Consequences of impaired 1-MDa TIC complex assembly for the abundance and composition of chloroplast high-molecular mass protein complexes.

Peter Schäfer,Daniel Köhler,Birgit Agne,Stefan Helm,Sacha Baginsky,Andrew Webber

doi:10.1371/journal.pone.0213364

Peter Schäfer, Daniel Köhler + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0213364

Copy DOI

Journal: PloS one	Publication Date: Mar 13, 2019
Citations: 10	License type: CC BY 4.0

Affiliation: Martin Luther University Halle-Wittenberg

Abstract

We report a systematic analysis of chloroplast high-molecular mass protein complexes using a combination of native gel electrophoresis and absolute protein quantification by MSE. With this experimental setup, we characterized the effect of the tic56-3 mutation in the 1-MDa inner envelope translocase (TIC) on the assembly of the chloroplast proteome. We show that the tic56-3 mutation results in a reduction of the 1-MDa TIC complex to approximately 10% of wildtype levels. Hierarchical clustering confirmed the association of malate dehydrogenase (MDH) with an envelope-associated FtsH/FtsHi complex and suggested the association of a glycine-rich protein with the 1-MDa TIC complex. Depletion of this complex leads to a reduction of chloroplast ATPase to approx. 75% of wildtype levels, while the abundance of the FtsH/FtsHi complex is increased to approx. 140% of wildtype. The accumulation of the major photosynthetic complexes is not affected by the mutation, suggesting that tic56-3 plants can sustain a functional photosynthetic machinery despite a significant reduction of the 1-MDa TIC complex. Together our analysis expands recent efforts to catalogue the native molecular masses of chloroplast proteins and provides information on the consequences of impaired accumulation of the 1-MDa TIC translocase for chloroplast proteome assembly.

Highlights

Protein/protein interactions determine complex biosynthetic activities such as the synthesis of metabolites, DNA, RNA and proteins
Extrapolating from a regression curve, the gel allowed separation of protein complexes up to around 4 MDa, which is similar to previous studies where gel-filtration was used for native complex isolation from chloroplasts [8]
We cut the gel in the region between 600 kDa and approximately 4 MDa into nine sized slices and identified and quantified the protein constituents of every gel slice by HD-MSE mass spectrometry, as previously described (Fig 1C) [20]

Summary

Introduction

Protein/protein interactions determine complex biosynthetic activities such as the synthesis of metabolites, DNA, RNA and proteins. We could show that a recently identified FtsH/FtsHi complex at the inner envelope membrane accumulates independently of the 1-MDa TIC complex, even though they form a supercomplex during protein translocation in wildtype chloroplasts [18]. We first assessed the abundance of protein complexes in chloroplasts using the wildtype data as a reference (Fig 1D).

Results

Conclusion