The RNA-binding protein RNP29 is an unusual Toc159 transport substrate

Abstract

The precursors of RNP29 and Ferredoxin (Fd2) were previously identified in the cytosol of ppi2 plant cells with their N-terminal amino acid acetylated. Here, we explore whether precursor accumulation in ppi2 is characteristic for Toc159 client proteins, by characterizing the import properties of the RNP29 precursor in comparison to Fd2 and other Toc159-dependent or independent substrates. We find specific accumulation of the RNP29 precursor in ppi2 but not in wild type or ppi1 protoplasts. With the exception of Lhcb4, precursor accumulation is also detected with all other tested constructs in ppi2. However, RNP29 is clearly different from the other proteins because only precursor but almost no mature protein is detectable in protoplast extracts. Co-transformation of RNP29 with Toc159 complements its plastid import, supporting the hypothesis that RNP29 is a Toc159-dependent substrate. Exchange of the second amino acid in the RNP29 transit peptide to Glu or Asn prevents methionine excision but not N-terminal acetylation, suggesting that different N-acetyltransferases may act on chloroplast precursor proteins in vivo. All different RNP29 constructs are efficiently imported into wild type but not into ppi2 plastids, arguing for a minor impact of the N-terminal amino acid on the import process.

Sequence Motifs in Transit Peptides Act as Independent Functional Units and Can Be Transferred to New Sequence Contexts

“…All four preproteins containing defective TPs were degraded at varying degrees, which is in agreement with the previous report showing that the stability of chloroplast preproteins in the cytosol can vary depending on the precursors (Supplemental Fig. S1; Grimmer et al, 2014). However, their targeting efficiencies were not improved in the presence of MG132, indicating that the low targeting efficiencies of these four hybrid TPs are not caused by the cytosolic quality control (Supplemental Fig.…”

“…Previous studies have shown that unimported precursors are subject to the proteasomemediated proteolytic degradation in the cytosol to prevent cytotoxicity to the cell (Lee et al, 2009b;Bischof et al, 2011;Grimmer et al, 2014;Tillmann et al, 2015). Moreover, many chloroplast precursor proteins are known to be modified by N-terminal acetylation in the cytosol, which may possibly mediate the proteasomal degradation in the cytosol (Hwang et al, 2010;Bischof et al, 2011;Grimmer et al, 2014). To test the possibility of proteasomal degradation of unimported hybrid preproteins, four defective hybrid TPs, R(1-22)C(23-80), Fig.…”

Importance of Translocon Subunit Tic56 for rRNA Processing and Chloroplast Ribosome Assembly

“…Bischof and colleagues analyzed the proteome and transcriptome of ppi2 plants and showed that their molecular phenotype can be largely attributed to transcriptional regulation . Import assays using ppi2 protoplasts showed an import defect for precursor proteins from different functional categories, and the strongest client protein preference of Toc159 was observed for the RNA-binding protein RNP29 (Grimmer et al, 2014). Similarly, in a large-scale screen for precursor/Toc159 interactions, Dutta and colleagues identified a diverse set of proteins with different functions to interact with Toc159 and Toc132 (Dutta et al, 2014).…”

Mild proteasomal stress improves photosynthetic performance in Arabidopsis chloroplasts

“…In the work presented here, we provide a deeper understanding of the processes that integrate the UPS with the regulation of chloroplast protein import and biogenesis. We have recently shown that plastid-precursor proteins accumulate in the cytosol of Toc159-deficient plastids (ppi2) 4,15 . Since precursor proteins are cleared from the cytosol by the UPS 3 , we analyzed the effect of proteasome impairment on precursor stability and plastid proteome assembly.…”