Manganese induces oligomerization to promote down-regulation of the intracellular trafficking receptor used by Shiga toxin.
Bottom Line: Alanine substitutions blocking Mn binding abrogated both oligomerization of GPP130 and GPP130 sorting from the Golgi to lysosomes.Further, oligomerization was sufficient because forced aggregation, using a drug-controlled polymerization domain, redirected GPP130 to lysosomes in the absence of Mn.These experiments reveal metal-induced oligomerization as a Golgi sorting mechanism for a medically relevant receptor for Shiga toxin.
Affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213.Show MeSH
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Mentions: Based on the transferability of the Mn response, it seemed possible that the GPP130 stem domain might bind Mn. As a test, uncoated or Mn-coated nitrilotriacetic acid (NTA)-agarose beads were incubated with the purified glutathione S-transferase (GST)–tagged GPP130 residues 36–247, and recovery after washing was determined by SDS–PAGE and Coomassie staining. As control proteins we used GST itself and GST fused to the stem domain of GP73. The control proteins showed low recovery on the beads, whereas the GST-tagged GPP130 stem domain (36–175) appeared to interact strongly and specifically with the Mn-coated beads (Figure 2A). To estimate the Mn concentration required for half-maximal binding, we repeated the experiment by adding the GPP130 protein (36–247) to increasing concentrations of Mn and then recovering any formed complexes using uncoated NTA-agarose beads. Under these conditions, 0.4 mM Mn yielded half-maximal binding (Figure 2B). The concentration of Golgi Mn in Mn-treated cells is unknown. Because Mn is actively pumped into the Golgi, it seems reasonable that Mn exposure results in Golgi levels of Mn sufficient to bind the GPP130 stem domain. On the basis of these results, we next investigated Mn binding of the GPP130 stem domain using mutations based on two nonresponsive GPP130 constructs found in the trafficking assay. Therefore we purified GST-tagged 36-87 and 36-175-88AAAA91 and compared the binding of these proteins to uncoated and Mn-coated beads with that of the wild-type 36–175 construct. Each construct showed some recovery on Mn-coated beads, but the binding was >4.5-fold higher for the wild-type construct (Figure 2C). Thus maximal Mn binding in the assay required the same residues that are required for Mn responsiveness.
Affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213.