Identification of intragenic mutations in the Hansenula polymorpha PEX6 gene that affect peroxisome biogenesis and methylotrophic growth.
Two interacting AAA ATPases, Pex1p and Pex6p, are indispensable for peroxisome biogenesis in different organisms. Mutations affecting corresponding genes are the most common cause of the peroxisome biogenesis disorders in humans. By UV mutagenesis of the Hansenula polymorpha pex6 mutant, deficient in peroxisome biogenesis, we isolated a conditional cold-sensitive strain with restored ability to grow in methanol medium at 37 degrees C but not at 28 degrees C. Sequencing of the pex6 allele revealed a point mutation in the first AAA module of the PEX6 gene that leads to substitution of a conserved amino acid residue (G737E). An additional intragenic mutation identified in the cold-sensitive pex6 allele leads to a conserved amino acid substitution in the second AAA domain (R1000G). Electron microscopic analysis revealed restored peroxisomes in methanol-induced cold-sensitive pex6 cells at both permissive and restrictive temperatures. If separated, the secondary mutation did not affect methylotrophic growth. Our data suggest that H. polymorpha Pex6p may have a complex function in peroxisome biogenesis in which identified amino acid residues are involved.
Stasyk
Oleh V
OV
Institute of Cell Biology, Drahomanov Str. 14/16, 79005, Lviv, Ukraine.
Nazarko
Volodymyr Y
VY
Pochapinsky
Olexiy D
OD
Nazarko
Taras Y
TY
Veenhuis
Marten
M
Sibirny
Andrei A
AA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D020675:Peroxisomes / Q000235:genetics* / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D016133:Polymerase Chain Reaction
D005656:Fungal Proteins