Cytosolic chaperones influence the fate of a toxin dislocated from the endoplasmic reticulum

Abstract

The plant cytotoxin ricin enters target mammalian cells by receptor-mediated endocytosis and undergoes retrograde transport to the endoplasmic reticulum (ER). Here, its catalytic A chain (RTA) is reductively separated from the cell-binding B chain, and free RTA enters the cytosol where it inactivates ribosomes. Cytosolic entry requires unfolding of RTA and dislocation across the ER membrane such that it arrives in the cytosol in a vulnerable, nonnative conformation. Clearly, for such a dislocated toxin to become active, it must avoid degradation and fold to a catalytic conformation. Here, we show that, in vitro, Hsc70 prevents aggregation of heat-treated RTA, and that RTA catalytic activity is recovered after chaperone treatment. A combination of pharmacological inhibition and cochaperone expression reveals that, in vivo, cytosolic RTA is scrutinized sequentially by the Hsc70 and Hsp90 cytosolic chaperone machineries, and that its eventual fate is determined by the balance of activities of cochaperones that regulate Hsc70 and Hsp90 functions. Cytotoxic activity follows Hsc70-mediated escape of RTA from an otherwise destructive pathway facilitated by Hsp90. We demonstrate a role for cytosolic chaperones, proteins typically associated with folding nascent proteins, assembling multimolecular protein complexes and degrading cytosolic and stalled, cotranslocational clients, in a toxin triage, in which both toxin folding and degradation are initiated from chaperone-bound states.

Fig. 1.

Deoxyspergualin (DSG) protects HeLa cells from ricin intoxication. (A) Cells were treated for 4 h with graded doses of ricin or diphtheria toxin (DTx) in growth medium containing 50 μg ml⁻¹ DSG/100 μg ml⁻¹ lactose (filled circles) or 100 μg ml⁻¹ lactose carrier only (open circles), and their subsequent ability to synthesize proteins was determined by measuring incorporation of [³⁵S]-Met into acid-precipitable material. Typical single assays are shown. (B) Control cells and cells pretreated (pre) with DSG/lactose were treated as in A, sensitivities to toxin (IC₅₀, toxin concentration required to reduce protein synthesis to 50% that of nontoxin treated controls) were determined, and fold protection (IC₅₀ DSG-treated cells: IC₅₀ control cells) is displayed. Means of three (DTx, ricin 16-h DSG pretreatment) or six (ricin, no DSG pretreatment) independent experiments are displayed. (Bars, ±1 S.D.; broken line, no protective effect over that of treatment with lactose carrier only). (C) Cells were treated with lactose (lac) or lactose/DSG only as appropriate for 4 h, and remaining protein synthesis ability was determined. (n = 3; bars, ±1 S.D.).

Fig. 2.

Geldanamycin (GA) and radicicol (RA) sensitize HeLa cells to ricin challenge. (A) Cells were treated (4 h) with increasing doses of ricin in medium containing GA/DMSO (black circles), RA/DMSO (gray circles), or DMSO vehicle only (white circles), and their subsequent ability to synthesize proteins was determined as in Fig. 1A. Typical single assays are shown. (B) Cells were challenged as in (A), IC₅₀ values were determined as in Fig. 1B, and the effects of GA and RA are displayed as fold sensitizations (IC₅₀ GA- or RA-treated cells: IC₅₀ control DMSO-treated cells). Also shown are the effects of treatment with NECA, and combined treatments with RA, DSG, and DMSO. Means of three independent experiments are displayed, except for NECA treatment, where n = 5. (Bars, ±1 S.D.; broken line, no effect over that of treatment with DMSO vehicle only) (C) Cells were treated with vehicle DMSO, DMSO/GA, or DMSO/RA as appropriate for 4 h, and remaining protein synthesis ability was determined. (n = 3; bars, ±1 S.D.) (D) Immunoblots of detergent soluble cell extracts (25 μg protein per lane) from cells incubated for various lengths of time (t) in growth medium containing 1 μM GA or 1 μM RA were probed for Hsp72 (upper strip), Hsp90 (middle strip), or γ-tubulin (lower strip).

