Polyglycine Acts as a Rejection Signal for Protein Transport at the Chloroplast Envelope

PLoS One. 2016 Dec 9;11(12):e0167802. doi: 10.1371/journal.pone.0167802. eCollection 2016.

Abstract

PolyGly is present in many proteins in various organisms. One example is found in a transmembrane β-barrel protein, translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75). Toc75 requires its N-terminal extension (t75) for proper localization. t75 comprises signals for chloroplast import (n75) and envelope sorting (c75) in tandem. n75 and c75 are removed by stromal processing peptidase and plastidic type I signal peptidase 1, respectively. PolyGly is present within c75 and its deletion or substitution causes mistargeting of Toc75 to the stroma. Here we have examined the properties of polyGly-dependent protein targeting using two soluble passenger proteins, the mature portion of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (mSS) and enhanced green fluorescent protein (EGFP). Both t75-mSS and t75-EGFP were imported into isolated chloroplasts and their n75 removed. Resultant c75-mSS was associated with the envelope at the intermembrane space, whereas c75-EGFP was partially exposed outside the envelope. Deletion of polyGly or substitution of tri-Ala for the critical tri-Gly segment within polyGly caused each passenger to be targeted to the stroma. Transient expression of t75-EGFP in Nicotiana benthamiana resulted in accumulation of c75-EGFP exposed at the surface of the chloroplast, but the majority of the EGFP passenger was found free in the cytosol with most of its c75 attachment removed. Results of circular dichroism analyses suggest that polyGly within c75 may form an extended conformation, which is disrupted by tri-Ala substitution. These data suggest that polyGly is distinct from a canonical stop-transfer sequence and acts as a rejection signal at the chloroplast inner envelope.

MeSH terms

  • Chloroplasts / chemistry
  • Chloroplasts / metabolism*
  • Green Fluorescent Proteins / analysis
  • Green Fluorescent Proteins / metabolism
  • Membrane Proteins / analysis
  • Membrane Proteins / metabolism
  • Peptides / analysis
  • Peptides / metabolism*
  • Pisum sativum / chemistry
  • Pisum sativum / metabolism*
  • Plant Proteins / analysis
  • Plant Proteins / metabolism*
  • Protein Transport
  • Ribulose-Bisphosphate Carboxylase / analysis
  • Ribulose-Bisphosphate Carboxylase / metabolism

Substances

  • Membrane Proteins
  • Peptides
  • Plant Proteins
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • polyglycine
  • Ribulose-Bisphosphate Carboxylase

Grants and funding

The initial phase of this work involving preparation of some DNA constructs was supported by the National Research Initiative of the United States Department of Agriculture Cooperative State Research, Education and Extension Service (grant no. 2003-02860) to KI. The assay using recombinant Plsp1 was supported by the Division of Chemical Sciences, Geosciences and Biosciences, Office of Basic Energy Sciences of the US Department of Energy (grant no. DE-FG02-08ER15963) to KI. Performance of all other experiments and data analysis was supported by the Division of Molecular and Cellular Biosciences at the US National Science Foundation (grant no. 1050602) to KI. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.