Figure 2.

The SAM domains of ProSAP1/Shank2 and of ProSAP2/Shank3 specifically bind zinc ions. (A) Full-length GFP-ProSAP1/Shank2, ProSAP2/Shank3 or their C-terminal SAM domains colocalize with Zn²⁺ stained with Zinquin (arrowheads) in HeLa cells. GFP-Shank1 or its C-terminal SAM domain do not colocalize with Zinquin. Mutations of the ProSAP2/Shank3 SAM domain within the zinc-binding site (H22A) or the site of oligomerization (M56E) (Baron et al, 2006) abolish Zinquin colocalization. (B) Gel filtration of purified maltose-binding protein (MBP)-Shank1 (upper panel) and MBP-ProSAP2/Shank3 (middle panel) after zinc preincubation (solid black line). The plots are overlayed with Zinquin fluorescence profile (red lines, open circles). Quantification of the Zinquin fluorescence signals per nmole of zinc-precipitated MBP-AH-Shank1 and MBP-AH-ProSAP2/Shank3 protein (lower panel). (C) Transfection of ProSAP2/Shank3-GFP shows that EGFP signals cocluster with the fluorescence zinc dye Zinquin in hippocampal neurons in culture. Similarly, Zinquin signals colocalize with endogenous ProSAP2/Shank3 staining. (D) Immunolabelling of excitatory synapses of cultured hippocampal neurons (14 DIV). The zinc signal (Newport Green) colocalizes with the PSD protein ProSAP1/Shank2 and is juxtaposed to the presynaptic marker Bassoon. (E) The postsynaptic enrichment of zinc can be visualized in living cells, using the FM dye 4–64 that labels recycling presynaptic vesicles and Zinquin.