Fig. 3.

In vitro interactions of RTA and Hsp40 and Hsc70 chaperones. (A) RTA (500 ng) was incubated at the indicated temperatures for 15 min in 20 μl of 20 mM Mops pH 7.2, 100 mM KCl, in the presence or absence of Hsp40, Hsc70, or ATP (shown below the panels). Aggregated (P) and soluble (S) fractions were separated by centrifugation, heated in reducing SDS sample buffer, and analyzed by SDS/PAGE and subsequent silver staining. *, proteolytic fragment of Hsc70 lacking the C-terminal regulatory domain. Proportions (%) of aggregated (gray) and soluble (white) RTA are shown. (Bars, ±1 S.D.) (B) Reaction mixtures where RTA was replaced by saporin were treated as in (A). (C) Dilutions of the soluble fractions from 375 ng of heat-treated RTA (upper panel), 375 ng of chaperone-stabilized RTA (lower panel), and 375 ng of native RTA were added to yeast ribosomes for 2 h at 30°C. After cleavage of any depurinated 28S rRNA with acetic-aniline, rRNAs were extracted, electrophoresed in denaturing conditions (1.2% agarose, 50% formamide), and gels were stained with ethidium bromide before quantifying (22).

Fig. 4.

Hsc70 cochaperone activity determines the fate of dislocated RTA in vivo. (A) Cells transiently transfected with an expression plasmid expressing Hop (Hop/Hop), with an equimolar mixture of plasmids expressing LacZ and Hop (Hop/LacZ), or with a plasmid expressing LacZ (LacZ/LacZ) were assayed for ricin sensitivity. Values were corrected for transfection efficiency, determined as 35% by epifluorescent examination of cells transiently transfected with a plasmid expressing GFP. Dotted line, no effect over that of LacZ transfection. (B) Cells were transfected, as in (A), with expression plasmids expressing LacZ, BAG-1S [the small (S) isomer of BAG-1], CHIP, Hip, or BAG-2 and were assayed for ricin sensitivity compared with cells transfected coevally with vector alone. Dotted line, no protective effect over vector transfection. (C–G) Interactions of cochaperones with Hsc70 and client protein (red line).

Fig. 5.

RTA is a substrate for CHIP. (A) RTA was added to a reaction mixture (complete), which can recapitulate CHIP activity (28), and to mixtures lacking E1 ubiquitin activating enzyme, E2 (UbcH5 ubiquitin conjugating enzyme), CHIP, or Hsp40/Hsc70 (40/70) as indicated. After heating (45°C, 10 min), reactions were activated by addition of 5 mM MgCl₂, 10 mM DTT, and 5 mM ATP, incubated (2h, 30°C), and products were identified by reducing SDS/PAGE and immunoblotting for Hsc70 and RTA. (B) Addition of E1 to a reaction mixture containing RTA, Hsp40, Hsc70, E2, CHIP, and ubiquitin as in A, and incubation at 37°C for 2 h results in CHIP-mediated ubiquitylation of Hsc70 (upper panel, Ub-Hsc70), CHIP (middle panel, Ub-CHIP), and both RTA 6K and RTA (lower panel, Ub-RTA) as revealed by immunoblots. (C) RTA (500 ng) was incubated (45°C or 37°C, 15 min) in the presence or absence of Hsp90 or ATP (shown below the panels). Aggregated (P) and soluble (S) fractions were separated by centrifugation, heated in reducing SDS sample buffer, and analyzed by SDS/PAGE and subsequent silver staining. Proportions (%) of aggregated (gray) and soluble (white) RTA are shown. (D) RTA was added to CHIP recapitulation mixtures as in A, but containing Hsp40 and Hsp70 (40/70), HOP, and Hsp90 as indicated below the panels, incubated (2 h, 37°C), and products were identified by reducing SDS/PAGE and immunoblotting for HOP and RTA, and by silver staining for Hsc70 and Hsp90. *, cross-reacting contaminant. (E) Proposed cytosolic triage of dislocated RTA.