Characterization of peroxisome-deficient mutants of Hansenula polymorpha
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X Tan1, V I Titorenko, I J van der Klei
1Department of Chemistry, Biochemistry, and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland 97291-1000, USA.
Abstract:
In the methylotrophic yeast Hansenula polymorpha, approximately 25% of all methanol-utilization-defective (Mut-) mutants are affected in genes required for peroxisome biogenesis (PER genes). Previously, we reported that one group of per mutants, termed Pim-, are characterized by the presence of a few small peroxisomes with the bulk of peroxisomal enzymes located in the cytosol. Here, we describe a second major group of per mutants that were observed to be devoid of any peroxisome-like structure (Per-). In each Per- mutant, the peroxisomal methanol-pathway enzymes alcohol oxidase, catalase and dihydroxyacetone synthase were present and active but located in the cytosol. Together, the Pim- and Per- mutant collections involved mutations in 14 different PER genes. Two of the genes, PER5 and PER7, were represented by both dominant-negative and recessive alleles. Diploids resulting from crosses of dominant per strains and wild-type H. polymorpha were Mut- and harbored peroxisomes with abnormal morphology. This is the first report of dominant-negative mutations affecting peroxisome biogenesis.
Insights
Researchers identified two new groups of peroxisome-deficient mutants in Hansenula polymorpha yeast. This study reveals novel genes essential for peroxisome assembly and introduces the first dominant-negative mutations impacting peroxisome biogenesis.
Area of Science:
- Cell Biology
- Yeast Genetics
- Molecular Biology
Background:
- Peroxisomes are vital organelles involved in various metabolic processes.
- In methylotrophic yeast Hansenula polymorpha, mutations affecting peroxisome biogenesis (PER genes) lead to methanol-utilization defects.
- Previous studies identified 'Pim-' mutants with few, small peroxisomes and cytosolic enzymes.
Purpose of the Study:
- To characterize a second major group of peroxisome-deficient (Per-) mutants in H. polymorpha.
- To identify novel PER genes involved in peroxisome assembly.
- To investigate the nature of dominant-negative mutations affecting peroxisome biogenesis.
Main Methods:
- Phenotypic analysis of methanol-utilization-defective (Mut-) mutants.
- Characterization of peroxisome structure and enzyme localization in mutant strains.
- Genetic analysis, including complementation tests and analysis of dominant and recessive alleles.
Main Results:
- A new class of Per- mutants, lacking any peroxisome-like structures, was identified.
- In Per- mutants, key methanol pathway enzymes (alcohol oxidase, catalase, dihydroxyacetone synthase) were active but cytosolic.
- Mutations in 14 distinct PER genes were identified across both Pim- and Per- mutant collections.
- PER5 and PER7 genes were found to have both dominant-negative and recessive alleles.
- Crosses involving dominant per alleles resulted in Mut- diploids with abnormal peroxisome morphology.
Conclusions:
- Two distinct mutant collections (Pim- and Per-) highlight diverse genetic requirements for peroxisome biogenesis in H. polymorpha.
- The identification of Per- mutants expands the known PER gene set.
- The discovery of dominant-negative mutations affecting peroxisome assembly provides new insights into organelle biogenesis regulation.
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