THE PICHIA-PASTORIS DIHYDROXYACETONE KINASE IS A PTS1-CONTAINING, BUTCYTOSOLIC, PROTEIN THAT IS ESSENTIAL FOR GROWTH ON METHANOL

Dihydroxyacetone kinase (DAK) is essential for methanol assimilation i n methylotrophic yeasts. We have cloned the DAK gene from Pichia pasto ris by functional complementation of a mutant that was unable to grow on methanol. An open reading frame of 1824 bp was identified that enco des a 65.3 kDa protein with high homology to DAK from Saccharomyces ce revisiae. Although DAK from P, pastoris contained a C-terminal tripept ide, TKL, which we showed can act as a peroxisomal targeting signal wh en fused to the green fluorescent protein, the enzyme was primarily cy tosolic. The TKL tripeptide was not required for the biochemical funct ion of DAK because a deletion construct lacking the DNA encoding this tripeptide was able to complement the P. pastoris dak Delta mutant. Pe roxisomes, which are essential for growth of P. pastoris on methanol, were present in the dak Delta mutant and the import of peroxisomal pro teins was not disturbed. The dak Delta mutant grew at normal rates on glycerol and oleate media. However, unlike the wild-type cells, the da k Delta mutant was unable to grow on methanol as the sole carbon sourc e 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 repor ted in this paper has been submitted to GenBank with Accession Number AF019198. (C) 1998 John Wiley & Sons, Ltd.