In eukaryotic cells, organelle biogenesis is pivotal for cellular function and cell survival. Chloroplasts are unique organelles with a complex internal membrane network. The mechanisms of the migration of imported nuclear-encoded chloroplast proteins across the crowded stroma to thylakoid membranes are less understood. Here, we identified two Arabidopsis ankyrin-repeat proteins, STT1 and STT2, that specifically mediate sorting of chloroplast twin arginine translocation (cpTat) pathway proteins to thylakoid membranes. STT1 and STT2 form a unique hetero-dimer through interaction of their C-terminal ankyrin domains. Binding of cpTat substrate by N-terminal intrinsically disordered regions of STT complex induces liquid-liquid phase separation. The multivalent nature of STT oligomer is critical for phase separation. STT-Hcf106 interactions reverse phase separation and facilitate cargo targeting and translocation across thylakoid membranes. Thus, the formation of phase-separated droplets emerges as a novel mechanism of intra-chloroplast cargo sorting. Our findings highlight a conserved mechanism of phase separation in regulating organelle biogenesis.

在真核细胞中，细胞器的生物发生对于细胞功能和细胞存活至关重要。叶绿体是具有复杂内部膜网络的独特细胞器。进口的核编码叶绿体蛋白跨过拥挤的基质迁移到类囊体膜的迁移机制尚不清楚。在这里，我们确定了两个拟南芥锚蛋白重复蛋白STT1和STT2，专门介导叶绿体双精氨酸易位（cpTat）途径蛋白到类囊体膜的分选。STT1和STT2通过其C末端锚蛋白域的相互作用形成独特的异二聚体。cpTat底物与STT络合物的N端固有无序区的结合引起液-液相分离。STT低聚物的多价性质对于相分离至关重要。STT-Hcf106相互作用使反相分离，并促进货物靶向和跨类囊体膜转运。因此，相分离液滴的形成作为叶绿体内部货物分拣的新机制而出现。我们的发现突出了调节细胞器生物发生的相分离的保守机制。