Detection of Dual Targeting and Dual Function of Mitochondrial Proteins in Yeast

Abstract

Eukaryotic cells are defined by the existence of subcellular compartments and organelles. The localization of a protein to a specific subcellular compartment is one of the most fundamental processes of a living cell. It is well documented that in eukaryotic cells molecules of a single protein can be located in more than one subcellular compartment, a phenomenon termed dual targeting, bimodal targeting, or dual localization. Recently, growing evidence started to accumulate for abundant dual targeting of mitochondrial proteins, which are localized to a second location in the cell, besides this specific organelle. We have termed these dual localized proteins echoforms or echoproteins (echo in Greek denotes repetition). As the research on dual targeting of proteins is developing and evidence is accumulating for high abundance of the phenomenon, there is a growing need for new methods that would allow the identification of dual localized proteins and analysis of their functions in each subcellular compartment. This is particularly critical for single translation products that are encoded by the same gene and are actually derived from the same protein but nevertheless distribute between different subcellular compartments. The above considerations have led us to develop several approaches for studying dual localized proteins and their dual function. These include an α-complementation-based assay, specific depletion, and selection of the individual echoproteins.

Genome-wide SWAp-Tag yeast libraries for proteome exploration

“…Several N’ proteins that localized to mitochondria also showed localization to another organelle in the same cell or localized differently when C’ tagged. This suggests that some of these proteins are dually targeted 28. We imaged a subset of these proteins from the NATIVEpr-GFP library under several growth conditions (Supplementary figure 5) and found that they indeed could reside in a variety of organelles depending on the medium.…”

Assigning mitochondrial localization of dual localized proteins using a yeast Bi-Genomic Mitochondrial-Split-GFP

“…However, resolving mitochondrial proteomes is challenging due to the difficulty of obtaining pure mitochondria and because many proteins transiently localize in mitochondria and are found elsewhere in cells. Up to 10–20% of the yeast mitoproteome was suggested to be composed of proteins with another location in cells ( i.e the cytosol, the nucleus, ER…) ( Ben-Menachem and Pines, 2017 ; Morgenstern et al, 2017 ). Our BiG Mito-Split-GFP system will be especially helpful to resolve these proteome complexities.…”

“…As a result, most of the proteins required for mitochondrial structure and functions are expressed from the nuclear genome (>99%) and synthetized as precursors targeted to the mitochondria by mitochondrial targeting signals (MTS), that in some case are cleaved upon import ( Chacinska et al, 2009 ). In the yeast S. cerevisiae , about a third of the mitochondrial proteins (mitoproteome) have been suggested to be dual localized ( Ben-Menachem et al, 2011 ; Dinur-Mills et al, 2008 ; Kisslov et al, 2014 ), and have been named echoforms (or echoproteins) to accentuate the fact that two identical or nearly identical forms of a protein, can reside in the mitochondria and another compartment ( Ben-Menachem and Pines, 2017 ). Due to these two coexisting forms and the difficulty to obtain pure mitochondria, determination of a complete mitoproteome remains challenging and gave rise to conflicting results ( Kumar et al, 2002 ; Morgenstern et al, 2017 ; Reinders et al, 2006 ; Sickmann et al, 2003 ).…”

The Mitochondrial Hsp90 TRAP1 and Alzheimer’s Disease

“…Alternatively, TRAP1 may not be fully targeted towards mitochondria. Dual targeting of mitochondrial proteins has a biological function for other proteins, too (Kalderon and Pines, 2014;Dik et al, 2016;Ben-Menachem and Pines, 2017).…”