Protein Import Motors in Chloroplasts: On the Role of Chaperones

Hsp93

Hsp93 (not the Ycf2-FtsHi-MDH complex) was the first “pulling AAA-ATPase motor” identified for chloroplast protein import. Hsp93 belongs to the family of class I (with two ATPase domains, as opposed to class II with only one ATPase domain) AAA+ proteins (ATPases associated with various cellular activities; White and Lauring, 2007; Sauer and Baker, 2011). Members of this family form double-ring hexameric complexes with a central pore. They use the energy of ATP hydrolysis to create pulses of pulling force (not a Brownian ratchet mechanism as suggested by Herrmann [2018]) to actively drive translocation of their substrate proteins through the central pore. Well-known family members include NSF (N-ethylmaleimide-Sensitive Factor) for SNARE complex disassembly and p97 (Cdc48) for pulling proteins out of the endoplasmic reticulum for degradation by the proteasome. Hsp93 was first reported as being present in the chloroplast protein import translocon in 1997 (Akita et al., 1997; Nielsen et al., 1997) and confirmed by at least three other laboratories (Kouranov et al., 1998; Chou et al., 2003; Rosano et al., 2011). Hsp93 also directly binds transit peptides of translocating preproteins at early stages of the import process (Rosano et al., 2011; Huang et al., 2016). In Arabidopsis (Arabidopsis thaliana), Hsp93 exists as two isoforms encoded by the genes HSP93-III and HSP93-V. Hsp93-V is the major functional form, with knockout mutants having pale green leaves and a reduced rate of protein import into chloroplasts. This import-deficient phenotype has been independently observed by at least four laboratories (Constan et al., 2004; Kovacheva et al., 2005, 2007; Su and Li, 2010; Flores-Pérez et al., 2016; Lee et al., 2018), while only one study reported that they observed no import defect (Sjögren et al., 2004). Knockout mutants of HSP93-III display no obvious growth or import-deficient phenotype, but knocking out both HSP93-III and HSP93-V results in embryo lethality (Kovacheva et al., 2007), indicating that the two Hsp93 isoforms play redundant but essential functions.

In addition to reports of Hsp93 functioning as an import motor, Hsp93 has been described to associate with members of the ClpP protease complex and to participate in protein homeostasis through its role in unfolding protein substrates for the ClpP protease complex (Nishimura and van Wijk, 2015). In the review article by Nakai (2018), the author presented three lines of evidence arguing that Hsp93 only functions in protein degradation and not in protein import. The author first cited the work of Moreno et al. (2018) and wrote that RNAi-mediated knockdown of HSP93 in tobacco (Nicotiana tabacum) “causes a significant enrichment, as opposed to a reduction, of a wide variety of imported proteins inside the chloroplasts.” However, although Moreno et al. (2018) showed enrichment of 123 chloroplast proteins, they also showed decreased levels of an additional 153 chloroplast proteins in the HSP93-knockdown plants. In fact, Moreno et al. (2018) specifically noted that the vast majority of down-regulated proteins were identified in the proteome of HSP93-knockdown plants, as opposed to proteomes of plants with reduced expression of other ClpP complex components, and the authors suggested that this result agreed with the reported function of Hsp93 in protein import.

Second, Nakai (2018) wrote that components of the ClpP complex were recruited to the inner membrane by Hsp93, which associates with the inner membrane via its binding to Tic110 (Sjögren et al., 2014; Flores-Pérez et al., 2016). It was further shown that an HSP93 mutant with reduced interaction with ClpP components failed to complement the phenotypes of pale green leaves and reduced rates of protein import, suggesting that failure to interact with the ClpP protease was the primary cause of hsp93 phenotypes (Flores-Pérez et al., 2016). However, it is not clear why reducing the amount of ClpP protease at the envelope would reduce the import rate of the minute amounts of radioactive preproteins used in the import experiments. Moreover, whether the Hsp93 mutation used in the experiment compromised the ATPase activity of Hsp93 needs to be tested. In any case, these findings do not exclude the possibility that Hsp93 has dual functions in protein import and protein degradation.

