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The Pichia pastoris PAS4 gene encodes a ubiquitin-conjugating enzyme required for peroxisome assembly
D I Crane1, J E Kalish, S J Gould
1Kennedy Krieger Institute, Baltimore, Maryland 21205.
Insights
The PAS4 gene is crucial for peroxisome assembly in yeast. This gene encodes a protein similar to ubiquitin-conjugating enzymes, indicating ubiquitination is essential for peroxisome formation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Peroxisome assembly is a complex process involving multiple proteins.
- The yeast Pichia pastoris is a model organism for studying peroxisome biogenesis.
Purpose of the Study:
- To clone and characterize the PAS4 gene involved in peroxisome assembly.
- To elucidate the function of the Pas4 protein in Pichia pastoris.
Main Methods:
- Gene cloning and characterization.
- Protein sequence analysis and homology searches.
- Site-directed mutagenesis to investigate enzyme activity.
- Detection of protein conjugates in vivo and in vitro.
Main Results:
- PAS4 gene cloned and characterized, encoding a 24-kDa protein (Pas4p).
- Pas4p shows similarity to ubiquitin-conjugating enzymes, with a conserved active-site cysteine.
- Mutating the active-site cysteine abolished PAS4 function.
- A Pas4p-ubiquitin conjugate was detected, confirming Pas4p's role in ubiquitination.
Conclusions:
- PAS4 is a member of the ubiquitin-conjugating enzyme gene family.
- Ubiquitination is a necessary step for peroxisome assembly in Pichia pastoris.
Abstract:
We report here the cloning and initial characterization of PAS4, a gene required for peroxisome assembly in the yeast Pichia pastoris. The PAS4 gene encodes a 24-kDa protein (Pas4p) that is located on the cytoplasmic surface of peroxisomes and is induced during peroxisome proliferation. Analysis of the Pas4p sequence revealed a high degree of similarity to ubiquitin-conjugating enzymes, particularly in the region surrounding the putative active-site cysteine residue with which ubiquitin forms a thioester bond. As expected for a ubiquitin-conjugating enzyme, substitution of alanine or serine for the conserved active-site cysteine residue abolished PAS4 function. In addition, a small amount of a 32 kDa form of Pas4p (the predicted size of a Pas4p-ubiquitin conjugate) was detected both in vivo and in vitro. This species was eliminated by reducing agents and was not detected in the cysteine to alanine substitution mutant, suggesting that it is a Pas4p-ubiquitin conjugate. Using a yeast strain that overexpresses a Myc-ubiquitin fusion protein, we demonstrate directly that this conjugate contains ubiquitin. We conclude from these observations that PAS4 is a member of the ubiquitin-conjugating enzyme gene family and that one or more ubiquitination reactions are required for peroxisome assembly.