The transcription factor interacting protein RCD1 contains a novel conserved domain

Abstract

The RCD1 protein is a regulator of both developmental and stress responses in Arabidopsis thaliana and it interacts with several transcription factors. Its closest homolog, SRO1, seems to be dispensable for proper plant responses but the hardly viable phenotype of the rcd1 sro1 double mutant reveals that it encodes a functional protein that can partially compensate for the loss of RCD1 in the single rcd1 mutant. Both RCD1 and SRO1 contain a WWE domain, the catalytic core of poly(ADP-ribose) polymerases and a novel conserved domain termed RST which is also found in the transcription initiation complex component TAF4.

Here we summarize recent findings on the protein-protein interactions mediated by RCD1 and highlight the different functional possibilities that form the basis of our future experiments concerning the biochemical function of RCD1.

The Radical-induced Cell Death1 (RCD1) protein is a key regulator of several ROS-and abiotic stress-related responses in Arabidopsis thaliana. The rcd1 mutant plant displays several pleiotrophic phenotypes including ozone and salt sensitivity, UV-B and methyl viologen tolerance, early flowering and senescence. Additionally, it is an NO overproducer and has alterations in its responses to ethylene, salicylic acid and methyl jasmonate.^1–5 The Arabidopsis genome encodes a close homolog of RCD1, called SRO1 (Similar to RCD1 One1). In contrast to rcd1, the sro1 single mutant displays only subtle phenotypes⁶ but the protein is necessary for plant development since the rcd1 sro1 double mutant is severely disturbed in its development.^6,7

RCD1 seems to be involved in extensive protein-protein interactions: One of the earliest findings was reported by Lin and Heaton in 2001,⁸ when RCD1 was recovered in a yeast 2-hybrid (Y2H) screen with a viral movement protein from turnip crinkle virus. Katiyar-Agarwal and coworkers⁵ described its interaction with the cytoplasmic tail of Salt Overly Sensitive1 (SOS1), a well-known component of salt tolerance pathway. Furthermore, Miao and colleagues⁹ discussed a putative glutathione peroxidase3- RCD1 interaction and hypothesized that RCD1 could be a plant equivalent of the yeast redox-regulated transcription factor Yap1. Their idea was inspired by the earlier Y2H results by Belles-Boix and coworkers¹⁰ where two transcription factors, DREB2A and STO were found to interact with RCD1. Our group extended on these findings by doing a large-scale pair-wise Y2H interaction testing against the REGIA TF collection,¹¹ and found a large number of RCD1-interacting TFs from several protein families, predominantly AP2/ERF, NAC and bHLH.⁷ All of the transcription factor interactions and the SOS1 interaction seemed to take place at the C-terminal end of the RCD1 protein.^5,12

Both RCD1 and SRO1 contain three distinguishable domains: an N-terminal WWE domain which in Drosophila is known to mediate protein-protein interactions,¹³ and a poly(ADP-ribose) polymerase (PARP) catalytic domain that could be involved in protein modification reactions. The third conserved domain was recently identified in the C-terminus of RCD1 and named RST for RCD1-SRO-TAF4, since in addition to RCD1 and SRO1, it was found in four other homologs of these proteins (SRO2-SRO5) as well as in TBP-associated factor4,⁷ (TAF4; Fig. 1). The RST domain was present in RCD1-like proteins throughout the plant kingdom and consists of about 70 amino acids. The presence of the RST domain in TAF4s, which are involved in the assembly of the multimeric general transcription factor complex TFIID,¹⁴ indicated that this domain was likely to be involved in specifying protein-protein interactions. Furthermore, the large number of RCD1-TF interactions and similarity to a component of the transcription initiation complex hint that RCD1 might be involved in a protein complex that somehow regulates the function of the RCD1-interacting TFs.

Figure 1.

The structure of Arabidopsis RST domain-containing proteins RCD 1, SRO1, TA F4 and TA F4b and the amino acid sequence of the individual RST domains. The degree of amino acid conservation between the proteins within the RST domain is highlighted.

RCD1 and SRO1 are very similar on amino acid level (61% of the amino acids in the proteins are identical and 74% similar) and based on the double mutant phenotype SRO1 is a functional protein. However, the absence of a phenotype in the sro1 single mutant indicated that there must be some critical difference(s) between the two proteins. Do they differ in some key amino acids that confer specificity in interaction or are they differentially regulated on a post-translational level? In Y2H, RCD1 interacted with more transcription factors than SRO1, this suggests that the critical difference between the proteins might lie in the RST domain (Fig. 1). We are currently addressing these questions by creating domain swap constructs and targeted mutations to the proteins and assessing their functionality in mutant complementation and TF interaction experiments.

Biochemical activity of RCD1 or SRO1 has not been experimentally verified. Although their domain structure indicates a possible poly(ADP-ribose) polymerase activity, we are inclined to believe this is not the case (Vainonen JP, Overmyer K, Jaspers P, Kangasjärvi J, unpublished results). Instead, RCD1 could have a mechanistically similar activity or the domain could represent a necessary conserved structure without any enzymatic activity. Alternatively, the PARP domain could be a cofactor binding domain through which the function of the rest of the protein is regulated. This is an especially tempting scenario keeping in mind that the PARP domain is accompanied by two different putative protein-protein interaction domains, WWE and RST. Thus RCD1 or SRO1 could act as a scaffold protein and assemble TFs for their post-translational modification, relocalization or degradation.

Abbreviations

RCD1

radical-induced cell death1

ROS

reactive oxygen species

NO

nitric oxide

SRO1

similar to RCD1 one1

Y2H

yeast two-hybrid

REGIA

regulatory gene initiative in arabidopsis

TF

transcription factor

PARP

poly(ADP-ribose) polymerase

RST

RCD1-SRO-TAF4

TAF4

TBP-associated factor4

Addendum to: Jaspers P, Blomster T, Brosché M, Salojärvi J, Ahlfors R, Vainonen JP, et al. Unequally redundant RCD1 and SRO1 mediate stress and developmental responses and interact with transcription factors. Plant J. 2009;60:268–279. doi: 10.1111/j.1365-313X.2009.03951.x..