DER containing two consecutive GTP-binding domains plays an essential role in chloroplast ribosomal RNA processing and ribosome biogenesis in higher plants

Young Jeon,Yeonhee Choi,Guen Tae Park,Jihwan Hwang,Hyun-Sook Pai,Chang Sook Ahn,Hyun Ju Jung,Hunseung Kang

doi:10.1093/jxb/ert360

Abstract

This study investigated protein characteristics and physiological functions of DER (Double Era-like GTPase) of higher plants. Nicotiana benthamiana DER (NbDER) contained two tandemly repeated GTP-binding domains (GD) and a C-terminal domain (CTD) that was similar to the K-homology domain involved in RNA binding. Both GDs possessed GTPase activity and contributed to the maximum GTPase activity of NbDER. NbDER fused to green fluorescent protein was localized primarily to chloroplast nucleoids. Arabidopsis der null mutants exhibited an embryonic lethal phenotype, indicating an essential function of DER during plant embryogenesis. Virus-induced gene silencing of NbDER resulted in a leaf-yellowing phenotype caused by disrupted chloroplast biogenesis. NbDER was associated primarily with the chloroplast 50S ribosomal subunit in vivo, and both the CTD and the two GD contributed to the association. Recombinant proteins of NbDER and its CTD could bind to 23S and 16S ribosomal RNAs in vitro. Depletion of NbDER impaired processing of plastid-encoded ribosomal RNAs, resulting in accumulation of the precursor rRNAs in the chloroplasts. NbDER-deficient chloroplasts contained significantly reduced levels of mature 23S and 16S rRNAs and diverse mRNAs in the polysomal fractions, suggesting decreased translation in chloroplasts. These results suggest that DER is involved in chloroplast rRNA processing and ribosome biogenesis in higher plants.

Highlights

GTPases are widely distributed among prokaryotes, archaea, and eukaryotes
Nicotiana benthamiana DER (NbDER) contained two tandemly repeated GTP-binding domains (GD) and a C-terminal domain (CTD) that was similar to the K-homology domain involved in RNA binding
The full-length NbDER was detected in the fractions containing the 50S subunit, whereas ∆GD1 and ∆GD2 deletion mutants were partially fractionated with the 30S small subunit. ∆GD1/2 mutant protein that lacks both GD1 and GD2 was not incorporated into ribosome fractions. ∆CTD lacking the C-terminal domain was co-fractionated with the 30S small subunit, 50S large subunit, and 70S monosome, suggesting a lack of interaction specificity. These results suggest that NbDER is associated primarily with 50S ribosomal subunits and that the specific association depends on the contribution of multiple domains, including GD1, GD2, and CTD

Summary

Introduction

GTPases are widely distributed among prokaryotes, archaea, and eukaryotes. They play critical roles in protein translation, protein translocation, and signal transduction (Caldon and March, 2003; Verstraeten et al, 2011). GTPases generally cycle between a GDP-bound inactive state and a GTPbound active state, and the accompanying conformational changes are crucial for their function as molecular switches in diverse cellular processes. GTPases contain conserved sequence motifs called the G motifs (G1–G5), which are crucially involved in guanine nucleotide binding, catalysis, and effector binding. The universally conserved bacterial GTPases play important roles in ribosome function, including ribosome biogenesis and protein translation (Caldon and March, 2003; Verstraeten et al, 2011). Except for a few GTPases that belong to translation machinery, the regulatory

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Conclusion