Energetic manipulation of chloroplast protein import and the use of chemical cross-linkers to map protein-protein interactions

Hitoshi Inoue, Fei Wang, Takehito Inaba, Danny J. Schnell

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Scopus citations

Abstract

Most chloroplast proteins are synthesized in the cytosol as preproteins with N-terminal cleavable transit peptides and are imported into the organelle through the TOC-TIC translocon system. Import involves a complex set of recognition and membrane translocation steps that ensure the fidelity and unidirectional transport of the polypeptide across the double-membrane chloroplast envelope. To understand the mechanism of import, the molecular interactions and energetics of each step must be defined. Here, we describe the methods for capturing intermediates in the import process through the manipulation of the energy state of chloroplasts, and the use of two different chemical cross-linking approaches to examine the molecular interactions that mediate the import process and to assess the assembly state of the translocons. These approaches can be employed to identify sequential protein-protein interactions, and thereby dissect the pathway and roles of import components during protein import into chloroplasts.

Original languageEnglish (US)
Title of host publicationChloroplast Research in Arabidopsis
Subtitle of host publicationMethods and Protocols, Volume 1
EditorsPaul Jarvis
Pages307-320
Number of pages14
DOIs
StatePublished - Sep 23 2011

Publication series

NameMethods in Molecular Biology
Volume774
ISSN (Print)1064-3745

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Keywords

  • ATP
  • Chloroplast
  • Cross-link
  • Early import intermediate
  • GTP
  • Import
  • TIC
  • TOC

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Inoue, H., Wang, F., Inaba, T., & Schnell, D. J. (2011). Energetic manipulation of chloroplast protein import and the use of chemical cross-linkers to map protein-protein interactions. In P. Jarvis (Ed.), Chloroplast Research in Arabidopsis: Methods and Protocols, Volume 1 (pp. 307-320). (Methods in Molecular Biology; Vol. 774). https://doi.org/10.1007/978-1-61779-234-2_18