Protein functions depend on their tertiary structures, which are dictated by their amino acid sequences, as demonstrated by Christian Anfinsen more than half a century ago (Anfinsen, 1973). Although protein folding is a spontaneous process in principle, folding frequently competes with the side process of aggregate formation, which is repressed by a variety of molecular chaperones (Dobson, 2003; Richter et al., 2010; Balchin et al., 2016). Indeed, a proteome-wide aggregation analysis of thousands of Escherichia coli proteins, using a reconstituted cell-free translation system, found that around 30% of proteins tend to aggregate without chaperones (Niwa et al., 2009), and the majority are saved by conserved chaperones such as the chaperonin GroEL/GroES (GroE) or DnaK/DnaJ/GrpE (DnaK system) (Niwa et al., 2012).

Chaperonins, a subclass of conserved chaperones, are responsible for promoting protein folding in cells (Thirumalai and Lorimer, 2001; Taguchi, 2005, 2015; Hayer-Hartl et al., 2016; Weiss et al., 2016; Thirumalai et al., 2020; Horovitz et al., 2022). The best-characterized chaperonin is E. coli GroE. GroE is a heat shock protein and the only indispensable chaperone for bacterial viability (Fayet et al., 1989), except for some species of Mollicutes such as Ureaplasma and Mycoplasma (Glass et al., 2000; Clark and Tillier, 2010; Schwarz et al., 2018). GroE helps fold numerous client proteins within cells using ATP. ATP binding to the GroEL rings induces drastic conformational changes leading to the formation of GroEL-GroES complexes, which have central cavities for client protein encapsulation (Xu et al., 1997; Kanno et al., 2009; Chen et al., 2013). The encapsulation of client proteins in the chaperonin cavity is essential to the growth of E. coli (Koike-Takeshita et al., 2006). An in vitro analysis revealed that the GroE cavity could accommodate proteins up to ∼60 kDa (Sakikawa et al., 1999). Although this review does not cover the detailed molecular mechanism of GroE, the basic role of GroEL is to bind to non-native monomeric proteins that arise after translation or during heat stress to prevent aggregation, and then promote folding in an ATP- and GroES-dependent manner.

In this review, we summarize the progress on the in vivo client proteins of the chaperonin GroEL and GroES (in vivo GroE clients) and discuss the roles of chaperonins in the cell. Other details on chaperonins have been summarized in recent excellent reviews (Weiss et al., 2016; Balchin et al., 2020; Horovitz et al., 2022).

One of the reasons why chaperones are necessary for the cell is that proteins often aggregate. Indeed, researchers handling proteins know empirically that some proteins tend to form aggregates. What fraction of proteins is aggregation-prone at the proteome level? In this regard, a comprehensive study was conducted in which more than 70% of E. coli proteins, 3,173 proteins, were translated under chaperone-free conditions to determine whether they formed aggregates or were soluble (Niwa et al., 2009). The large-scale analysis using a reconstituted cell-free E. coli translation system (PURE system) revealed that the distribution of solubilities was bimodal, indicating that the E. coli proteome is divided into two groups: Soluble and aggregation-prone (Figure 1A). The analysis also showed that one-third of the proteins were aggregated when translated in the absence of chaperones, supporting the empirical view that proteins are aggregation-prone. A bioinformatics analysis demonstrated that protein solubility correlates better with cellular abundance, rather than gene-expression levels (Castillo et al., 2011). Subsequent analyses with the DnaK system or GroE showed that the addition of the chaperones during the translation of those aggregation-prone proteins alleviated aggregation overall, indicating the necessity of chaperones at the proteome level (Niwa et al., 2012).

Aggregation formation is associated with impaired folding. Recent global refolding experiments using an E. coli lysate, combined with mass spectrometry-based proteomics, revealed that one-third of the E. coli proteome is not intrinsically refoldable (To et al., 2021).

As these large-scale studies suggest, a certain fraction of proteins does not fold easily and often aggregates, supporting the notion that chaperones are essential to maintain the protein homeostasis (proteostasis) in the cell.