Third, it was demonstrated that Hsp93 can be directly cross-linked to transit peptides of different preproteins during early stages of active import (Huang et al., 2016). This finding was criticized by Nakai (2018) as lacking sufficient specificity because, apart from the preprotein-Hsp93 adducts, some un-cross-linked preproteins were also immunoprecipitated by the anti-Hsp93 antibody. Un-cross-linked preproteins were, by far, the dominant radioactive signal in the samples before immunoprecipitation. When the gels were exposed for a longer period to observe weaker cross-linked species, background bands of un-cross-linked preproteins were often observed. This type of result has been seen by many laboratories (Akita et al., 1997; Geissler et al., 2002; Yamamoto et al., 2002; Banerjee et al., 2015; Richardson et al., 2018). When this same cross-linking approach was used in the Kikuchi et al. (2018) study to demonstrate direct cross-linking of FtsHi1 to a preprotein, the region of the gel where the un-cross-linked preproteins would be located was not shown.

Hsp90C

The evidence for a specific role for Hsp90C is limited to one report (Inoue et al., 2013). Hsp90C was identified in complexes containing translocating preprotein intermediates in the later stages of protein import, and it was later shown to associate with known TIC complex components, including Tic110, cpHsp70, and Hsp93 (Inoue et al., 2013). Its potential role in import was demonstrated using the reversible Hsp90 ATPase inhibitor, radicicol. Radicicol treatment of isolated chloroplasts did not inhibit binding of model preproteins at the TOC complex, but it did inhibit the ATP-dependent translocation of the polypeptide across the inner membrane (Inoue et al., 2013). These data, in addition to the known interaction of cpHsp70 and Hsp90C in complexes containing additional cochaperones in Arabidopsis (Willmund and Schroda, 2005; Willmund et al., 2008), led to the proposal that Hsp90C participates with other stromal chaperones as part of the import motor. Hsp90C is essential for plastid biogenesis, and null mutants are embryo-lethal in Arabidopsis (Feng et al., 2014). We do realize that additional molecular genetic data are required to test the role of Hsp90C in import and will need to rely on the identification of specific, viable point mutants that inhibit protein import while maintaining other essential functions of the chaperone in plastid biogenesis.

cpHsp70

cpHsp70 is a member of the well-known family of 70-kD heat shock proteins (Hsp70) that serve as molecular chaperones. This family, initially postulated to mediate protein folding (Pelham, 1986)—and it certainly does—was recognized early on as having a major role in protein targeting (Chirico et al., 1988; Deshaies et al., 1988). It is now well established that the motor driving the posttranslational import of proteins into mitochondria and the endoplasmic reticulum are trans-side Hsp70 chaperones (mtHsp70 and BiP, respectively). Marshall and Keegstra (1992) were the first to detect Hsp70 homologs in chloroplasts, with multiple versions residing in the stroma, the receiving compartment for protein import across the envelope membranes. This led to the prediction that one or multiple stromal Hsp70s, referred to hereafter as cpHsp70, constitute the import motor coupling ATP hydrolysis to chloroplast protein import (Marshall and Keegstra, 1992). While a motor role was long attributed to Hsp93 (see above), two articles from our laboratories published in 2010 (Shi and Theg, 2010; Su and Li, 2010) and a third published in 2014 (Liu et al., 2014) provided compelling evidence that stromal cpHsp70s contribute significantly to the activity of the import motor. The most relevant experiments are described below and listed in Table 1.

The theoretical underpinnings behind the idea that a chloroplast cpHsp70 participates in powering plastid protein import was provided by the Bruce lab through experiments monitoring the interaction of chloroplast transit peptides with cpHsp70 chaperones. This group not only demonstrated that two well-characterized chloroplast transit peptides had binding sites for both DnaK and Hsp70 (Ivey et al., 2000), they also showed that those transit peptides lacking such binding sites did not target proteins to chloroplasts (Chotewutmontri and Bruce, 2015).

