Msp1 and Pex19-Pex3 cooperate to achieve correct localization of Pex15 to peroxisomes.

Yeast Msp1 is a dual-localized AAA-ATPase on the mitochondrial outer membrane (OM) and peroxisomal membrane. We previously showed that Msp1 transfers mistargeted tail-anchored (TA) proteins from mitochondria to the endoplasmic reticulum (ER) for degradation or delivery to their original destinations. However, the mechanism by which Msp1 in mitochondria and peroxisomes handles authentic peroxisomal TA proteins remains unclear. We show that newly synthesized Pex15 is targeted to peroxisomes primarily via the Pex19- and Pex3-dependent pathway. Mistargeted Pex15 on the mitochondrial OM is extracted by mitochondrial Msp1 and transferred to the ER via the guided-entry of TA proteins pathway for degradation or to peroxisomes via the Pex19-Pex3 pathway. Intriguingly, endogenous Pex15 localized in peroxisomes is also extracted from the membranes by peroxisomal Msp1 but returns to peroxisomes via the Pex19-Pex3 pathway. These results suggest that correct Pex15 localization to peroxisomes relies on not only the initial targeting by Pex19-Pex3 but also the constant re-routing by Msp1 and Pex19-Pex3.