Intraplastidial trafficking of a phage-type RNA polymerase is mediated by a thylakoid RING-H2 protein

Abstract

The plastid genome of dicotyledonous plants is transcribed by three different RNA polymerases; an eubacterial-type enzyme, PEP; and two phage-type enzymes, RPOTp and RPOTmp. RPOTp plays an important role in chloroplast transcription, biogenesis, and mesophyll cell proliferation. RPOTmp fulfills a specific function in the transcription of the rrn operon in proplasts/amyloplasts during seed imbibition/germination and a more general function in chloroplasts during later developmental stages. In chloroplasts, RPOTmp is tightly associated with thylakoid membranes indicating that functional switching of RPOTmp is connected to thylakoid association. By using the yeast two-hybrid system, we have identified two proteins that interact with RPOTmp. The two proteins are very similar, both characterized by three N-terminal transmembrane domains and a C-terminal RING domain. We show that at least one of these proteins is an intrinsic thylakoid membrane protein that fixes RPOTmp on the stromal side of the thylakoid membrane, probably via the RING domain. A model is presented in which light by triggering the synthesis of the RING protein determines membrane association and functional switching of RPOTmp.chloroplast ͉ RING finger T ranscription of this small organellar genome is accomplished by three different RNA polymerases in a developmentally regulated manner (1, 2). One of the three RNA polymerases is of the eubacterial type, and its subunits are encoded on the plastid genome [plastid-encoded RNA polymerase (PEP)]. The two other RNA polymerases are nucleus-encoded [nucleus encoded plastid RNA polymerase (NEP)] and of the phage type (3, 4). Three different genes have been identified in Arabidopsis coding for NEP proteins that are localized in mitochondria (RPOTm, At1g68990), plastids (RPOTp, At2g24120), or in both organelles (RPOTmp, At5g15700; refs. 5 and 6). However, only two genes (RPOTp and RPOTm) have been detected in monocotyledons (4,7,8), thus raising the question of the function of RPOTmp in plastids of dicotyledon plants. Recent results have shown that RPOTp can be regarded as the principal NEP enzyme in chloroplasts playing an important role in chloroplast transcription, biogenesis, and mesophyll cell proliferation (9, 10). Results obtained with RPOTmp indicate partial replacement of RPOTp in RPOTp mutants (10, 11), an early function in lightinduced accumulation of several plastid mRNAs (12, 13), and specific transcription of the rrn operon of Arabidopsis from the PC promoter during seed imbibition and germination (10).Most of the plastid transcription units are preceded by NEP as well as PEP promoters and could be transcribed by the two types of RNA polymerase. However, with a few exceptions, NEP transcripts are barely detectable in mature chloroplasts, and most of the so-far-determined NEP promoters have been analyzed in PEP-deficient photosynthetically inactive plant material (14-17). These results led to an initial model of plastid transcription attributing special importance to NEP for the transcription of ho...

New insights into plastid nucleoid structure and functionality

“…Switching between the ''core DNA'' conformation and the ''surrounding DNA'' conformation could be mediated by chromatin remodeling proteins like SWIB-4 that are depicted here exemplarily at the interface between the two layers. Other membrane-associated proteins like the NEP interacting protein NIP can selectively control the activity of the NEP polymerase by preventing its association with plastid DNA (Azevedo et al 2008) an according classification has been discussed (Dillon and Dorman 2010). In this review we follow a simplified classification into enzymatic ptNAPs (including transcription, post-transcriptional levels of gene expression, DNA replication and repair) and architectural ptNAPs (including membrane anchoring and DNA compaction).…”

“…On the other hand, at least one type of plastid RNA polymerase, namely the NEP encoded by the RPOTmp gene seems to be sensitive to membrane binding and was proposed to be switched off when it is brought into close proximity of the thylakoid membranes. This sensitivity might be conferred by a thylakoid membrane-embedded RING protein, NIP1 (NEP interacting protein 1), that showed specific interaction with RPOTmp (Azevedo et al 2008), reflecting the complex interplay between structure, distribution and activity.…”

The prolific ATL family of RING-H2 ubiquitin ligases

“…A model proposes that AthATL25/NIP2 accumulates and attaches to the membrane during light-dependent differentiation when the thylakoid membranes are assembled. 41 weigelworld.org/expviz/expviz.jsp), suggesting that it may be a pseudogene, or that its expression is highly specific.…”

“…RPOTmp is present in plastids and mitochondria of eudicotyledonous plants, and has been found to be soluble in nonphotosynthetic cells, but associates with thylakoid membranes of chloroplasts. 40 NEP-Interacting Protein 2 (NIP2) was identified in a yeast two-hybrid screen from a study searching for proteins that may assist in the association of RPOTmp with the thylakoid membrane 41 ; NIP2 corresponds to AthATL25. A series of biochemical tests led to the identification of AthATL25/NIP2 is an integral protein of the thylakoid membrane, which may localize to the stromal side.…”

Genetic Dissection of Chloroplast Biogenesis and Development: An Overview

“…This includes both nuclear-encoded polymerase and plastidencoded polymerase machinery. Plastidic RNA polymerase sigma (sig) factors are required as transcription factors for chloroplast genes with the loss of sig2 and sig6 delaying chloroplast biogenesis (Ishizaki et al, 2005;Chi et al, 2010) and nuclear-encoded RNA polymerases, such as rpoT, has been shown to affect chloroplast biogenesis (Azevedo et al, 2008).…”