Msp1 is a conserved AAA ATPase in budding yeast localized to mitochondria where it prevents accumulation of mistargeted tail-anchored (TA) proteins, including the peroxisomal TA protein Pex15. Msp1 also resides on peroxisomes but it remains unknown how native TA proteins on mitochondria and peroxisomes evade Msp1 surveillance. We used live-cell quantitative cell microscopy tools and drug-inducible gene expression to dissect Msp1 function. We found that a small fraction of peroxisomal Pex15, exaggerated by overexpression, is turned over by Msp1. Kinetic measurements guided by theoretical modeling revealed that Pex15 molecules at mitochondria display age-independent Msp1 sensitivity. By contrast, Pex15 molecules at peroxisomes are rapidly converted from an initial Msp1-sensitive to an Msp1-resistant state. Lastly, we show that Pex15 interacts with the peroxisomal membrane protein Pex3, which shields Pex15 from Msp1-dependent turnover. In sum, our work argues that Msp1 selects its substrates on the basis of their solitary membrane existence.

中文翻译：

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AAA 蛋白 Msp1 介导从过氧化物酶体膜清除多余的尾锚定蛋白

Msp1 是出芽酵母中一种保守的 AAA ATP 酶，定位于线粒体，可防止错误靶向的尾锚 (TA) 蛋白（包括过氧化物酶体 TA 蛋白 Pex15）的积累。Msp1 也存在于过氧化物酶体上，但线粒体和过氧化物酶体上的天然 TA 蛋白如何逃避 Msp1 监视仍然未知。我们使用活细胞定量细胞显微镜工具和药物诱导基因表达来剖析 Msp1 功能。我们发现过氧化物酶体 Pex15 的一小部分，被过度表达夸大，被 Msp1 翻转。由理论模型指导的动力学测量表明，线粒体中的 Pex15 分子显示出与年龄无关的 Msp1 敏感性。相比之下，过氧化物酶体上的 Pex15 分子迅速从最初的 Msp1 敏感状态转变为 Msp1 抗性状态。最后，我们显示 Pex15 与过氧化物酶体膜蛋白 Pex3 相互作用，它保护 Pex15 免受 Msp1 依赖性转换。总之，我们的工作认为 Msp1 基于其孤立膜的存在来选择其底物。