Presentation description

Cryo-electron tomography (cryo-ET) is a powerful technique for investigating protein structure and localization within cells. This study focused on two systems: mitochondrial protein quality control in yeast and calcium signaling at endoplasmic reticulum (ER)-mitochondria contact sites in mammalian cells. In yeast, CIS1 acts as a recruiter that directs the AAA-ATPase MSP1 to the TOM complex, facilitating the removal of mis-localized tail-anchored (TA) proteins from the outer mitochondrial membrane under import stress. To study CIS1 localization, an EGFP tag was introduced via homologous recombination, followed by CCCP treatment to induce mitochondrial stress in yeast. Further, cells were stained with DAPI and Mito-Tracker Deep Red and analyzed by fluorescence microscopy. In the second part of the study, we investigated components of the ER-mitochondria calcium signaling complex in mammalian cells, which includes the inositol 1,4,5-trisphosphate receptor (IP3R) on the ER, glucose-regulated protein 75 (GRP75) as a tethering factor, and voltage-dependent anion channel 1 (VDAC1) on the outer mitochondrial membrane. Tagging GRP75 with the red fluorescent protein Scarlet represents a promising strategy to enable visualization of ER-mitochondria contact sites via fluorescence microscopy.