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: Figure 1 shows the localization of a few key constructs before and after Mn (Figure 1A), as well as schematic depictions of the tested GPP130 segments together with their quantified presence in peripheral punctae (Figure 1B). Consistent with our previous work (Mukhopadhyay et al., 2010), residues 36–175 produced a strong response, shifting from fewer than 5 non-Golgi puncta/cell in the absence of Mn to >30 puncta/cell in the presence of Mn. As noted before (Mukhopadhyay et al., 2010), most of the puncta contained redistributed endogenous GPP130, whereas not all endogenous GPP130 puncta were positive for the transfected construct. C-terminal deletion generated constructs 36–107, 36–100, 36–95, and 36–87. All of these except 36–87 retained activity, indicating that residues 88–95 were critical. A similar set of N-terminal truncations indicated that the first 14 residues (36–49) were dispensable, with larger deletions causing loss of Mn-response activity. This left 50–95 as the minimal mapped region and the sequence stretch 88–95 as a tractable segment for point mutation. Several individual and combined alanine substitutions within 88–95 were without effect, but alanine substitution of the sequence 88DFLV91 to generate 36-95-88AAAA91 potently blocked activity. This nonresponsive construct was stable and well localized and did not prevent the endogenous GPP130 response in the same cells. Insertion of the quadruple alanine substitution into the original construct to generate 36-175-88AAAA91 also strongly blocked its response (Figure 1). In summary, our mapping studies narrowed a transferable Mn responsive sequence to ∼50 residues and identified a required sequence stretch that, when mutated, could be used as a critical negative control in subsequent experiments.
Affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213.