BiFC Method Based on Intraorganellar Protein Crowding Detects Oleate-Dependent Peroxisomal Targeting of Malate Dehydrogenase.

The maintenance of intracellular NAD/NADH homeostasis across multiple, subcellular compartments requires the presence of NADH-shuttling proteins, which circumvent the lack of permeability of organelle membranes to these cofactors. Very little is known regarding these proteins in the methylotrophic yeast, . During the study of the subcellular locations of these shuttling proteins, which often have dual subcellular locations, it became necessary to develop new ways to detect the weak peroxisomal locations of some of these proteins. We have developed a novel variation of the traditional Bimolecular Fluorescence Complementation (BiFC), called divergent BiFC, to detect intraorganellar colocalization of two noninteracting proteins based on their proximity-based protein crowding within a small subcellular compartment, rather than on the traditional protein-protein interactions expected for BiFC. This method is used to demonstrate the partially peroxisomal location of one such NADH-shuttling protein, malate dehydrogenase B, only when cells are grown in oleate, but not when grown in methanol or glucose. We discuss the mode of NADH shuttling in and the physiological basis of the medium-dependent compartmentalization of MdhB.