Abstract

The NanoLuc split luciferase assay has proven to be a powerful tool for the analysis of protein translocation. Its flexibility has enabled in vivo, ex vivo, and in vitro studies—including systems reconstituting protein transport from pure components. The assay has been particularly useful in the characterization of bacterial secretion and mitochondrial protein import. In the latter case, MitoLuc has been developed for the investigation of the TIM23-pathway via import into the matrix of isolated yeast mitochondria. Subsequent analysis identified three distinct phases of import, rather than in a single continuous step. The assay has also been developed to monitor import into the mitochondrial matrix of intact cultured cells. This latter innovation has laid the foundations for further analysis of the import process in humans, including the consequences of interactions with cytosolic factors and neighboring organelles. The versatility of the MitoLuc assay is conducive for its adaptation to also monitor import into the inter-membrane space (MIA-pathway), and into the inner-membrane via the TIM22- and TIM23-complexes. Here, we present detailed protocols for the application of MitoLuc to mitochondria isolated from yeast and to those within cultured human cells.

Original languageEnglish
Title of host publicationMitochondrial Translocases Part A
PublisherAcademic Press Inc.
Pages407-436
Number of pages30
ISBN (Print)9780443293306
DOIs
StatePublished - Jan 2024

Publication series

NameMethods in Enzymology
Volume706
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • Cell culture
  • Luciferase
  • Mitochondrial biogenesis
  • Mitochondrial protein import
  • MitoLuc assay
  • NanoLuc
  • Protein translocation
  • Yeast

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