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The Hansenula polymorpha per6 mutant is affected in two adjacent genes which encode dihydroxyacetone kinase and a
I J van der Klei1, M van der Heide, R J Baerends
1Eukaryotic Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, NL-9751 NN Haren, The Netherlands. IJVDKLEI@BIOL.RUG.NL
Abstract:
The Hansenula polymorpha per6-210 mutant is impaired in respect of growth on methanol (Mut-) and is characterized by aberrant peroxisome formation. The functionally complementing DNA fragment contains two open reading frames. The first encodes dihydroxyacetone kinase (DAK), a cytosolic enzyme essential for formaldehyde assimilation; the second ORF codes for a novel protein (Pak1p). We have demonstrated that per6-210 cells lack DAK activity, causing the Mut- phenotype, and have strongly reduced levels of Pak1p, resulting in peroxisomal defects. Sequence analysis revealed that per6-210 contains a mutation in the 3' end of the DAK coding region, which overlaps with the promoter region of PAK1. Possibly this mutation also negatively affects PAK1 expression.
Insights
The Hansenula polymorpha per6-210 mutant shows impaired methanol growth and peroxisome defects due to a mutation affecting dihydroxyacetone kinase (DAK) and Pak1p. This genetic defect impacts formaldehyde assimilation and peroxisome formation.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- The yeast Hansenula polymorpha utilizes methanol as a carbon source, a process involving peroxisomes.
- Mutations can disrupt metabolic pathways and cellular structures, impacting yeast growth and function.
Purpose of the Study:
- To investigate the genetic basis of the per6-210 mutant's impaired methanol utilization (Mut-) and aberrant peroxisome formation.
- To identify the genes responsible for these phenotypes and elucidate their roles.
Main Methods:
- Genetic complementation of the per6-210 mutant using a DNA fragment containing two open reading frames (ORFs).
- Enzyme activity assays to measure dihydroxyacetone kinase (DAK) levels.
- Analysis of Pak1p protein levels.
- DNA sequencing to identify mutations.
Main Results:
- The complementing DNA fragment contained genes for dihydroxyacetone kinase (DAK) and a novel protein, Pak1p.
- The per6-210 mutant cells lacked DAK activity, explaining the Mut- phenotype.
- Reduced levels of Pak1p were observed, correlating with peroxisomal defects.
- Sequence analysis revealed a mutation in the DAK gene's 3' end, potentially affecting PAK1 expression.
Conclusions:
- A single mutation in H. polymorpha per6-210 affects both DAK activity and Pak1p levels, leading to impaired methanol growth and peroxisomal abnormalities.
- The DAK gene and the novel Pak1p protein are crucial for methanol assimilation and peroxisome biogenesis in H. polymorpha.