Figure 1. SUMOylation of GluR6 in brain and cultured hippocampal neurons.

a, The SUMO-conjugating enzyme Ubc9 and the SUMO ligase PIAS3 selectively bind to the C-terminal domain of GluR6a in the yeast two-hybrid assay. The C termini of none of the other ionotropic glutamate receptor subunits tested interact strongly with both enzymes, as determined by β-galactosidase reporter activation. b, A series of overlapping truncation mutants of the C terminus of GluR6a tested in the yeast two-hybrid assay showed that the domain for both SUMOylation enzymes contains the consensus SUMOylation motif (ΨKXE, where Ψ is the hydrophobic residue). c, Immunoblot (IB) analysis of synaptic SUMOylated proteins after rat brain fractionation. Multiple SUMOylated substrates are present in the synaptic fractions. Equal amount of protein (40 μg protein per lane) from each fraction was loaded. P1, crude nuclear fraction; P2, post-nuclear crude membrane fraction; S3, cytosol; P3, microsomes; My, myelin; Mt, mitochondria; Syn, synaptosomes; PSD, post-synaptic density fraction. Immunoblot probed with anti-PSD95 antibody is shown below (2 μg protein per lane). We note that the PSD95 blot is loaded with very low levels of protein to avoid saturation of the signal in the highly enriched synaptic and postsynaptic density fraction lanes. d, Immunoblots showing SUMOylated and non-SUMOylated forms of GluR6a in brain homogenate. Note that, in the absence of the cysteine protease inhibitor NEM, no detectable band corresponding to SUMOylated GluR6a was observed, which confirms that the SUMO adduct is highly labile and sensitive to de-conjugation. e, Immunoblots showing SUMOylated and non-SUMOylated forms of GluR6a in cultured hippocampal neurons. Immunoprecipitation (IP) experiments using anti-SUMO-1 antibody reveal that a fraction of GluR6a is SUMOylated in cultured hippocampal neurons (right panel). IgG, immunoglobulin. The blots are representative of at least four separate experiments. f, g, Infrequent but punctate colocalization of surface (f) and total GluR6 (g) with total SUMO (in green) in unstimulated cultured hippocampal neurons. We note that SUMO is highly concentrated in the nuclear compartment, consistent with its described role in nuclear function, but also clearly show a punctate distribution along the dendrites. Arrowheads denote colocalization. Scale bar, 20 μm. h, Quantification of surface and total GluR6 receptor population that colocalize with total SUMO-1 in unstimulated cultured hippocampal neurons as shown in f and g. Puncta corresponding to GluR6 that colocalized with the SUMO-1 labelling were quantified using the LSM510 version 3.2 software (Zeiss). Analysis was made from at least eight cells for each condition and given as a percentage ± s.e.m.