Hansenula polymorpha ass3 mutants are characterized by the accumulation of inactive alcohol oxidase (AO) monomers in the cytosol, whereas other peroxisomal matrix proteins are normally activated and sorted to peroxisomes. These mutants also have a glutamate or aspartate requirement on minimal media. Cloning of the corresponding gene resulted in the isolation of the H. polymorpha PYC gene that encodes pyruvate carboxylase (HpPyc1p). HpPyc1p is a cytosolic, anapleurotic enzyme that replenishes the tricarboxylic acid cycle with oxaloacetate. The absence of this enzyme can be compensated by addition of aspartate or glutamate to the growth media. We show that HpPyc1p protein but not the enzyme activity is essential for import and assembly of AO. Similar results were obtained in the related yeast Pichia pastoris. In vitro studies revealed that HpPyc1p has affinity for FAD and is capable to physically interact with AO protein. These data suggest that in methylotrophic yeast pyruvate carboxylase plays a dual role in that, besides its well-characterized metabolic function as anapleurotic enzyme, the protein fulfils a specific role in the AO sorting and assembly process, possibly by mediating FAD-binding to AO monomers.
INTRODUCTIONThe yeast Hansenula polymorpha is able to use methanol as sole carbon and energy source. Growth on this compound is accompanied by the induction of peroxisomes that contain the key enzymes of methanol metabolism. Alcohol oxidase (AO) is a major constituent of these organelles and catalyzes the oxidation of methanol into formaldehyde and hydrogen peroxide. Inactive AO monomers are synthesized in the cytosol and posttranslationally imported into the target organelle, where the protein is activated. The active enzyme is an octamer, containing eight identical subunits, which each contains a FAD molecule as cofactor (reviewed by van der Klei et al., 1991a).Both in vivo and in vitro experiments suggested that assembly of AO into active octamers is most likely not a spontaneous process (Distel et al., 1987;van der Klei et al., 1989b). Several independent experiments suggested that specific helper proteins (tentatively called assembly factors) are required to mediate AO assembly. Studies on an H. polymorpha riboflavin (Rf) auxotrophic mutant revealed that Rf limitation interfered with the assembly and the import of AO (Evers et al., 1994) and suggested that cofactor binding, oligomerization, and translocation of AO are tightly coupled processes. However, in all H. polymorpha peroxisome-deficient (pex) mutants analyzed so far AO is normally assembled and active in the cytosol. This suggests that AO assembly does not require the specific (acidic) microenvironment of the peroxisomal matrix (van der Klei et al., 1991c).Previous biochemical approaches to identify AO assembly factors failed so far. We therefore sought to isolate these components by a genetic approach. To this end we have isolated a collection of mutants that displayed reduced AO activities (van Dijk et al., 2002).Here, we report the functio...
