Transmembrane protein insertion orientation in yeast depends on the charge difference across transmembrane segments, their total hydrophobicity, and its distribution
Transmembrane protein insertion orientation in yeast depends on the charge difference across transmembrane segments, their total hydrophobicity, and its distribution
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DOI：
10.1074/jbc.273.38.24963
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发表时间：
1998-09-18
影响因子：
4.8
通讯作者：
Tipper, DJ
中科院分区：
文献类型：
--
作者：
Harley, CA;Holt, JA;Tipper, DJ
内质网跨膜蛋白插入方向的决定因素已在酿酒酵母中使用源自 α 因子受体 Ste2p N 末端的 III 型（天然外表面 N 末端 (N-exo)）跨膜融合蛋白的变体进行了研究。跨膜部分附近的小正电荷和负电荷对方向具有相同和相反的影响，并且这种影响与 N 端或 C 端位置无关，与响应机制的纯静电相互作用一致。在没有电荷差异的情况下观察到的 N-exo 插入的 3:1 偏向反映了较长跨膜片段赋予的 N-exo 偏向。方向与总疏水性而不是长度的相关性最好，但它也受到跨膜片段内疏水性分布的强烈影响，最疏水的末端优先易位，因此插入方向取决于对至少三个参数的响应的整合：跨膜片段的电荷差、其总疏水性及其疏水性梯度。估计了相对信号强度，并讨论了拓扑预测的后果。对跨膜序列的响应可能取决于蛋白质-易位子相互作用，但对电荷差异的响应可能由阴离子磷脂提供的静电场介导。
The determinants of transmembrane protein insertion orientation at the endoplasmic reticulum have been investigated in Saccharomyces cerevisiae using variants of a Type III (naturally exofacial N terminus (N-exo)) transmembrane fusion protein derived from the N terminus of Ste2p, the alpha-factor receptor. Small positive and negative charges adjacent to the transmembrane segment had equal and opposite effects on orientation, and this effect was independent of N- or C-terminal location, consistent with a purely electrostatic interaction with response mechanisms. A 3:1 bias toward N-exo insertion, observed in the absence of a charge difference, was shown to reflect the N-exo bias conferred by longer transmembrane segments. Orientation correlated best with total hydrophobicity rather than length, but it was also strongly affected by the distribution of hydrophobicity within the transmembrane segment, The most hydrophobic terminus was preferentially translocated, Insertion orientation thus depends on integration of responses to at least three parameters: charge difference across a transmembrane segment, its total hydrophobicity, and its hydrophobicity gradient. Relative signal strengths were estimated, and consequences for topology prediction are discussed. Responses to transmembrane sequence may depend on protein-translocon interactions, but responses to charge difference may be mediated by the electrostatic field provided by anionic phospholipids.