Transient residence of the repulsive client Shutdown in Yb bodies plays a critical role in Piwi-piRISC biogenesis and maintaining fertility.

The formation of biomolecular condensates, such as Yb bodies in Drosophila ovarian somatic cells, is significantly contributed to by phase separation of scaffold proteins. Client proteins transiently accumulate via interactions with these scaffolds; however, how client flux is regulated remains unclear. Here, we investigate Shutdown, a client protein of Yb bodies-the site of Piwi-piRNA-induced silencing complex (piRISC) precursor (pre-Piwi-piRISC) formation-and show that cytosolic Shutdown connects Armitage and Piwi to promote Piwi deposition into Yb bodies before quickly returning to the cytosol. This return allows Armitage to transfer pre-Piwi-piRISC to mitochondria for maturation. Shutdown's acidic N terminus mediates self-repulsion, conferring its transient residence within Yb bodies. A point mutation in armitage, analogous to a human armitage/MOV10L1 mutation associated with azoospermia, traps Shutdown in Yb bodies, blocking Piwi-piRISC generation. This study reveals the mechanism underlying Shutdown's transient localization in Yb bodies and its essential role in Piwi-piRISC biogenesis and fertility.