ThePichia pastoris dihydroxyacetone kinase is a PTS1-containing, but cytosolic, protein that is essential for growth on methanol
✍ Scribed by Lüers, Georg H.; Advani, Raj; Wenzel, Thibaut; Subramani, Suresh
- Book ID
- 101226367
- Publisher
- John Wiley and Sons
- Year
- 1998
- Tongue
- English
- Weight
- 376 KB
- Volume
- 14
- Category
- Article
- ISSN
- 0749-503X
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✦ Synopsis
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 mutant. Peroxisomes, which are essential for growth of P. pastoris on methanol, were present in the dak mutant and the import of peroxisomal proteins was not disturbed. The dak mutant grew at normal rates on glycerol and oleate media. However, unlike the wild-type cells, the dak 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 mutant on dihydroxyacetone is discussed.