D050994:Voltage-Dependent Anion Channels; D000255:Adenosine Triphosphate