THE ABSENCE OF PMP47, A PUTATIVE YEAST PEROXISOMAL TRANSPORTER, CAUSES A DEFECT IN TRANSPORT AND FOLDING OF A SPECIFIC MATRIX ENZYME

Candida boidinii Pmp47, an integral peroxisomal membrane protein, belo ngs to a family of mitochondrial solute transporters (e.g,, ATP/ADP ex changer), and is the only known peroxisomal member of this family. How ever, its physiological and biochemical functions have been unrevealed because of the difficulties in the molecular genetics of C. boidinii. In this study, we first isolated the PMP47 gene, which was the single gene encoding for Pmp47 in a gene-engineerable strain S2 of C, boidin ii. Sequence analysis revealed that it was very similar to PMP47A and PMP47B genes from a polyploidal C. boidinii strain (ATCC32195). Next, the PMP47 gene was disrupted and the disruption strain (pmp47 Delta) w as analyzed. Depletion of Pmp47 from strain S2 resulted in a retarded growth on oleate and a complete loss of growth on methanol, Both growt h substrates require peroxisomal metabolism. EM observations revealed the presence of peroxisomes in methanol- and oleate-induced cells of p mp47 Delta, but in reduced numbers, and the presence of material of hi gh electron density in the cytoplasm in both cases, Methanol-induced c ells of pmp47 Delta were investigated in detail. The activity of one o f the methanol-induced peroxisome matrix enzymes, dihydroxyacetone syn thase (DHAS), was not detected in pmp47 Delta. Further biochemical and immunocytochemical experiments revealed that the DHAS protein aggrega ted in the cytoplasm as an inclusion body, while two other peroxisome matrix enzymes, alcohol oxidase (AOD) and catalase, were active and fo und in peroxisomes, Two peroxisome-deficient mutants, strains M6 and M 13 (described in previous studies), retained DHAS activity although it was mislocalized to the cytoplasm and the nucleus, We disrupted PMP47 in these peroxisome-deficient mutants. In both strains, M6-pmp47 Delt a and M13-pmp47 Delta, DHAS was enzymatically active and was located i n the cytoplasm and the nucleus, We suggest that an unknown small mole cule, which PMP47 transports, is necessary for the folding or the tran slocation machinery of DHAS within peroxisomes, Pmp47 does not catalyz e folding directly because active DHAS is observed in the M6-pmp47 Del ta and M13-pmp47 Delta strains, Since both AOD and DHAS have the PTS1 motif sequences at their carboxyl terminal, our results first show tha t depletion of Pmp47 could dissect the peroxisomal import pathway (PTS 1 pathway) of these proteins.