Abstract：
The neuronal glycine transporter GlyT2 removes glycine from the synaptic cleft through active Na+, Cl-, and glycine cotransport contributing to the termination of the glycinergic signal as well as supplying substrate to the presynaptic terminal for the maintenance of the neurotransmitter content in synaptic vesicles. Patients with mutations in the human GlyT2 gene (SLC6A5), develop hyperekplexia or startle disease (OMIM 149400), characterized by hypertonia and exaggerated startle responses to trivial stimuli that may have lethal consequences in the neonates as a result of apnea episodes. Post-translational modifications in cysteine residues of GlyT2 are an aspect of structural interest we analyzed. Our study is compatible with a reversible and short-lived S-acylation in spinal cord membranes, detectable by biochemical and proteomics methods (acyl-Rac binding and IP-ABE) confirmed with positive and negative controls (palmitoylated and non-palmitoylated proteins). According to a short-lived modification, direct labeling using click chemistry was faint but mostly consistent. We have analyzed the physiological properties of a GlyT2 mutant lacking the cysteines with high prediction of palmitoylation and the mutant is less prone to be included in lipid rafts, an effect also observed upon treatment with the palmitoylation inhibitor 2-bromopalmitate. This work demonstrates there are determinants of lipid raft inclusion associated with the GlyT2 mutated cysteines, which are presumably modified by palmitoylation.
摘要：
神经元甘氨酸转运蛋白GlyT2通过活性Na+从突触间隙去除甘氨酸，Cl-,和甘氨酸共转运有助于甘氨酸能信号的终止，并为突触前末端提供底物，以维持突触小泡中的神经递质含量。人GlyT2基因（SLC6A5）突变的患者，发展为中风或惊厥病（OMIM149400），其特征是高张力和对琐碎刺激的过度惊吓反应，这些刺激可能由于呼吸暂停发作而对新生儿造成致命后果。GlyT2的半胱氨酸残基中的翻译后修饰是我们分析的结构兴趣的一个方面。我们的研究与脊髓膜中可逆和短寿命的S-酰化反应相容，通过生物化学和蛋白质组学方法（酰基-Rac结合和IP-ABE）可检测到阳性和阴性对照（棕榈酰化和非棕榈酰化蛋白）。根据一个短暂的修改，使用点击化学直接标记是微弱的，但大多是一致的。我们已经分析了缺乏半胱氨酸的GlyT2突变体的生理特性，具有高的棕榈酰化预测，并且该突变体不太容易被包括在脂筏中。在用棕榈酰化抑制剂2-溴棕榈酸酯处理时也观察到效果。这项工作证明了与GlyT2突变的半胱氨酸相关的脂筏包含的决定因素，可能是通过棕榈酰化修饰的。
