Ricin A Chain Insertion into Endoplasmic Reticulum Membranes Is Triggered by a Temperature Increase to 37 °C

Abstract

After endocytic uptake by mammalian cells, the heterodimeric plant toxin ricin is transported to the endoplasmic reticulum (ER), where the ricin A chain (RTA) must cross the ER membrane to reach its ribosomal substrates. Here, using gel filtration chromatography, sedimentation, fluorescence, fluorescence resonance energy transfer, and circular dichroism, we show that both fluorescently labeled and unlabeled RTA bind both to ER microsomal membranes and to negatively charged liposomes. The binding of RTA to the membrane at 0 -30°C exposes certain RTA residues to the nonpolar lipid core of the bilayer with little change in the secondary structure of the protein. However, major structural rearrangements in RTA occur when the temperature is increased. At 37°C, membrane-bound toxin loses some of its helical content, and its C terminus moves closer to the membrane surface where it inserts into the bilayer. RTA is then stably bound to the membrane because it is nonextractable with carbonate. The sharp temperature dependence of the structural changes does not coincide with a lipid phase change because little change in fluorescence-detected membrane mobility occurred between 30 and 37°C. Instead, the structural rearrangements may precede or initiate toxin retrotranslocation through the ER membrane to the cytosol. The sharp temperature dependence of these changes in RTA further suggests that they occur optimally in mammalian targets of the plant toxin.Ricin is a potent A-B cytotoxin composed of an RNA-specific N-glycosidase (A chain or RTA) 4 disulfide bonded to a cell binding lectin (B chain or RTB). The interaction of holotoxin with galactosylated surface components of mammalian cells is mediated by RTB and is followed by endocytic uptake (reviewed in Ref. 1). There is evidence that a tiny fraction of toxin then reaches the endoplasmic reticulum (ER) lumen (2) where it can be reduced to liberate RTA (3) in preparation for retrotranslocation across the membrane. However, RTA is not thought to penetrate the ER membrane directly. Instead, it appears to exploit protein-conducting translocons (4) as a non-native species (5, 6) in a manner akin to misfolded ER proteins that are dispatched by proteasomal degradation via the ER-associated degradation (ERAD) pathway (7, 8). There is evidence to suggest that once released to the cytosol, non-native RTA can uncouple from the ERAD pathway by virtue of its low lysine content (9). This would reduce the chance for polyubiquitylation and subsequent proteasomal degradation and thereby provide opportunities for refolding in a way that is not normally possible for terminally misfolded ERAD substrates whose dislocation is inextricably linked to degradation. Experimental evidence that toxins exploit various components of the ERAD pathway to reach the cytosol has been provided for ricin (9, 10), cholera toxin (11-14), pertussis toxin (15, 16), Shiga toxin (17,18),. It is generally assumed that RTA must make specific interactions with ER components to accomplish the unfolding that is required ...

How Ricin and Shiga Toxin Reach the Cytosol of Target Cells: Retrotranslocation from the Endoplasmic Reticulum

“…Supporting this interpretation is the partitioning of RTA, but not holoricin, in the detergent phase after Triton-X114 extraction, and the spontaneous structural changes that occur in RTA in the presence of negatively charged phospholipids Mayerhofer et al 2009). (Mayerhofer et al 2009).…”

“…Supporting this interpretation is the partitioning of RTA, but not holoricin, in the detergent phase after Triton-X114 extraction, and the spontaneous structural changes that occur in RTA in the presence of negatively charged phospholipids Mayerhofer et al 2009). (Mayerhofer et al 2009). Insertion into the membrane is temperaturedependent: at low temperatures, RTA binds membranes, and as the temperature increases, structural changes associated with membrane entry become more apparent (Mayerhofer et al 2009).…”

“…(Mayerhofer et al 2009). Insertion into the membrane is temperaturedependent: at low temperatures, RTA binds membranes, and as the temperature increases, structural changes associated with membrane entry become more apparent (Mayerhofer et al 2009). Isolated purified RTA is temperature-sensitive, forming a molten globule at 45ºC (Argent et al 2000), but even at 37 ºC, it is relatively unstable, and prone to aggregation (Spooner et al 2008a).…”

Protein-disulfide Isomerase Displaces the Cholera Toxin A1 Subunit from the Holotoxin without Unfolding the A1 Subunit

“…This conformational shift would identify PTS1 as an ERAD substrate and thereby facilitate PTS1 passage into the cytosol. Ricin A chain is more stable than either CTA1 or PTS1 (52), but an interaction with anionic phospholipids of the ER membrane at 37°C induces ricin A chain to assume a disordered conformation that would be treated as an ERAD substrate (53,54). Furthermore, the interaction between PDI and ricin mirrors the interaction between PDI and CT: PDI is responsible for disassembly of the ricin holotoxin but does not unfold ricin A chain (55,56).…”

Lipid Rafts Alter the Stability and Activity of the Cholera Toxin A1 Subunit

“…Thus, although it is not required for ERAD-mediated toxin translocation (54), the A1 3 subdomain still plays a functional role in host-toxin interactions. C-terminal domains from the catalytic subunits of Shiga toxin and ricin, two other ER-translocating toxins, also interact with lipid bilayers and play important roles in intoxication (62)(63)(64)(65). For ricin A chain, the C terminus has been shown to mediate an interaction with the negatively charged phospholipids of the ER membrane which results in toxin unfolding (62,63).…”