Supplementary Materials Appendix EMBJ-39-e103457-s001. SEZ6 decreased surface levels of GluK2/3 in primary neurons and reduced kainate\evoked currents in CA1 pyramidal neurons in acute hippocampal slices. Mechanistically, loss of SEZ6 and prevented modification of GluK2/3 with the human natural killer\1 (HNK\1) glycan, a modulator of GluK2/3 function. SEZ6 interacted with GluK2 through its ectodomain and promoted post\endoplasmic reticulum transport of GluK2 in the secretory pathway in heterologous cells and primary neurons. Taken together, SEZ6 acts as a new trafficking factor for GluK2/3. This novel function may help to better understand the role of SEZ6 in neurologic and psychiatric diseases. (DIV2) with lentiviral CRE recombinase or GFP to obtain neurons lacking (SEZ6KO) or maintaining SEZ6 (WT), respectively (workflow in Fig?1A). Metabolic labeling occurred from DIV5 to DIV7. At DIV7, surface proteins were biotinylated, enriched with streptavidin agarose, and analyzed (workflow in Fig?1A). The sample preparation workflow showed little variation between samples generally, as indicated with relationship coefficients of bigger than 0.94 between different examples (Appendix?Fig S1). Using SUSPECS, SEZ6 was recognized on the top of WT neurons regularly, rather than recognized in the SEZ6KO neurons regularly, consistent with a competent Cre\mediated SEZ6KO (Figs?2A and EV1). 3,209 proteins had been recognized in 3 out of 3 tests from the SUSPECS evaluation, and 571 had been glycosylated, relating to UniProt annotation (Fig?1B and Dataset EV1). 40% of the many proteins recognized, and 90% from the glycosylated proteins had been categorized as membrane proteins relating to UniProt keywords (Fig?1B), showing our technique enriched for membrane proteins. Proteins had been considered as strikes if their proteins level in SEZ6KO vs. WT neurons was less than log2 percentage(SEZ6KO/WT)?=??0.5 (0.7 fold modification) or more than log2 percentage(SEZ6KO/WT)?=?0.5 (1.4 collapse modification) and if the in mouse brains In WT neurons, the GluK2/3 immunoreactivity in European blots was viewed as two co\migrating rings closely, however in SEZ6KO neurons, the upper band appeared BIBS39 reduced and merging with the lower one, suggesting that N\glycosylation of GluK2 and/or GluK3 may be impaired in SEZ6KO neurons BIBS39 (Fig?4A). In fact, GluK2 and GluK3 BIBS39 have multiple N\glycosylation sites (Parker in primary neurons. To test whether maturation of GluK2 and/or GluK3 is also affected (Fig?4A), the upper one of the two GluK2/3 bands under control conditions (no EndoH treatment) was reduced in the SEZ6KO brain and this BIBS39 effect was even more clearly visible after EndoH treatment, where again the uppermost, mature glycoform shifted to a lower apparent molecular weight (Fig?4B for brain homogenates and ?and4E4E for synaptosomes and model in Fig?EV2B). In contrast to the primary neurons, total levels of the GluK2/3 in the brain samples were not significantly decreased and this was also seen for a control protein, the GluA2 subunit of AMPA receptors (Figs?4B and D, and EV2C). Although SEZ6 has two homologs, SEZ6L BIBS39 and SEZ6L2, which have a similar domain structure as SEZ6, there was no compensatory change in SEZ6 expression nor an effect on mature glycosylation of the GluK2/3 band in SEZ6L and SEZ6L2 single knock\out brain synaptosomes (Figs?4E and EV2D). Moreover, the reduced maturation of the GluK2/3 band was not further reduced in synaptosomes from triple knock\out mice lacking SEZ6 and both of its homologs (Fig?4E). This demonstrates that specifically SEZ6, but not its homologs, is required for mature glycosylation of GluK2 and/or Rabbit Polyclonal to OR5K1 GluK3. The relevance of SEZ6 for GluK2/3 maturation was not only seen at very young ages, when SEZ6 expression is high [(Kim test, no biotin vs. 20?min **system of acute hippocampal slices from.