Experimental determination of organelle targeting-peptide cleavage sites using transient expression of green fluorescent protein translational fusions

https://doi.org/10.1016/j.ab.2012.10.040Get rights and content

Abstract

The majority of nuclear-encoded organellar proteins contain a cleavable presequence, which is necessary for protein targeting and import into the correct cellular compartment. Knowledge about targeting-peptide cleavage sites is essential for the structural and functional characterization of the mature organellar proteins as well as for a deeper understanding of the import process. Because of the low consensus and high variability of presequences, bioinformatics of targeting-peptide cleavage fails to predict the length of the targeting peptide with high confidence. Therefore, we have developed a rapid and robust method to experimentally determine the cleavage site of the transit peptide for proteins imported into mitochondria or plastids. The protein precursor with green fluorescent protein (GFP) fused to its C-terminus is transiently expressed in cells (for animal proteins) or protoplasts (for plant proteins), allowing translocation into organelles and removal of the transit peptide. After lysis, the matured protein is immunopurified using an anti-GFP antibody coupled to magnetic beads. The N-terminal amino sequence is then determined by Edman microsequencing or mass spectrometry. The method has been validated using proteins with known targeting-peptide sequences and is suitable for animal and plant organelle-targeted proteins.

Section snippets

Plant material and growth conditions

Arabidopsis plants (Arabidopsis thaliana (L.) Heynh, Col-0 ecotype) were grown in Jiffy7 pots (Jiffy Products International, Oslo, Norway) in a growth chamber (23 °C, 75% relative humidity, 16 h light with an intensity of 80–100 μmol m−2 s−1).

DNA constructs

Full-length cDNA clones were ordered from OriGene (NM-000284, human PDHA1) and from the Arabidopsis Biological Resource Center (At3g10920.1, MSD1; At5g38430.1, RBCS; At4g05180.1, PSBQ2; At2g42540.2, COR15A; At2g42530.1, COR15B). Plasmids purified from clones

Results and discussion

The method we describe here to determine the targeting-peptide cleavage sites for proteins imported into mitochondria or plastids is shown schematically in Fig. 1. It takes advantage of the high level of accumulation of GFP translational fusions that typically occurs during transient expression in transfected animal cells or plant protoplasts. Proteins of interest are genetically fused upstream of GFP, in order not to mask the N-terminal targeting peptide. During the expression period the

Acknowledgments

We are very grateful to Jennifer Alban for technical assistance with human cell transfection, to Mathilde Joint for expert help with mass spectrometry experiments, and to Marjorie Juchaux (IMAC) for assistance with confocal microscopy. A.C. and P.P. acknowledge the support of Ph.D. fellowships from the Région-Pays-de-la-Loire/INRA and Angers Loire Metropole, respectively. We are thankful to Professor David Logan for careful proofreading and helpful suggestions.

References (36)

  • M.F. Hoylaerts et al.

    Mammalian alkaline phosphatase catalysis requires active site structure stabilization via the N-terminal amino acid microenvironment

    Biochemistry

    (2006)
  • X. Wang et al.

    A multi-label predictor for identifying the subcellular locations of singleplex and multiplex eukaryotic proteins

    PLoS One

    (2012)
  • A.H. Millar et al.

    Exploring the function–location nexus: using multiple lines of evidence in defining the subcellular location of plant proteins

    Plant Cell

    (2009)
  • O. Emanuelsson et al.

    Locating proteins in the cell using TargetP, SignalP and related tools

    Nat. Protoc.

    (2007)
  • F. Rebeille et al.

    The role of plant mitochondria in the biosynthesis of coenzymes

    Photosynth. Res.

    (2007)
  • J. Bardel et al.

    A survey of the plant mitochondrial proteome in relation to development

    Proteomics

    (2002)
  • D. Wellner et al.

    Sequencing of peptides and proteins with blocked N-terminal amino-acids—N-acetylserine or N-acetylthreonine

    Proc. Natl. Acad. Sci. U.S.A.

    (1990)
  • B. Zybailov et al.

    Sorting signals, N-terminal modifications and abundance of the chloroplast proteome

    PLoS One

    (2008)
  • Cited by (20)

    • Folding and Lipid Composition Determine Membrane Interaction of the Disordered Protein COR15A

      2018, Biophysical Journal
      Citation Excerpt :

      The analyses presented in this study, together with previous experimental and computational results, allow us to propose the most detailed mechanism of action for any LEA protein thus far. COR15A is induced under cold but nonfreezing conditions at the transcriptional level (72) and is imported into the chloroplast stroma, where it resides as an intrinsically disordered, soluble protein (34–36). Upon freezing, ice crystallization takes place in the apoplastic space and cells are dehydrated (73).

    • The Use of Mass Spectrometry to Examine IDPs: Unique Insights and Caveats

      2018, Methods in Enzymology
      Citation Excerpt :

      Specifically they are involved in the stabilization of chloroplast membranes in the leaf during freezing. COR15A (used as an example in this chapter) and COR5B are COR proteins, found in Arabidopsis thaliana, which are critical to the plants adaptation to cold temperatures and specifically to dehydration (Candat et al., 2013; Lin & Thomashow, 1992; Nakayama et al., 2007). The COR15 proteins are found to be relatively disordered in hydrophilic conditions and undergo conformational transition to helical forms upon dehydration.

    • Intrinsically Disordered Stress Protein COR15A Resides at the Membrane Surface during Dehydration

      2017, Biophysical Journal
      Citation Excerpt :

      This is further into the bilayer than the water molecules penetrate, and indicates that the protein is not simply a freely dissolved solute in the water phase, but directly interacting with the lipid phase. COR15A is accumulated in the chloroplasts of Arabidopsis leaves when the plants are exposed to low temperatures (12–14). This accumulation is necessary for the plants to obtain their full, cold-acclimated freezing tolerance (16).

    • No plastidial calmodulin-like proteins detected by two targeted mass-spectrometry approaches and GFP fusion proteins

      2016, New Negatives in Plant Science
      Citation Excerpt :

      Primers sequences were as follows: Arabidopsis protoplasts transformation and confocal microscopy were carried out according to [46]. Arabidopsis plants, Wassilewskija background (Ws), were grown in culture chambers at 23 °C (12-h light cycle) with a light intensity of 150 μmol.m−2 s−1 in standard conditions [47].

    View all citing articles on Scopus
    1

    These authors have equally contributed to this work.

    View full text