Localizing mRNAs encoding mitochondrial proteins in yeast by fluorescence microscopy and subcellular fractionation

Mitochondria are thought to have evolved from ancestral proteobacteria and, as a result of symbiosis, became an indispensable organelle in all eukaryotic cells. Mitochondria perform essential functions that provide the cell with ATP, amino acids, phospholipids, and both heme and iron-sulfur clusters. However, only 1% of mitochondrial proteins are encoded by the mitochondrial genome, while the remaining 99% are encoded in the nucleus. This raises a logistical challenge to the cell, as these nuclear-encoded proteins have to be translated, delivered to the mitochondrial surface, and translocated to its various compartments. Over the past decade, it was shown that subsets of mRNAs encoding mitochondrial proteins (mMPs) are localized to the mitochondrial surface in both yeast and mammalian cells. Moreover, factors (e.g., RNA-binding proteins) have been discovered that facilitate mMP targeting, and their loss leads to RNA mislocalization and defects in mitochondrial function (e.g., deficient respiration). Therefore, there is a demand in the field of mitochondrial biology to accurately measure mMP localization to the mitochondrial surface. In this chapter, we describe two techniques that allow for the visualization of mMPs using single-molecule fluorescent in situ hybridization and preparation of a highly enriched mitochondrial fraction followed by quantitative real-time PCR. Together, these techniques constitute powerful tools to link changes in mMP trafficking to defects in mitochondrial physiology.