The Pichia pastoris dihydroxyacetone kinase is a PTS1-containing, but cytosolic, protein that is essential for growth on methanol.
Dihydroxyacetone kinase (DAK) is essential for methanol assimilation in methylotrophic yeasts. We have cloned the DAK gene from Pichia pastoris by functional complementation of a mutant that was unable to grow on methanol. An open reading frame of 1824 bp was identified that encodes a 65.3 kDa protein with high homology to DAK from Saccharomyces cerevisiae. Although DAK from P. pastoris contained a C-terminal tripeptide, TKL, which we showed can act as a peroxisomal targeting signal when fused to the green fluorescent protein, the enzyme was primarily cytosolic. The TKL tripeptide was not required for the biochemical function of DAK because a deletion construct lacking the DNA encoding this tripeptide was able to complement the P. pastoris dak delta mutant. Peroxisomes, which are essential for growth of P. pastoris on methanol, were present in the dak delta mutant and the import of peroxisomal proteins was not disturbed. The dak delta mutant grew at normal rates on glycerol and oleate media. However, unlike the wild-type cells, the dak delta mutant was unable to grow on methanol as the sole carbon source but was able to grow on dihydroxyacetone at a much slower rate. The metabolic pathway explaining the reduced growth rate of the dak delta mutant on dihydroxyacetone is discussed. The nucleotide sequence reported in this paper has been submitted to GenBank with Accession Number AF019198.
Lüers
G H
GH
Department of Biology, University of California at San Diego, La Jolla 92093-0322, USA.
Advani
R
R
Wenzel
T
T
Subramani
S
S
D000595:Amino Acid Sequence; D002458:Cell Fractionation; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D004098:Dihydroxyacetone / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D000432:Methanol / Q000378:metabolism; D008830:Microbodies / Q000201:enzymology / Q000378:metabolism; D008969:Molecular Sequence Data; D019301:Oleic Acid / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics* / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019301:Oleic Acid; D017853:Phosphotransferases (Alcohol Group Acceptor); C023132:glycerone kinase; D004098:Dihydroxyacetone; D005990:Glycerol; D000432:Methanol