Monitoring organelle turnover in yeast using fluorescent protein tags

Cellular degradative processes including proteasomal and vacuolar/lysosomal autophagic degradation, as well as the activity of proteases (both cytosolic and mitochondrial), provide for a continuous turnover of damaged and obsolete macromolecules and organelles. Mitochondria are essential for oxidative energy production in aerobic eukaryotic cells, where they are also required for multiple biosynthetic pathways to take place. Mitochondrial homeostasis also plays a crucial role in aging and programmed cell death, and recent data have suggested that mitochondrial degradation is a strictly regulated process. A recent study has shown that in yeast cells subjected to nitrogen starvation, degradation of mitochondria by autophagy occurs by both a selective process (termed mitophagy) and a nonselective process. This chapter provides an overview of the techniques that enable the study of mitophagy. Fluorescent proteins targeted to mitochondria can be used to follow mitochondrial sequestration within vacuoles. Degradation of mitochondria can be assayed using a mitochondrially targeted alkaline phosphatase (ALP) reporter test in which the delivery of mitochondrial N-terminal truncated Pho8Delta60 to the vacuole results from mitophagy. Degradation of mitochondrial proteins can also be followed by Western immunoblot analyses. Finally, electron microscopy observations permit the discrimination between selective mitophagy and nonselective mitochondrial degradation.