Ricin trafficking in cells

Abstract

The heterodimeric plant toxin ricin binds exposed galactosyls at the cell surface of target mammalian cells, and, following endocytosis, is transported in vesicular carriers to the endoplasmic reticulum (ER). Subsequently, the cell-binding B chain (RTB) and the catalytic A chain (RTA) are separated reductively, RTA embeds in the ER membrane and then retrotranslocates (or dislocates) across this membrane. The protein conducting channels used by RTA are usually regarded as part of the ER-associated protein degradation system (ERAD) that removes misfolded proteins from the ER for destruction by the cytosolic proteasomes. However, unlike ERAD substrates, cytosolic RTA avoids destruction and folds into a catalytic conformation that inactivates its target ribosomes. Protein synthesis ceases, and subsequently the cells die apoptotically. This raises questions about how this protein avoids the pathways that are normally sanctioned for ER-dislocating substrates. In this review we focus on the molecular events that occur with non-tagged ricin and its isolated subunits at the ER-cytosol interface. This focus reveals that intra-membrane interactions of RTA may control its fate, an area that warrants further investigation.

Figure 1

Crystal structure (left) and cartoon representation (right) of ricin holotoxin. The crystal structure (PDB code 2AAI [11]) was viewed in Chimera (UCSF) and is shown from the side with the receptor-binding surfaces of ricin toxin B chain (RTB, green) facing downwards. Ricin toxin A chain (RTA, red) and RTB are held together by hydrophobic interactions and a disulphide bond (orange).

Figure 2

Intracellular trafficking of ricin. Ricin binds N-glycosylated molecules with available β1→4 linked galactosyls at the plasma membrane and after internalization by endocytosis, traffics via early endosomes, the TGN and the Golgi stack to the endoplasmic reticulum (ER), where reductive separation of the toxic ricin toxin A (RTA) from the cell-binding ricin toxin B (RTB) occurs. Free RTA retrotranslocates (dislocates), refolds in the cytosol and inhibits protein synthesis by catalytic removal of a key adenine residue on the 28S ribosomal subunit, at the site of EF-2 complex interaction [15]. Following a ribotoxic stress response and activation of multiple signaling pathways [16], ricin-treated cells die apoptotically [17].

Figure 3

Comparison of S. cerevisiae p24 proteins. (A): Amino acid comparisons were made in ClustalW [60], and are shown as a tree prepared in TREEVIEW [61]; (B): The striking similarity of Erp2 and Erp4 proteins. Overlined, GOLD domain; underlined, coiled-coil domain; boxed, trans-membrane domain; amino-acid similarity (*:.), identity, conserved, and less-conserved respectively; white triangle, junction of gene fusions between the GOLD and coiled-coil domains.

Figure 4

Requirement of p24 proteins for toxicity of RTA in S. cerevisiae. (A): Upper panels: growth of wt (BY4741) and p24 deletion strains (upper panels) transformed with an expression plasmid (URA selection) for ER-targeted RTA on agar containing glucose (glc, repressing, left-hand panels, 3d growth) and galactose (gal, inducing, right-hand panels, 4d growth). Lower panels; growth of wt (RSY1848) and its deletion derivative (p24Δ8) lacking all eight p24 genes, otherwise as in (A,B) Growth of these strains in liquid medium. Circles: white, BY4741; increasing tone, Δerp1, Δerp2, Δerp3, Δerp4, and Δerp5, respectively. Squares: black, Δerp6; dark grey, Δemp24; mid-grey, Δerv25. Triangles: white, RSY1848; black, p24Δ8. Inset: growth of these strains transformed with vector on galactose. a, RSY1484, p24Δ8; b, all other strains. Symbols as in main panel. (C): Upper panels: growth of wt (RSY1484) and p24Δ8 yeast transformed with an expression vector for ERP2 (LEU selection) or its vector control (pRS405, LEU selection) on glucose (left) and galactose (right) agar. Lower panels: growth of the strains from the upper panels, all transformed with an expression vector for RTA. (D): Growth of all strains from C in liquid selective medium containing glucose or galactose.

Figure 5

Overexpression of ERP2 and ERP2-ERP4 gene fusions. (A): Structures of the ERp2p and ERp4p proteins and the predicted structures of the reciprocal hybrids following domain swap (Epp2G4CCp and Erp4G2CCp), and their relative growth (determined from (B)). SP, signal peptide (dashed boxes); GOLD, luminal GOLD domain; CC, coiled-coli domain; TM, trans-membrane domain; C, cytosolic domain, dotted box, ER membrane, white triangle, junction of the GOLD-CC domain swap; (B): Upper panels: growth of the Δerp2 strain transformed separately with expression vectors for ERP2, ERP4 and the reciprocal hybrids (all LEU selection) on glucose and galactose agar. Lower panels: the same strains subsequently transformed with an expression vector for ER-targeted RTA (URA selection). C. Growth of the strains in (B) in liquid selective media.

Figure 6

Proposed cycling of ricin toxins A (RTA) chain between the endoplasmic reticulum (ER) and Golgi. Erp1p, Erp5p, Emp24p and Erv25p (1, 5, 24, 25 respectively) prevent entry into COPII coated ER membrane buds, and reduce toxicity: Erp3p, Erp4p and Erp6p play little or no role (3, 4 and 6 respectively). In contrast, in the presence of other p24 proteins, dislocation of RTA is an Erp2p (2)-dependent process with key roles close to or within the membrane. In the absence of all p24 proteins, ER-targeted RTA is toxic to yeast, suggesting unfettered access to the budding vesicle. GOLD, CC: GOLD and coiled-coil domains, respectively.

Figure 7

Post-dislocation scrutiny by a network of chaperones determines the cytosolic fate of RTA [38,40,81]. Following extraction from the ER by the RPT4 subunit of the proteasome, interactions with RPT5 allow RTA to recover activity. The role of Hsc70/Hsp40 interactions may be continuous scrutiny of RTA, preventing aggregation, and inactivation and activation (folding) fates follow from release of RTA from this complex. Transfer to Hsp90 via the Hsc70-Hsp90 operating protein HOP leads to CHIP-mediated ubiquitylation (Ub, ubiquitin) and inactivation of RTA. The Hsc70-interacting protein HIP stabilizes the Hsc70:RTA complex, and subsequent release by the BAG family guanine nucleotide exchange factors BAG-1 and BAG-2 leads to inactivation or folding respectively.