INVESTIGATION Evolutionary Conservation and Emerging Functional Diversity of the Cytosolic Hsp70:J Protein Chaperone Network of Arabidopsis thaliana Amit K. Verma,* Danish Diwan,* Sandeep Raut,* Neha Dobriyal,* Rebecca E. Brown, † Vinita Gowda,* Justin K. Hines, † and Chandan Sahi* ,1 *Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, Madhya Pradesh 462066, India and † Department of Chemistry, Lafayette College, Easton, Pennsylvania 18042-1768 ORCID IDs: 0000-0003-4928-8458 (A.K.V.); 0000-0002-4821-9736 (C.S.) ABSTRACT 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 housekeep- ing 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. KEYWORDS Arabidopsis thaliana Hsp40 Hsp70 J protein yeast evolution Being sessile, plants have to deal with complex environmental cues including a variety of stresses. They have evolved with specific mech- anisms that help them regulate their cellular proteome with the changing external environment (Kosova et al. 2011; Kurepa et al. 2009). Molec- ular chaperones are a diverse group of proteins that play critical roles in maintaining cellular proteostasis in all organisms, including plants, under normal as well as stress conditions (Boston et al. 1996; Bukau et al. 2006; Hartl et al. 2011; Miernyk 1999; Wang et al. 2004). The Hsp70 class of molecular chaperones is a large and evolutionary con- served family of proteins known to perform myriad cellular functions (Bukau and Horwich 1998; Kampinga and Craig 2010). Hsp70s never work alone. They always partner with multiple, structurally diverse J proteins (Hsp40s) to constitute the Hsp70:J protein chaperone network (Kampinga and Craig 2010). J proteins interact with Hsp70s through their conserved and signature J domain and stimulate their otherwise weak intrinsic ATPase activity. ATP hydrolysis results in profound conformational changes in the client binding domain (CBD), thereby modulating substrate executions and thus driving Hsp70’s functions (Kampinga and Craig 2010). In this way, Hsp70s, along with their obligate cochaperones, the J proteins, form a formidable chaperone network that performs various protein folding, remodelling, and qual- ity control functions. The J domain, the defining feature of all J proteins, is a compact tetrahelical domain of 70 aa residues with a highly conserved and functionally critical histidine, proline, and aspartic acid (HPD) tripep- tide motif. Although J domains are critical for J protein function, often the regions outside the J domain determine the interaction of J proteins with their clients or affect their subcellular localization and thereby dictate the functional specificity of a J protein (Sahi and Craig 2007). Historically, J proteins have been classified into three classes based on Copyright © 2017 Verma et al. doi: https://doi.org/10.1534/g3.117.042291 Manuscript received January 22, 2017; accepted for publication April 17, 2017; published Early Online April 21, 2017. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Supplemental material is available online at www.g3journal.org/lookup/suppl/ doi:10.1534/g3.117.042291/-/DC1. 1 Corresponding author: Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal Bypass Rd., Bhauri, Bhopal, Madhya Pradesh 462066, India. E-mail: sahi@iiserb.ac.in Volume 7 | June 2017 | 1941