Plasma membrane calcium pump (PMCA) differential exposure of hydrophobic domains after calmodulin and phosphatidic acid activation.
Plasma membrane calcium pump (PMCA) differential exposure of hydrophobic domains after calmodulin and phosphatidic acid activation.
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钙调蛋白和磷脂酸激活后，质膜钙泵 (PMCA) 疏水域的差异暴露。
DOI：
10.1074/jbc.m110.210088
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Rossi,JuanPabloFC
中科院分区：
文献类型：
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作者：
Mangialavori,Irene;Villamil-Giraldo,AnaMaría;Pignataro,MaríaF;Ferreira-Gomes,Mariela;Caride,ArielJ;Rossi,JuanPabloFC
通过测量可光激活的磷脂酰胆碱类似物[125I]TID-PC/16与蛋白质的掺入来评估质膜钙泵(PMCA)对周围磷脂的暴露。在钙离子存在下，钙调素(CaM)和磷脂酸(PA)均显著降低[125I]TID-PC/16对PMCA的掺入。用V8酶对PMCA进行蛋白水解会产生三个主要片段：N，包括跨膜片段M1和M2；M，包括M3和M4；C，包括M5到M10。CaM降低了[125I]TID-PC/16对片段M和C的掺入水平，而磷脂酸则降低了[125I]TID-PC/16对N和M片段的掺入，这表明CaM或PA结合引起的构象变化延伸到相邻的跨膜区。有趣的是，这一结果也表明了CaM和PA产生的活性构象之间的差异。为了验证这一点，我们测量了在ATP结合位点标记了曙红异硫氰酸酯的PMCA与PMCA胶束中包含的磷脂RhoPE之间的共振能量转移。CaM降低了这两种探针之间的能量传递效率，而PA则没有。这一结果表明，CaM的激活增加了ATP结合位点与膜之间的距离，但PA不影响这一距离。我们的结果揭示了CaM和PA诱导的PMCA构象的主要差异，并表明这些差异涉及跨膜区。
The exposure of the plasma membrane calcium pump (PMCA) to the surrounding phospholipids was assessed by measuring the incorporation of the photoactivatable phosphatidylcholine analog [125I]TID-PC/16 to the protein. In the presence of Ca2+both calmodulin (CaM) and phosphatidic acid (PA) greatly decreased the incorporation of [125I]TID-PC/16 to PMCA. Proteolysis of PMCA with V8 protease results in three main fragments: N, which includes transmembrane segments M1 and M2; M, which includes M3 and M4; and C, which includes M5 to M10. CaM decreased the level of incorporation of [125I]TID-PC/16 to fragments M and C, whereas phosphatidic acid decreased the incorporation of [125I]TID-PC/16 to fragments N and M. This suggests that the conformational changes induced by binding of CaM or PA extend to the adjacent transmembrane domains. Interestingly, this result also denotes differences between the active conformations produced by CaM and PA. To verify this point, we measured resonance energy transfer between PMCA labeled with eosin isothiocyanate at the ATP-binding site and the phospholipid RhoPE included in PMCA micelles. CaM decreased the efficiency of the energy transfer between these two probes, whereas PA did not. This result indicates that activation by CaM increases the distance between the ATP-binding site and the membrane, but PA does not affect this distance. Our results disclose main differences between PMCA conformations induced by CaM or PA and show that those differences involve transmembrane regions.