Related Experiment Video

Updated: Aug 12, 2026

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry
08:04

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

Published on: March 13, 2014

Comprehensive survey of proteins targeted by chloroplast thioredoxin

K Motohashi1, A Kondoh, M T Stumpp

  • 1Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8503, Japan.

Proceedings of the National Academy of Sciences of the United States of America
|September 13, 2001
PubMed

Insights

Researchers developed a novel method to identify chloroplast thioredoxin (Trx) targets. This technique immobilizes Trx to capture proteins involved in cellular redox networks, aiding in understanding plant cell regulation.

Area of Science:

  • Plant Biochemistry
  • Molecular Biology
  • Redox Signaling

Background:

  • Chloroplast thioredoxins (Trxs) are crucial for regulating plant cellular processes through redox control.
  • Identifying Trx target proteins is essential for understanding these complex redox networks.

Purpose of the Study:

  • To develop and validate a method for identifying novel chloroplast Trx target proteins.
  • To investigate the redox regulation of proteins within spinach chloroplast stroma.

Main Methods:

  • Immobilization of a mutant m-type thioredoxin onto cyanogen bromide-activated resin.
  • Affinity capture of proteins forming mixed-disulfide intermediates with immobilized Trx.
  • Identification of captured proteins using SDS-PAGE, N-terminal sequencing, and homology searches.

Main Results:

  • Successfully identified known Trx targets like Rubisco activase and 2-Cys-type peroxiredoxin (Prx).
  • Also identified Calvin cycle enzymes (GAPDH, SBpase) and novel potential targets including glutamine synthetase, cyclophilin, Prx-Q, and Rubisco small subunit.
  • Confirmed Trx susceptibility for Arabidopsis thaliana cyclophilin and Prx-Q.

Conclusions:

  • The developed immobilization method is effective for identifying chloroplast Trx targets.
  • This approach facilitates the investigation of diverse redox networks and related enzymes in plant cells.
  • Provides a valuable tool for advancing the study of thioredoxin-mediated regulation in chloroplasts.

More Related Videos

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves11:10

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Published on: March 9, 2014

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics10:28

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics

Published on: October 19, 2018

Related Concept Videos

Translocation of Proteins into the Mitochondria01:19

Translocation of Proteins into the Mitochondria

Mitochondrial precursors are translocated to the internal subcompartments via independent mechanisms involving distinct protein machineries called translocases.
Sorting of outer membrane proteins:
Mitochondrial outer membrane proteins are of two types: the transmembrane, beta-barrel porins, and the membrane-anchored, alpha-helical proteins. Beta-barrel porin precursors are translocated by the TOM complex and inserted into the outer mitochondrial membrane by the SAM complex. In contrast,...
Protein Transport to the Stroma01:24

Protein Transport to the Stroma

Chloroplasts are triple membrane structures with an outer membrane, an inner membrane, and a thylakoid membrane, each containing distinct metabolite transporters, membrane translocons, and enzymes. Appropriate sorting and translocating these proteins to their correct membrane systems is essential for chloroplast function.
Protein complexes called the translocon of the outer chloroplast membrane or TOC complex, and the translocon of the inner chloroplast membrane or TIC complex mediate the...
Protein Transport to the Outer Chloroplast Membrane01:11

Protein Transport to the Outer Chloroplast Membrane

Chloroplast outer membrane proteins encoded by the nucleus are synthesized in the cytosol. Soon after synthesis, they bind cytosolic factors such as 14-3-3 protein and the Hsp70 chaperones that keep these precursors in an unfolded state until their translocation.
Two models describe the mechanism of precursor recognition and entry across the outer membrane through the TOC complex. Model 1 suggests the newly synthesized precursor binds to the TOC receptor 159 and forms a complex.
Protein Transport to the Thylakoids01:22

Protein Transport to the Thylakoids

Thylakoids are membrane-bound sac-like structures within the chloroplast that serve as sites for photosynthesis. Thylakoid lumen contains many electron transport proteins and is enclosed by a thylakoid membrane rich in the light-harvesting complex. Proteins targeted to the thylakoids are transported as precursors and are sorted by the general TOC/TIC import pathway. Once the precursor reaches the stroma, stromal processing peptidases remove their transit signal and expose thylakoid signal...
Protein Transport to the Inner Chloroplast Membrane01:18

Protein Transport to the Inner Chloroplast Membrane

Proteins targeted to the inner chloroplast membrane, or plastid proteins, are transported by two general pathways: the stop-transfer and the re-insertion or post-import pathways. Most plastid proteins carry N-terminal transit sequences and internal import sequences targeting it to the specific chloroplast subcompartment. Proteins targeted by the stop-transfer pathway have internal hydrophobic sequences that inhibit their translocation into the stroma. As a result, these precursors are arrested...