It has been hypothesised that RTA exploits Hsc70 at some point proximal to its retrotranslocation of the ER membrane. RTA putatively uses this interaction to increase the yield of successfully reactivated toxin in the cytosol. A subsequent recurrent, transient interaction of RTA with Hsc70 after having acquired a cytosolic localisation might also significantly extend its toxic half-life therein, enabling the toxin subunit to deactivate more ribosomes than it would be able to otherwise. Often, if a client has a persistent association with Hsc70 it would result in its degradation via polyubiquitination (McDonough & Patterson, 2003; Jiang et al., 2001), but as RTA has a dearth of lysines this side-effect of the interaction might be diminished (Deeks et al., 2002).
As an alternative suggestion for the observations of the previous chapter, Hsc70 could contribute to the mechanism by which the retrotranslocation step itself occurs. This would not be without analogous precedents in the cell, as Hsc70-type chaperones are required in the post-translational import of polypeptides across a variety of organelle membranes (Höhfeld & Hartl, 1994; Plath & Rapoport, 2000; Young et al., 2003). In such post-translational import pathways, cytosolic Hsc70 binds to clients on the cis-side of the membrane being crossed, typically before the cargo polypeptide has completely folded. This interaction ensures that the polypeptide is kept in a conformationally-malleable, transport-competent state (Corsi & Schekman, 1996). Arguably more interesting to this study, however, Hsc70-type chaperones are also thought to act as motors operating upon the trans-side of the membrane being crossed (Tomkiewicz et al., 2007). In this position they are the hypothesised mediators of “Brownian ratchet” or “power-stroke” mechanisms which are linked to the ATPase cycle of the chaperone, and which effectively help to pull the translocating protein through the pore in the target membrane (Tomkiewicz et al., 2007; De Los Rios et al., 2006). In mammals, a mitochondrial Hsp70 and the ER-localised BiP have both been proposed to operate in this way (Jehnsen & Johnson, 1999; Tomkiewicz et al., 2007). It is therefore a consideration that the cytosolic counterpart of BiP and mitochondrial Hsp70, i.e. cytosolic Hsc70, could exhibit such a function in the dislocation of RTA into the cytosol.
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If cytosolic Hsc70 does help drive extraction of RTA from the ER membrane, it would be an unusual mechanism by which an otherwise ERAD-like pathway is resolved. For many bona fide ERAD substrates, including ApoB and CFTR∆F508, the AAA protein-containing 19S proteasomal cap and the p97-containing complex are thought to drive extraction (reviewed in Meusser et al., 2005; for ApoB see Fisher et al., 2008; and see Carlson et al., 2006, for CFTR∆F508). Unlike RTA, however, the typical substrates of these putative translocation motors become polyubiquitinated during their ERAD, a modification which is thought to provide a physical anchor which may be bound and pulled upon (Meusser et al., 2005). The results of Deeks et al. (2002), Li et al., (2010) and the previous chapter, however, clearly illustrate that canonical lysine-ubiquitination is not necessary for the retrotranslocation process that RTA undertakes. Previously, Marshall et al. (2008) showed that the p97- homologue in Nicotiana tabacum (known as Cdc48), can facilitate extraction of RTA0K from the ER of tobacco protoplasts. However, Li et al. (2010) showed that this was not the case in yeast. Speculatively, if RTAWT is extracted by p97 in mammals, its retarded polyubiquitination might diminish the avidity with which it is bound. This attenuated interaction might permit chaperones like Hsc70 to displace otherwise typical mediators of ERAD.
If any of the mechanisms hypothesised above were true, then a demonstrable interaction between RTA and Hsc70 should exist. This chapter, therefore, ultimately aims to investigate whether Hsc70 interacts with RTA in a direct and functional way.
4.1 Experimental approach
As described in the previous chapter, a co-immunoprecipitation approach to this investigation was precluded because of the low cytosolic concentrations of RTA that are found during intoxication. Instead, this chapter describes the development of a method to test for an interaction of Hsc70 with RTA in vitro. Ideally, this assay would be facile, reliable and able to show a functional association between Hsc70 and RTA amid a myriad of other proteins and chemicals.
