Protein Import Assay into Mitochondria Isolated from Human Cells

Lena M Murschall; Esra Peker; Thomas MacVicar; Thomas Langer; Jan Riemer

doi:10.21769/BioProtoc.4057

Protein Import Assay into Mitochondria Isolated from Human Cells

Bio Protoc. 2021 Jun 20;11(12):e4057. doi: 10.21769/BioProtoc.4057.

Authors

Lena M Murschall¹, Esra Peker¹, Thomas MacVicar², Thomas Langer^{2

3}, Jan Riemer^{1

3}

Affiliations

¹ Institute for Biochemistry, Redox Biochemistry, University of Cologne, Zuelpicher Str. 47a/R. 3.49, 50674 Cologne, Germany.
² Department of Mitochondrial Proteostasis, Max Planck Institute for Biology of Ageing, 50931 Cologne, Germany.
³ Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD) and Center for Molecular Medicine (CMMC), University of Cologne, 50931 Cologne, Germany.

Abstract

Mitochondria are essential organelles containing approximately 1,500 proteins. Only approximately 1% of these proteins are synthesized inside mitochondria, whereas the remaining 99% are synthesized as precursors on cytosolic ribosomes and imported into the organelle. Various tools and techniques to analyze the import process have been developed. Among them, in vitro reconstituted import systems are of importance to study these processes in detail. These experiments monitor the import reaction of mitochondrial precursors that were previously radiolabeled in a cell-free environment. However, the methods described have been mostly performed in mitochondria isolated from S. cerevisiae. Here, we describe the adaptation of this powerful assay to import proteins into crude mitochondria isolated from human tissue culture cells. Graphic abstract: Overview of the assay to monitor protein import into mitochondria isolated from human cells.

Keywords: Cell-free protein synthesis; Human tissue culture cells; In organello import; Isolated mitochondria; Radiolabeled proteins.