LAT is a critical regulator of T cell development and function. It organises signalling events at the plasma membrane. However, the mechanism, which controls LAT localisation at the plasma membrane is not fully understood. Here, we studied the impact of helix-breaking amino acids, two prolines and one glycine, in the transmembrane segment on localisation and function of LAT. Using in silico analysis, confocal and superresolution imaging and flow cytometry we demonstrate that central proline residue destabilises transmembrane helix by inducing a kink. The helical structure and dynamics is further regulated by glycine and another proline residue in the luminal part of LAT transmembrane domain. Replacement of these residues with aliphatic amino acids reduces LAT dependence on palmitoylation for sorting to the plasma membrane. However, surface expression of these mutants is not sufficient to recover function of non-palmitoylated LAT in stimulated T cells. These data indicate that geometry and dynamics of LAT transmembrane segment regulate its localisation and function in immune cells.

中文翻译：

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脯氨酸和甘氨酸在LAT跨膜结构域中的作用

LAT是T细胞发育和功能的关键调节因子。它在质膜上组织信号传递事件。但是，尚不完全了解控制LAT在质膜上定位的机制。在这里，我们研究了跨膜区段中螺旋断裂氨基酸，两个脯氨酸和一个甘氨酸对LAT定位和功能的影响。使用计算机分析，共聚焦和超分辨率成像以及流式细胞仪，我们证明了中央脯氨酸残基通过诱导扭结破坏了跨膜螺旋的稳定性。螺旋结构和动力学进一步受到LAT跨膜结构域腔部分甘氨酸和另一个脯氨酸残基的调控。用脂肪族氨基酸取代这些残基可减少LAT对棕榈酰化的依赖性，从而可以分选到质膜上。然而，这些突变体的表面表达不足以恢复刺激的T细胞中非棕榈酰化LAT的功能。这些数据表明，LAT跨膜区段的几何形状和动力学调节其在免疫细胞中的定位和功能。