FLAVIN ADENINE-DINUCLEOTIDE BINDING IS THE CRUCIAL STEP IN ALCOHOL OXIDASE ASSEMBLY IN THE YEAST HANSENULA-POLYMORPHA

We have studied the role of flavin adenine dinucleotide (FAD) in the i n vivo assembly of peroxisomal alcohol oxidase (AO) in the yeast Hanse nula polymorpha. In previous studies, using a riboflavin (Rf) autotrop hic mutant, an unequivocal judgement could not be made, since Rf-limit ation led to a partial block of AO import in this mutant. This resulte d in the accumulation of AO precursors in the cytosol where they remai ned separated from the putative peroxisomal AO assembly factors. In or der to circumvent the peroxisomal membrane barrier, we have now studie d AO assembly in a peroxisome-deficient/Rf-autotrophic double mutant ( Delta per1.rif1) of H. polymorpha. By sucrose density centrifugation a nd native gel electrophoresis, three conformations of AO were detected in crude extracts of Delta per1.rif1 cells grown under Rf-limitation, namely active octameric AO and two inactive, monomeric forms. One of the latter forms lacked FAD; this form was barely detectable in extrac ts wild-type and Delta per1 cells, but had accumulated in the cytosol of rif1 cells. The second form of monomeric AO contained FAD; this for m was also present in Delta per1 cells but absent/very low in wild-typ e and rif1 cells. In vivo only these FAD-containing: monomers associat e into the active, octameric protein. We conclude that in H. polymorph a FAD binding to the AO monomer is mediated by a yet unknown peroxisom al factor and represents the crucial and essential step to enable AO o ligomerization; the actual octamerization and the eventual crystalliza tion in peroxisomes most probably occurs spontaneously.