UC San Diego

Peroxisomes proliferate in media whose utilization requires peroxisomal metabolic pathways. In methylotrophic yeast, such as Pichia pastoris, oleate and methanol are the most common carbon sources used for peroxisome proliferation studies. When grown in these conditions, peroxisome metabolism is essential for carbon assimilation and energy production. In Saccharomyces cerevisiae, during growth in oleate, NADH shuttling from the peroxisome to mitochondria, via the cytosol, maintains the cellular redox balance during fatty acid β-oxidation and contributes to energy production. In P. pastoris, during growth in methanol, NADH produced by methanol oxidation shuttles to the mitochondria becoming the only source of energy; however, the NADH shuttling mechanism, which typically requires enzymes in peroxisomes, cytosol and mitochondria, has not been studied yet in this yeast. We used fluorescence microscopy to determine the subcellular localization of the homologous P. pastoris NADH shuttling proteins (malate dehydrogenases and glycerol 3-phosphate dehydrogenases). Surprisingly, none of the NADH shuttling proteins fused to GFP showed peroxisomal localization, although this was expected. To improve the detection, we developed a divergent bimolecular fluorescence complementation (BiFC) assay to detect low levels of peroxisomally localized proteins which could be masked by a strong cytosolic localization. Using this assay, we confirmed that one of the malate dehydrogenases has a dual localization, cytosolic and peroxisomal, but only when grown in oleate, but it was exclusively cytosolic when grown in methanol. These localizations can be rationalized in terms of the NADH produced by oleate metabolism in the peroxisome matrix and in the cytosol during methanol metabolism. Finally, we elucidated the pathway responsible for targeting the malate dehydrogenase to the peroxisome; however, no obvious peroxisomal targeting signal was found in the enzyme suggesting an alternative translocation mechanism, such as piggyback import with a peroxisomal protein containing a peroxisomal targeting signal.