Perhaps the more compelling evidence in favor of cpHsp70 as an import motor comes from experiments from two of our laboratories, performed without knowledge of each other’s efforts, with different plant species and published essentially simultaneously. We showed first that relevant genes for cpHsp70 are essential (Shi and Theg, 2010; Su and Li, 2010). While this does not implicate cpHsp70 directly in chloroplast protein trafficking, it is a hallmark of organellar targeting mutants in other systems (Altmann and Westermann, 2005; Zhang and Ren, 2015). We then demonstrated that chloroplasts isolated from knockout mutants of either of the two Arabidopsis cpHsp70 isoforms, or from Physcomitrella cphsp70 temperature-sensitive mutant plants, were compromised in their ability to import proteins. This extended to a knockdown mutant of the cpHsp70 cochaperone CGE, encoding a chloroplast homolog of bacterial GrpE that exhibits Hsp70-specific nucleotide-exchange activity. Significantly, the ATPase activity of the moss cpHsp70-2 showed a strong preference for the moss CGE, demonstrating that the cochaperones targeted their cognate chaperones (Liu et al., 2014). We went on to show that cpHsp70 interacted with known components of the TOC and TIC complexes and that they immunoprecipitated preproteins in the act of being transported (Shi and Theg, 2010; Su and Li, 2010). Such experiments are the gold standard for identifying protein components involved in organellar protein import. In fact, the same approach contributes significantly to the conclusions of Kikuchi et al. (2018) as well as in the earlier and still controversial article calling into question the composition of the TIC complex (Kikuchi et al., 2013). In light of our experiments, we hold that the statement by Kikuchi et al. (2018) that “no direct physical interaction between the TIC complex and the stromal Hsp70 has so far been observed” is incorrect.

The experiments published by Liu et al. (2014) also provide persuasive evidence that cpHsp70 is a major part of the import motor. In that work, we made two point mutations in the moss cpHsp70-2 predicted to increase the K_m for ATP hydrolysis (based on knowledge acquired from bovine Hsp70). Our rationale was that decreasing cpHsp70-2 affinity for ATP should translate into an increased requirement for ATP for in vitro protein import, and this is exactly what happened. While the K_m for ATP hydrolysis in the mutant cpHsp70-2s was increased 2.6-fold over that of the wild type, the K_m for ATP utilization during the protein import reaction increased threefold to fourfold, depending on the preproteins examined. We concluded that while cpHsp70-2 is not necessarily the only ATPase contributing to protein import, it dominates the energetics, and thus must be part of the motor.

CONCLUDING REMARKS

We believe that it is not possible to explain our experimental results, taken collectively, without invoking a role for the three chaperones (Hsp93, cpHsp70, and Hsp90C) in preprotein translocation across the chloroplast envelope. The sentence quoted above from Kikuchi et al. (2018) ends with “…the idea that the stromal Hsp70 acts as the import motor remains an open question.” We submit that there are many interesting open questions concerning the individual roles that these chaperones play in the import motor, but whether they participate at all is not one of them. To this point, Huang et al. (2016) have attempted to study the different contributions of the chaperones and showed that Hsp93 directly binds to translocating preproteins only at early stages before and during transit peptide processing, while cpHsp70 is associated with preprotein complexes throughout the import process. It is likely that structural data for the complexes and biochemical experiments with reconstituted systems will be required to fully understand the underlying functional mechanisms. However, we can speculate now that initial pulling by Hsp93, accompanied by continuous translocation through Brownian ratchet motions provided by cpHsp70, will be required for efficient preprotein import.

The Ycf2-FtsHi-MDH may indeed perform some motor function in chloroplasts of Arabidopsis and tobacco, but this does not exclude Hsp93, cpHsp70, and Hsp90C from being part of an import motor; the extensive experimental evidence we list herein indicates just the opposite. It is unclear why the work on Ycf1/2 and the Hsp93/70/90 chaperone ATPase complexes must be mutually exclusive. We hold that the most productive way forward is to engage in efforts to generate a unifying model that reconciles all of the accumulated data. This will be facilitated by in-depth analysis of the specific relevance and function of each component as well as the methodology used to describe their role along the protein import process.