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 Citation (Scopus)

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
Pages307-320
Number of pages14
Volume774
DOIs
StatePublished - Sep 23 2011

Publication series

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

Fingerprint

Chloroplast Proteins
Protein Interaction Maps
Chloroplasts
Peptides
Proteins
Membranes
Organelles
Cytosol

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Medicine(all)

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 Chloroplast Research in Arabidopsis: Methods and Protocols, Volume 1 (Vol. 774, pp. 307-320). (Methods in Molecular Biology; Vol. 774). https://doi.org/10.1007/978-1-61779-234-2_18

Energetic manipulation of chloroplast protein import and the use of chemical cross-linkers to map protein-protein interactions. / Inoue, Hitoshi; Wang, Fei; Inaba, Takehito; Schnell, Danny J.

Chloroplast Research in Arabidopsis: Methods and Protocols, Volume 1. Vol. 774 2011. p. 307-320 (Methods in Molecular Biology; Vol. 774).

Research output: Chapter in Book/Report/Conference proceedingChapter

Inoue, H, Wang, F, Inaba, T & Schnell, DJ 2011, Energetic manipulation of chloroplast protein import and the use of chemical cross-linkers to map protein-protein interactions. in Chloroplast Research in Arabidopsis: Methods and Protocols, Volume 1. vol. 774, Methods in Molecular Biology, vol. 774, pp. 307-320. https://doi.org/10.1007/978-1-61779-234-2_18
Inoue H, Wang F, Inaba T, Schnell DJ. Energetic manipulation of chloroplast protein import and the use of chemical cross-linkers to map protein-protein interactions. In Chloroplast Research in Arabidopsis: Methods and Protocols, Volume 1. Vol. 774. 2011. p. 307-320. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-61779-234-2_18
Inoue, Hitoshi ; Wang, Fei ; Inaba, Takehito ; Schnell, Danny J. / Energetic manipulation of chloroplast protein import and the use of chemical cross-linkers to map protein-protein interactions. Chloroplast Research in Arabidopsis: Methods and Protocols, Volume 1. Vol. 774 2011. pp. 307-320 (Methods in Molecular Biology).
@inbook{ec560de6623c4dc9bef14ad526277bbd,
title = "Energetic manipulation of chloroplast protein import and the use of chemical cross-linkers to map protein-protein interactions",
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.",
keywords = "ATP, Chloroplast, Cross-link, Early import intermediate, GTP, Import, TIC, TOC",
author = "Hitoshi Inoue and Fei Wang and Takehito Inaba and Schnell, {Danny J.}",
year = "2011",
month = "9",
day = "23",
doi = "10.1007/978-1-61779-234-2_18",
language = "English (US)",
isbn = "9781617792335",
volume = "774",
series = "Methods in Molecular Biology",
pages = "307--320",
booktitle = "Chloroplast Research in Arabidopsis",

}

TY - CHAP

T1 - Energetic manipulation of chloroplast protein import and the use of chemical cross-linkers to map protein-protein interactions

AU - Inoue, Hitoshi

AU - Wang, Fei

AU - Inaba, Takehito

AU - Schnell, Danny J.

PY - 2011/9/23

Y1 - 2011/9/23

N2 - 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.

AB - 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.

KW - ATP

KW - Chloroplast

KW - Cross-link

KW - Early import intermediate

KW - GTP

KW - Import

KW - TIC

KW - TOC

UR - http://www.scopus.com/inward/record.url?scp=80052938274&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=80052938274&partnerID=8YFLogxK

U2 - 10.1007/978-1-61779-234-2_18

DO - 10.1007/978-1-61779-234-2_18

M3 - Chapter

C2 - 21822846

AN - SCOPUS:80052938274

SN - 9781617792335

VL - 774

T3 - Methods in Molecular Biology

SP - 307

EP - 320

BT - Chloroplast Research in Arabidopsis

ER -