Preparation and Utilization of a Versatile GFP-Protein Trap-Like System for Protein Complex Immunoprecipitation in Plants.

Protein complex immunoprecipitation (co-IP) is an in vitro technique used to study protein-protein interaction between two or more proteins. This method relies on affinity purification of recombinant epitope-tagged proteins followed by western blotting detection using tag-specific antibodies for the confirmation of positive interaction. The traditional co-IP method relies on the use of porous beaded support with immobilized antibodies to precipitate protein complexes. However, this method is time-consuming, labor-intensive, and provides lower reproducibility and yield of protein complexes. Here, we describe the implementation of magnetic beads and high-affinity anti-green fluorescent protein (GFP) antibodies to develop an in vitro GFP-protein trap-like system. This highly reproducible system utilizes a combination of small sample size, versatile lysis buffer, and lower amounts of magnetic beads to obtain protein complexes and aggregates that are compatible with functional assays, Western blotting, and mass spectrometry. In addition to protein-protein interactions, this versatile method can be employed to study protein-nucleic acid interactions. This protocol also highlights troubleshooting and includes recommendations to optimize its application.

J-like protein family of Arabidopsis thaliana: the enigmatic cousins of J-domain proteins.

KEY MESSAGE: J-like proteins (JLPs) are emerging as ancillaries to the cellular chaperone network. They modulate functions of Hsp70:J-domain protein (JDP) systems in novel ways thereby having key roles in diverse plant processes. J-domain proteins (JDPs) form an obligate co-chaperone partnership with Hsp70s with their highly conserved J-domain to steer protein quality control processes in the cell. The HPD motif between helix II and helix III of the J-domain is crucial for JDP's interaction with Hsp70s. According to the most recent classification, J-like proteins (JLPs) form an extended class of the JDP family possessing a degenerate J-domain with the HPD motif non-conservatively replaced by other amino acid residues and hence are not able to interact with Hsp70s. Considering this most updated and acceptable JLP classification, we identified 21 JLPs in Arabidopsis thaliana that share a structurally conserved J-like domain (JLD), but lack the HPD motif. Analysis of publicly available gene expression data as well as real-time quantitative PCR performed for a few selected JLPs implicated some of these proteins in growth, development and stress response. Here, we summarize the current state of knowledge on plant JLPs and their involvement in vital plant cellular/metabolic processes, including chloroplast division, mitochondrial protein import and flowering. Finally, we propose possible modes of action for these highly elusive proteins and other DnaJ-related proteins (DNAJRs) in regulating the Hsp70 chaperone network.

A Quantitative Arabidopsis IRE1a Ribonuclease-Dependent in vitro mRNA Cleavage Assay for Functional Studies of Substrate Splicing and Decay Activities.

The unfolded protein response (UPR) is an adaptive eukaryotic reaction that controls the protein folding capacities of the endoplasmic reticulum (ER). The most ancient and well-conserved component of the UPR is Inositol-Requiring Enzyme 1 (IRE1). Arabidopsis IRE1a (AtIRE1) is a transmembrane sensor of ER stress equipped with dual protein kinase and ribonuclease (RNase) activities, encoded by its C-terminal domain. In response to both physiological stresses and pathological perturbations, AtIRE1a directly cleaves bZIP60 (basic leucine zipper 60) mRNA. Here, we developed a quantitative in vitro cleavage assay that combines recombinant AtIRE1a protein that is expressed in Nicotiana benthamiana and total RNA isolated from Arabidopsis leaves. Wild-type AtIRE1a as well as its variants containing point mutations in the kinase or RNase domains that modify its cleavage activity were employed to demonstrate their contributions to cleavage activity levels. We show that, when exposed to total RNA in vitro, the AtIRE1a protein cleaves bZIP60 mRNA. Depletion of the bZIP60 transcript in the reaction mixture can be precisely quantified by a qRT-PCR-mediated assay. This method facilitates the functional studies of novel plant IRE1 variants by allowing to quickly and precisely assess the effects of protein mutations on the substrate mRNA cleavage activity before advancing to more laborious, stable transgenic approaches in planta. Moreover, this method is readily adaptable to other plant IRE1 paralogs and orthologs, and can also be employed to test additional novel mRNA substrates of plant IRE1, such as transcripts undergoing degradation through the process of regulated IRE1-dependent decay (RIDD). Finally, this method can also be modified and expanded to functional testing of IRE1 interactors and inhibitors, as well as for studies on the molecular evolution of IRE1 and its substrates, providing additional insights into the mechanistic underpinnings of IRE1-mediated ER stress homeostasis in plant tissues.

Multilevel regulation of endoplasmic reticulum stress responses in plants: where old roads and new paths meet.

The sessile lifestyle of plants requires them to cope with a multitude of stresses in situ. In response to diverse environmental and intracellular cues, plant cells respond by massive reprogramming of transcription and translation of stress response regulators, many of which rely on endoplasmic reticulum (ER) processing. This increased protein synthesis could exceed the capacity of precise protein quality control, leading to the accumulation of unfolded and/or misfolded proteins that triggers the unfolded protein response (UPR). Such cellular stress responses are multilayered and executed in different cellular compartments. Here, we will discuss the three main branches of UPR signaling in diverse eukaryotic systems, and describe various levels of ER stress response regulation that encompass transcriptional gene regulation by master transcription factors, post-transcriptional activities including cytoplasmic splicing, translational control, and multiple post-translational events such as peptide modifications and cleavage. In addition, we will discuss the roles of plant ER stress sensors in abiotic and biotic stress responses and speculate on the future prospects of engineering these signaling events for heightened stress tolerance.

Evolutionary Conservation and Emerging Functional Diversity of the Cytosolic Hsp70:J Protein Chaperone Network of Arabidopsis thaliana.

Heat shock proteins of 70 kDa (Hsp70s) partner with structurally diverse Hsp40s (J proteins), generating distinct chaperone networks in various cellular compartments that perform myriad housekeeping and stress-associated functions in all organisms. Plants, being sessile, need to constantly maintain their cellular proteostasis in response to external environmental cues. In these situations, the Hsp70:J protein machines may play an important role in fine-tuning cellular protein quality control. Although ubiquitous, the functional specificity and complexity of the plant Hsp70:J protein network has not been studied. Here, we analyzed the J protein network in the cytosol of Arabidopsis thaliana and, using yeast genetics, show that the functional specificities of most plant J proteins in fundamental chaperone functions are conserved across long evolutionary timescales. Detailed phylogenetic and functional analysis revealed that increased number, regulatory differences, and neofunctionalization in J proteins together contribute to the emerging functional diversity and complexity in the Hsp70:J protein network in higher plants. Based on the data presented, we propose that higher plants have orchestrated their "chaperome," especially their J protein complement, according to their specialized cellular and physiological stipulations.