Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Dept. of Chemistry, Faculty of Science, Utrecht University, Padualaan 8, 3584CH, Utrecht, The Netherlands.
Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, Padualaan 8, 3584CH, Utrecht, The Netherlands.
Nat Commun. 2017 Nov 3;8(1):1280. doi: 10.1038/s41467-017-01461-z.
Palmitoylation affects membrane partitioning, trafficking and activities of membrane proteins. However, how specificity of palmitoylation and multiple palmitoylations in membrane proteins are determined is not well understood. Here, we profile palmitoylation states of three human claudins, human CD20 and cysteine-engineered prokaryotic KcsA and bacteriorhodopsin by native mass spectrometry. Cysteine scanning of claudin-3, KcsA, and bacteriorhodopsin shows that palmitoylation is independent of a sequence motif. Palmitoylations are observed for cysteines exposed on the protein surface and situated up to 8 Å into the inner leaflet of the membrane. Palmitoylation on multiple sites in claudin-3 and CD20 occurs stochastically, giving rise to a distribution of palmitoylated membrane-protein isoforms. Non-native sites in claudin-3 indicate that membrane-protein function imposed evolutionary restraints on native palmitoylation sites. These results suggest a generic, stochastic membrane-protein palmitoylation process that is determined by the accessibility of palmitoyl-acyl transferases to cysteines on membrane-embedded proteins, and not by a preferred substrate-sequence motif.
棕榈酰化影响膜的分区、运输和膜蛋白的活性。然而，膜蛋白中棕榈酰化和多个棕榈酰化的特异性是如何确定的还不是很清楚。在这里，我们通过天然质谱法对三种人类紧密连接蛋白、人类 CD20 和工程化的原核 KcsA 和菌紫质的棕榈酰化状态进行了分析。克劳丁-3、KcsA 和菌紫质的半胱氨酸扫描表明，棕榈酰化与序列基序无关。在蛋白质表面暴露的半胱氨酸和位于膜内层 8Å 以内的位置都观察到了棕榈酰化。克劳丁-3 和 CD20 上的多个位点的棕榈酰化是随机发生的，导致了棕榈酰化膜蛋白同工型的分布。克劳丁-3 中的非天然位点表明，膜蛋白的功能对天然棕榈酰化位点施加了进化限制。这些结果表明，一种通用的、随机的膜蛋白棕榈酰化过程是由棕榈酰基转移酶与膜嵌入蛋白上的半胱氨酸的可及性决定的，而不是由首选的底物序列基序决定的。
