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.

Msp1是位于线粒体中的发芽酵母中的一种保守的AAA ATPase，可防止错误定位的尾锚定（TA）蛋白（包括过氧化物酶体TA蛋白Pex15）积聚。Msp1也存在于过氧化物酶体上，但仍不清楚线粒体和过氧化物酶体上的天然TA蛋白如何逃避Msp1监视。我们使用活细胞定量细胞显微镜工具和药物诱导性基因表达来剖析Msp1功能。我们发现，过氧化物酶体Pex15的一小部分（由过表达夸大了）由Msp1上交。理论建模指导下的动力学测量表明，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分子从最初的Msp1敏感状态迅速转变为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，从而使Pex15免受Msp1依赖性转换的影响。总而言之，我们的工作认为Msp1根据其孤立膜的存在选择其底物。