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: Because Mn alters GPP130 oligomerization status in the Golgi contemporaneous with Mn-induced movement of GPP130 to lysosomes, we next tested whether oligomerization is sufficient to cause sorting of GPP130 to lysosomes. Versions of GPP130 were constructed harboring an array of three copies of a modified version of a FKBP12 domain termed FM (after its F36M substitution) as previously described for the Golgi enzyme mannosidase 1 (Rizzo et al., 2013). The FM domain self-interacts in a manner that is inhibited by the drug AP21998 (AP), an analogue of rapamycin. In the absence of AP, self-interactions are expected to form large complexes due to multiplexing of the three FM domains. In the previous work only 15 min of AP washout was needed to shift the FM-tagged mannosidase 1 from recovery in supernatant to pellet fractions (indicating polymerization), and this correlated with the construct's movement from cis- to trans-Golgi (Rizzo et al., 2013). In the case of GPP130-1-247-FM, its expression in the presence of AP yielded stable Golgi localization, and within 30 min of drug washout, it was evident in endosome-like peripheral punctae (Figure 6A). The redistribution continued until GPP130-1-247-FM had disappeared altogether, indicating degradation in lysosomes. Quantification of the redistribution to peripheral structures (Figure 6B) and of the loss of fluorescence (Figure 6C) confirmed a rapid and complete response. By comparison, giantin in the same cells remained Golgi localized throughout. Thus induced oligomerization of GPP130 is sufficient to alter its sorting such that it traffics out of the Golgi and is degraded.
Affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213.