Pyruvate carboxylase is an essential protein in the assembly of yeast peroxisomal oligomeric alcohol oxidase.
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.
Ozimek
Paulina
P
Eukaryotic Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Haren, The Netherlands.
van Dijk
Ralf
R
Latchev
Kantcho
K
Gancedo
Carlos
C
Wang
Dong Yuan
DY
van der Klei
Ida J
IJ
Veenhuis
Marten
M
D001224:Aspartic Acid / Q000378:metabolism; D015153:Blotting, Western; D003600:Cytosol / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D009154:Mutation; D020675:Peroxisomes / Q000378:metabolism*; D010843:Pichia / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011766:Pyruvate Carboxylase / Q000737:chemistry / Q000502:physiology*; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D001224:Aspartic Acid; D018698:Glutamic Acid; D006639:Histidine; D011766:Pyruvate Carboxylase