Proline 146 is critical to the structure, function and targeting of sod2, the Na+/H+ exchanger of Schizosaccharomyces pombe.
Sod2 is the Na(+)/H(+) exchanger of the fission yeast Schizosaccharomyces pombe that is principally responsible for salt tolerance. We examined the role of nine polar, membrane associated amino acids in the ability of the protein to confer salt tolerance in S. pombe. Wild type sod2 protein with a C-terminal GFP tag effectively rescued salt tolerance in S. pombe with deleted endogenous sod2. Sod2 protein with the mutations P163A, P183A, D298N, D389N, E390Q, E392Q and E397Q also conveyed salt tolerance as effectively as the wild type sod2 protein. In contrast, the mutation P146A resulted in a protein that did not convey salt tolerance nearly as effectively as the wild type and did not extrude Na(+) as well as the wild type. Mutation of Pro(146) to Ser, Asp or Lys had an intermediate effect. Mutation of Thr(142) to Ser resulted in a slightly defective protein. Western blot analysis showed that all mutant proteins were expressed at similar levels as wild type sod2 protein. Examination of the localization of the proteins showed that wild type and most sod2 mutants were present in the plasma membrane while the P146A mutant had an intracellular localization. Limited tryptic digestion suggested that the P146A sod2 protein had a change in conformation in comparison to the wild type protein. The results suggest that Pro(146) is an amino acid critical to sod2 structure, function and localization.
Ndayizeye
Maxime
M
Department of Biochemistry, Faculty of Medicine, University of Alberta, 347 Medical Science Building, Edmonton, Alberta, Canada T6G 2H7.
Touret
Nicolas
N
Fliegel
Larry
L
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D055592:Biophysical Phenomena; D004271:DNA, Fungal / Q000235:genetics; D019084:Fluorescent Antibody Technique, Indirect; D005800:Genes, Fungal; D017136:Ion Transport; D018613:Microscopy, Confocal; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000737:chemistry; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013054:Spectrophotometry, Atomic; D014357:Trypsin
D004271:DNA, Fungal; D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins; C501077:Sod2 protein, S pombe; D017923:Sodium-Hydrogen Exchangers; D011392:Proline; D012964:Sodium; D014357:Trypsin