Fig. 5.
Roles of palmitoylation and phosphorylation in SR binding to the membrane. (A) Palmitoylation inhibition blocks SR translocation in primary neuronal cultures. The neuronal cultures were preincubated for 2.5 h in NB medium containing 7 mg/ml fatty-acid-free albumin and in the presence or in the absence of 100 μM 2-Br-palmitate. Subsequently, 100 μM NMDA was added for 30 min and the levels of membrane-bound SR were monitored by Western blotting (Upper). Lower panel depicts β-tubulin levels in each sample. (B) Treatment with 100 μM 2Br-palmitate for 12 h decreases the amount of membrane-bound SR (Mem) but does not change the cytosolic levels (Cyt) of SR in transfected SH-SY5Y cells. Statistical analysis shows a 2-fold increase in cytosol/membrane SR ratio (Right; n = 6). Cytosolic fractions were analyzed using 5 μg protein, whereas membrane fractions contained 50 μg protein. (C) Phosphorylation of Thr-227 is exclusively found in the membrane-bound SR. Cytosolic and membrane-bound HA-SR from SH-SY5Y cells were purified, in-gel digested with trypsin, and the peptides analyzed by LC-MS (see SI Methods). The extracted ion chromatogram (m/z 737.1 ± 0.1) of the membrane-bound HA-SR shows elution of the monophosphorylated peptide 222–241 after 28.8 min (Left), whereas no corresponding peptide peak was detected in the cytosolic HA-SR (Right). (D) Mutation of Thr-227 to Ala (T227A) does not affect the acylation levels of HA SR by [3H]palmitate in transfected HEK 293 cells (Upper). Lower panel depicts the SDS-PAGE loading control. (E) T227A SR mutant exhibits lower binding to the membrane in transfected HEK293 cells. Statistical analysis shows a 4- to 5-fold increase in cytosol/membrane ratio when T227A is compared with wild-type SR (n = 6). (F) Levels of membrane-bound SR under basal (−NMDA) or stimulated (+NMDA) conditions in primary neuronal culture transduced with recombinant lentivirus containing either HA-SR WT (Left) or HA-SR T227A (Right). HA-SR was detected with anti-HA. Lower panel depicts TfR loading control in the membrane fraction. **Different from control at P < 0.01; ***Different from control at P < 0.001.