PPR-SMRs: ancient proteins with enigmatic functions

Abstract

A small subset of the large pentatricopeptide repeat (PPR) protein family in higher plants contain a C-terminal small MutS-related (SMR) domain. Although few in number, they figure prominently in the chloroplast biogenesis and retrograde signaling literature due to their striking mutant phenotypes. In this review, we summarize current knowledge of PPR-SMR proteins focusing on Arabidopsis and maize proteomic and mutant studies. We also examine their occurrence in other organisms and have determined by phylogenetic analysis that, while they are limited to species that contain chloroplasts, their presence in algae and early branching land plant lineages indicates that the coupling of PPR motifs and an SMR domain into a single protein occurred early in the evolution of the Viridiplantae clade. In addition, we discuss their possible function and have examined conservation between SMR domains from Arabidopsis PPR proteins with those from other species that have been shown to possess endonucleolytic activity.

Keywords: Arabidopsis thaliana; Zea mays; chloroplast; endonuclease; genomes uncoupled; mitochondria; pentatricopeptide repeat protein; plastid; small MutS-related domain.

Figure 1. Proteins containing an SMR domain in the model plant Arabidopsis thaliana. A non-redundant set of 12 proteins was identified by searching the Universal Protein knowledgebase (UniProt; www.uniprot.org) for Arabidopsis proteins that contain the InterPro domain IPR002625 (Smr protein/MutS2 C-terminal domain). Proteins are denoted by their corresponding Arabidopsis Genome Identifier (AGI; ATXGXXXXX) and, if applicable, followed by their common name (e.g., GUN1). Protein domain structure is shown alongside each AGI to demonstrate presence and location of the pentatricopeptide repeat (PPR), small MutS-related (SMR), domain of unknown function (DUF) 1771, and MutS domains. The schematics of protein domain structure were created by combining TPRpred to predict PPR domains (those with P > 0.01 were excluded), InterProScan to identify other domains and DOG 1.0 for visualization of their respective positions.

Figure 2. Bayesian phylogenetic tree of PPR-SMR protein sequences from a range of different species. Sequences of PPR-SMR proteins were obtained from BLAST searches and InterPro domain searches (IPR002625 and IPR002885) and aligned using MUSCLE. A phylogenetic tree was constructed using MrBayes version 3.2.1 which employs Markov Chain Monte Carlo (MCMC) sampling to approximate the posterior probabilities of phylogenies (shown above the branches). MrBayes 3.2.1 was run in parallel on the Fornax supercomputer (located at iVEC@UWA) utilizing the BEAGLE library with a mixed model of molecular evolution (determined using jModelTest⁵⁶), utilizing 12 chains for 50 million generations and trees sampled every 1000 generations. All runs reached a plateau in likelihood score, which was indicated by the standard deviation of split frequencies (0.0015), and the potential scale reduction factor was close to one, indicating the MCMC chains converged. Sequences are color shaded based on their lineage as indicated.

Figure 3. SMR domain alignment to assess amino acid sequence conservation. The SMR domains of the eight Arabidopsis PPR-SMR proteins were aligned with SMR domains from proteins that have been experimentally demonstrated to have endonucleolytic activity.^,– The sequences are denoted by the SMR subfamily type (1_, 2_, or 3_) followed by the AGI (for Arabidopsis proteins) or alternative identifier (Tt_MutS2 – Thermus thermophilus MutS2 protein; Hs_B3BP – Homo sapiens BCL3 binding protein; Ld_CSBP – Leishmania donovani cycling sequence binding protein; Ec_YdaL – Escherichia coli YdaL protein), and the length of the SMR domain (e.g., /1–93). Alignment was performed using MUSCLE and visualized using Jalview (www.jalview.org) with ClustalX coloring by conservation. The positions of previously described conserved regions are indicated on the alignment: the LDXH motif present in subfamily 2 SMR domains and the centrally located HGXG/TGXG (subfamilies 1 and 3/subfamily 2) are bounded by the red and blue boxes, respectively.