Tetraspanin CD9、CD81和CD63是细胞外囊泡（EVs）的主要组成部分。然而，它们对EVs成分的影响尚未进行充分的研究。在MCF7乳腺癌细胞系中，CD63主要存在于胞内，符合预期。相比之下，CD9和CD81在质膜上强烈共定位，尽管不同的位置具有不同的比例，这可能解释了CD81在EVs中更高的富集度。通过定量质谱分析，这些四跨膜蛋白对EVs的蛋白质组成几乎没有影响。我们还分析了CD9和CD81的共同敲除对细胞的影响，因为这两种四跨膜蛋白在多种细胞过程中发挥类似的作用，并且直接与两个Ig结构域蛋白CD9P-1/EWI-F/PTGFRN和EWI-2/IGSF8结合。在CD9和CD81双缺失细胞的EVs中，这两个蛋白是唯一明显减少的。在EWI-2的情况下，主要是由于细胞表达水平下降引起的。总之，本研究表明，常用作EV蛋白标记物的CD9、CD81和CD63在决定MCF7细胞释放的EVs的蛋白质组成中起到边缘作用，并突出了CD9和CD81调控CD9P-1和EWI-2的表达水平和/或转运的机制。© 2023 The Authors. Journal of Extracellular Vesicles由Wiley Periodicals, LLC代表国际细胞外囊泡学会发布。

The tetraspanins CD9, CD81 and CD63 are major components of extracellular vesicles (EVs). Yet, their impact on EV composition remains under-investigated. In the MCF7 breast cancer cell line CD63 was as expected predominantly intracellular. In contrast CD9 and CD81 strongly colocalized at the plasma membrane, albeit with different ratios at different sites, which may explain a higher enrichment of CD81 in EVs. Absence of these tetraspanins had little impact on the EV protein composition as analysed by quantitative mass spectrometry. We also analysed the effect of concomitant knock-out of CD9 and CD81 because these two tetraspanins play similar roles in several cellular processes and associate directly with two Ig domain proteins, CD9P-1/EWI-F/PTGFRN and EWI-2/IGSF8. These were the sole proteins significantly decreased in the EVs of double CD9- and CD81-deficient cells. In the case of EWI-2, this is primarily a consequence of a decreased cell expression level. In conclusion, this study shows that CD9, CD81 and CD63, commonly used as EV protein markers, play a marginal role in determining the protein composition of EVs released by MCF7 cells and highlights a regulation of the expression level and/or trafficking of CD9P-1 and EWI-2 by CD9 and CD81.© 2023 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.