In seminal experiments expounding the function of Hsc70, Minami et al. (1996) compared the degree to which luciferase irreversibly aggregated in conditions with or without chaperones. They demonstrated that Hsc70 and its loading co-factor, Hsp40, could prevent the aggregation of thermally-denatured luciferase during a 42°C heat-treatment in vitro, a feat that was maximally effective in the presence of ATP. They observed this by two methods:
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(1) In real-time, by measuring the dynamic light scattering from particles of aggregated luciferase.
(2) By separating an incubation of luciferase and chaperones by centrifugation and analysing the relative distribution of protein between pellet and soluble fractions.
If such assays could be recapitulated with RTA, it would provide evidence for a direct interaction between the two proteins – with RTA as client and Hsc70 functioning as a bona fide chaperone. These observations could then help to reconcile the in vivo effects of chaperone inhibition. With this intention, this chapter describes a series of experiments investigating whether the general parameters known to govern protein stability lead to measurable changes in the assays mentioned above, and whether these changes are consistent with what would be expected of a good assay for measuring protein folding.
The parameters tested to this end included: pH, temperature, electrolyte concentration, macromolecular crowding and the effect of a small molecule chaperone. With this foundation in place, assays which examined the influence of the Hsc70/Hsp40 chaperone pathway were later introduced.
Mimicking the retrotranslocating or post-retrotranslocation state - As it is hypothesised that
RTA interacts with Hsc70/Hsp40 in at least a partially-unfolded state after it has crossed the ER membrane, thermal denaturation was enlisted as a tool to denature RTA (as per Argent et al., 2000). This would render RTA in a state more representative of a post-retrotranslocation conformation (i.e. in which significant hydrophobic stretches may be solvent-exposed). It is worth mentioning, however, that this is not the physiological mechanism by which RTA is unfolded during intoxication. Rather, it is thought that factors in the ER environment promote the toxin subunit‟s change in conformation, e.g. negatively-charged phospholipids of the ER membrane. Liposomes comprising such phospholipid induce measurable changes to RTA‟s secondary structure when they are co-incubated in vitro (Day et al., 2002). This same effect is thought to promote co-option of the toxin subunit onto an ERAD-like pathway in vivo (Day et al., 2002; Mayerhofer et al., 2009).
Thermal versus lipid-based denaturation - Importantly for the interpretation of data
presented in this chapter and the next, the ways in which thermal denaturation and interaction with negatively-charged phospholipids influence the structure of RTA are certain to be qualitatively different. Thermal denaturation forces the polypeptide chain into a higher- energy state. This breaks non-covalent, intra-chain bonds by increasing the kinetic energy of
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the polypeptide backbone and its side-chains, expanding the diversity of conformations the protein population will inhabit. On the other hand, phospholipid-based disruption of the secondary structure would presumably replace non-covalent intra-peptide bonds with non- covalent phospholipid interactions instead. This promotes the partial insertion of RTA into liposomes in vitro (Mayerhofer et al., 2009). This insertion of RTA into the ER membrane might constrain the polypeptide into a relatively narrower array of conformations, promoting a correspondingly finer set of folding outcomes. These differences may alter the nature of the interactions that so-treated RTA can thereafter make in the cell when the polypeptide emerges on the cytoplasmic side of the membrane (such as with Hsc70). This physiologically relevant point is given consideration in the development of the assay and in the discussion of the results.
Screens for factors contributing to both unfolding and folding of RTA - Lastly, the folding
state of RTA is deemed critical to the events which govern both co-option onto an ERAD- like pathway in the ER and subsequent success of the toxin subunit in the cytosol (Beaumelle et al., 2002; Argent et al., 1994; Mayerhofer et al., 2009). As such, it was a secondary hypothesis that RTA may have evolved to exploit qualities of the lumenal and cytosolic environments to maximise the success of its retrotranslocation and subsequent reactivation. Therefore, a final objective of this chapter was to use the developed assay to screen for factors which might contribute to instability of RTA in the ER lumen (promoting co-option onto an ERAD pathway), and which might support its relative stability in the cytosol (extending its catalytic half-life therein).