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: Next we used fluorescence recovery after photobleaching (FRAP) to assess whether we could visualize an Mn-induced change in GPP130 diffusion in the Golgi. Cells were first transfected with a GFP-tagged GPP130 construct comprising the cytoplasmic, transmembrane, and stem domains and then were either untreated or treated with Mn. Again, a short-duration Mn exposure was used to initiate GPP130 redistribution but leave a substantial Golgi pool for analysis. A small region of this Golgi fluorescence was bleached, and recovery was determined over the next 10 min. Remarkably, the rapid recovery of GPP130 fluorescence in the Golgi of untreated control cells was significantly delayed in Mn-treated cells (Figure 5A). Quantification of fluorescence recovery in multiple independent trials confirmed that Mn induced a significant delay in GPP130 diffusion (Figure 5B). Note that in all other aspects, the Golgi fluorescence patterns of treated and untreated cells appeared indistinguishable. To test whether the blocking alanine substitutions would also prevent this Mn-induced delay in recovery, we repeated the experiments with the wild-type and alanine-substituted GP73-GPP130 chimeric constructs containing the GPP130 segment 36–175 followed by GFP. As expected, the wild-type construct rapidly recovered from photobleaching in the absence of Mn and showed a strong delay in recovery in Mn-treated cells (Figure 5C). In striking contrast to wild type, the 88AAAA91 substitution completely abrogated the Mn-induced delay in diffusion, such that both untreated and treated cells exhibited identical recoveries (Figure 5D). The final extent of recovery for the alanine substitution also matched that of the wild-type construct in untreated cells. For an unknown reason, the extent of recovery of these chimeric constructs was lower than that observed for the GPP130 construct. Thus, in living cells, Mn induces a change in GPP130 diffusion in the Golgi that depends on the same residues implicated in Mn binding and Mn-induced oligomerization.
Affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213.