In E. coli, three major chaperone systems, trigger factor (TF), the DnaK system, and the chaperonin GroE, are considered to contribute to the folding of newly synthesized polypeptides. These three chaperone systems work together in a cooperative manner, with TF and DnaK both playing similar roles during co-translational processes in vivo (Deuerling et al., 1999; Teter et al., 1999; Ferbitz et al., 2004), whereas it is thought that GroEL plays a role in folding polypeptides after they have left the ribosome, although there have also been reports of GroEL potentially being involved in the co-translational process (Genevaux et al., 2004; Vorderwülbecke et al., 2004; Ying et al., 2005, 2006). An important goal in understanding the role of GroE in the cell is to identify in vivo obligate GroE clients that absolutely require GroE for folding in cells. The determination of the obligate GroE clients should clarify GroE’s unique role among chaperones, provide insight into the structural characteristics of the obligate clients, and illuminate the role of GroE in protein evolution.

After the in vivo obligate GroE clients have been identified, the next challenge is to distinguish the determinants that define such GroE dependence. Although these determinants are not yet fully understood, some attempts are introduced here.

The identification of the obligate GroE clients revealed that more than 70% (42 out of 57) are likely to be involved in metabolic reactions. What is the relationship between GroE and metabolic enzymes? A bioinformatics analysis of the metabolic pathways revealed that, as the GroE dependency increases, the clients are more laterally distributed in the metabolic network (Takemoto et al., 2011). In addition, a comparative genome analysis showed that the degree of conservation of GroE clients decreases with the GroE dependence, and the Class I GroE clients are most conserved as compared to other GroE client classes (Takemoto et al., 2011).

These findings could be discussed in the context of protein evolution. Most protein mutations have a negative effect since the protein stability is, in general, marginal (Tokuriki et al., 2008). It has been proposed that chaperones play a role in facilitating protein evolution by buffering the destabilizing mutations that cause misfolding (Rutherford and Lindquist, 1998). This concept, which was originally developed from studies on Hsp90 (Rutherford and Lindquist, 1998), has been extended to GroE (Fares et al., 2002; Tokuriki and Tawfik, 2009; Bogumil and Dagan, 2012). Directed evolution experiments revealed that GroE overexpression could buffer the destabilizing mutations, thereby promoting the folding of compromised proteins to improve their enzyme activities (Tokuriki and Tawfik, 2009; Wyganowski et al., 2013). Indeed, the evolved enzymes in GroE-overexpressing E. coli gained aggregation-prone properties and were converted to GroE-dependent proteins (Tokuriki and Tawfik, 2009), consistent with the MetK case described above (Ishimoto et al., 2014). Therefore, the role of GroE in buffering the aggregation-prone mutations helps the destabilized proteins to function in the cellular environment. The buffering effect would allow the destabilized proteins to exhibit novel cellular functions in a chaperone-dependent manner, eventually contributing to the molecular evolution of the protein (Takemoto et al., 2011).

The possible evolutional role of GroE prompted us to investigate whether GroE clients are conserved among species. If the obligate GroE clients (Class IV) have evolutionarily acquired some function or other traits more recently by mutations of existing proteins than other proteome members, then we would expect that the GroE clients are not conserved. So far, an extensive survey to identify in vivo obligate GroE-dependent clients has only been conducted in E. coli. For verification, we await a study to identify the in vivo clients in other bacteria, at the level of that in E. coli.

Even though there have been great breakthroughs in highly accurate protein structure prediction, as represented by AlphaFold2 (Jumper et al., 2021) and rational de novo protein design (Huang et al., 2016), we still do not fully understand the protein folding process. One of the obstacles is protein aggregation. Protein aggregation is a notorious problem when handling proteins and has often been ignored as an unwanted side reaction in protein folding. The chaperone studies unveiled the previously unrecognized role of aggregation-prone proteins in the protein world. Since Anfinsen demonstrated the basic principle of protein folding (Anfinsen, 1973), extensive efforts to elucidate the protein folding mechanism have been performed for more than half a century (Dill and MacCallum, 2012). However, the “folding” mechanisms of “recalcitrant” proteins, which are not spontaneously folded under chaperone-free conditions, remain enigmatic. Most physicochemical methods such as stopped-flow kinetic experiments and calorimetric measurements are not amenable to aggregated proteins. Approaches to tackle the “folding” mechanisms of recalcitrant proteins are necessary to understand the wide spectrum of protein folding in the cell.