The Conserved N-Terminal Extension of AtKEA1 Is Largely Dispensable for Plastid Function but Contributes to Potassium Homeostasis

Abstract

Members of the K efflux antiporter (KEA) family fulfill key roles in plastids and the endomembrane system. Plants and green algae possess at least one KEA mediating K/H exchange across the plastid inner envelope (IE) membrane. Recently, IE KEAs were shown to be essential for plastid gene expression (PGE), chloroplast development, and photosynthesis. Plants lacking these antiporters exhibit reduced stromal protein synthesis and accumulation of unprocessed rRNA precursors. KEA proteins comprise a conserved monovalent cation/proton antiporter 2 (CPA2) domain and a regulatory K transport and NAD-binding (KTN) domain. IE KEAs are distinguished by an additional [~]500-amino-acid N-terminal extension containing a coiled-coil (CC) domain embedded within a largely intrinsically disordered region (IDR). Intrigued by this unusual architecture, we performed phylogenetic analyses, revealing that this N-terminal fusion arose early and has been conserved throughout the green lineage. We then investigated the oligomeric state, native distribution, and function of the N-terminal domain. Using Arabidopsis thaliana, we found that IE KEAs localize to discrete clusters within the inner envelope membrane and assemble into complexes of approximately 600 kDa. Finally, complementary approaches using a functional KEA1 variant lacking the core N-terminal domains (KEA1{Delta}N) indicate that this extension plays a regulatory rather than an essential role. Our findings uncover an evolutionarily ancient regulatory module that shapes the molecular organization and function of IE KEAs, advancing our understanding of plastid ion and pH homeostasis and plastid ribosome integrity. One-sentence summaryPlastid KEA1/2 proteins feature a unique N-terminal extension that modulates potassium transport activity in a yet unknown manner but is not essential for normal plant growth under ambient conditions.