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: Of interest, the purified GPP130 stem domain constructs 36–247 and 36–175 appeared to oligomerize in response to Mn addition. In the absence of added Mn, GST-GPP13036-247 was recovered roughly in the middle of velocity gradients, whereas addition of 1 mM MnCl2 shifted ∼60% of the protein toward the bottom of the gradients (Figure 3A). Mn-induced aggregation of the protein was also apparent simply by measuring absorbance at 350 nm, indicating light scattering (Cromwell et al., 2006). Whereas the two purified control proteins, GST and GST-tagged GP73 stem, failed to show an increase, the GST-GPP13036-247 protein yielded a robust increased absorbance clearly evident at 1 mM MnCl2 (Figure 3B). This effect was reversible because it was abrogated by a subsequent incubation with equimolar EDTA to chelate the Mn (Figure 3C). Of importance, the alanine substitutions that blocked GPP130 responsiveness in cells and reduced binding of Mn in the GST-GPP130 stem construct (36-17588AAAA91) also reduced the Mn-induced light scattering activity of this construct (Figure 3D). These results suggest that Mn binding might promote altered GPP130 trafficking by binding and inducing GPP130 oligomerization.
Affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213.