Protocol for engineering and validating a synthetic mitochondrial intermembrane bridge in mammalian cells

Gunjan Purohit; Martonio Ponte Viana; Oleh Khalimonchuk

doi:10.1016/j.xpro.2022.101454

Protocol for engineering and validating a synthetic mitochondrial intermembrane bridge in mammalian cells

STAR Protoc. 2022 Jun 14;3(3):101454. doi: 10.1016/j.xpro.2022.101454. eCollection 2022 Sep 16.

Authors

Gunjan Purohit¹, Martonio Ponte Viana², Oleh Khalimonchuk³

Affiliations

¹ Department of Biochemistry, University of Nebraska, Lincoln, NE 68503, USA. Electronic address: gpurohit2@unl.edu.
² Department of Biochemistry, University of Nebraska, Lincoln, NE 68503, USA.
³ Department of Biochemistry, University of Nebraska, Lincoln, NE 68503, USA; Nebraska Redox Biology Center, University of Nebraska, Lincoln, NE 68503, USA; Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68105, USA; Nebraska Center for Integrated Biomolecular Communication, University of Nebraska, Lincoln, NE, USA. Electronic address: okhalimonchuk2@unl.edu.

Abstract

Membrane contact sites are recognized as critical means of intercompartmental communication. Here, we describe a protocol for engineering and validating a synthetic bridge between the inner and outer mitochondrial membranes to support functioning of the endogenous mitochondrial contact site and cristae organizing system (MICOS). A chimeric protein, MitoT, is stably expressed in cultured mammalian cells to bridge the mitochondrial membranes. This approach can be a valuable tool to study the function of the MICOS complex and associated proteins. For complete details on the use and execution of this protocol, please refer to Viana et al. (2021).

Keywords: Biotechnology and bioengineering; Cell Biology; Cell Membrane; Cell culture; Cell isolation; Flow Cytometry/Mass Cytometry; Metabolism; Microscopy; Molecular Biology.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

Animals
Mammals
Mitochondria* / genetics
Mitochondrial Membranes* / metabolism
Proteins / metabolism

Substances

Proteins

Abstract

Publication types

MeSH terms

Substances

Grants and funding