pH Regulation by NHX-Type Antiporters Is Required for Receptor-Mediated Protein Trafficking to the Vacuole in Arabidopsis

Abstract

Protein trafficking requires proper ion and pH homeostasis of the endomembrane system. The NHX-type Na + /H + antiporters NHX5 and NHX6 localize to the Golgi, trans-Golgi network, and prevacuolar compartments and are required for growth and trafficking to the vacuole. In the nhx5 nhx6 T-DNA insertional knockouts, the precursors of the 2S albumin and 12S globulin storage proteins accumulated and were missorted to the apoplast. Immunoelectron microscopy revealed the presence of vesicle clusters containing storage protein precursors and vacuolar sorting receptors (VSRs). Isolation and identification of complexes of VSRs with unprocessed 12S globulin by 2D blue-native PAGE/SDS-PAGE indicated that the nhx5 nhx6 knockouts showed compromised receptor-cargo association. In vivo interaction studies using bimolecular fluorescence complementation between VSR2;1, aleurain, and 12S globulin suggested that nhx5 nhx6 knockouts showed a significant reduction of VSR binding to both cargoes. In vivo pH measurements indicated that the lumens of VSR compartments containing aleurain, as well as the trans-Golgi network and prevacuolar compartments, were significantly more acidic in nhx5 nhx6 knockouts. This work demonstrates the importance of NHX5 and NHX6 in maintaining endomembrane luminal pH and supports the notion that proper vacuolar trafficking and proteolytic processing of storage proteins require endomembrane pH homeostasis.

CAX‐ing a wide net: Cation/H+ transporters in metal remediation and abiotic stress signalling

“…This close relationship observed between CAX and pH may signify a role in modulating endomembrane pH levels similar to the pH regulation by NHX‐type antiporters required for protein trafficking (Pittman 2012; Reguera et al . 2015). …”

AtNHX5 and AtNHX6: Roles in protein transport

“…[4][5][6] Accumulating evidence indicates that AtNHX5 and AtNHX6 play an important role in protein transport. 5,[7][8][9] AtNHX5 and AtNHX6 have diversified roles in protein transport Reguera et al examined the role of AtNHX5 and AtNHX6 in protein transport. 5 They found that nhx5 nhx6 produced large, heavy seeds with a dark coat.…”

“…5,[7][8][9] AtNHX5 and AtNHX6 have diversified roles in protein transport Reguera et al examined the role of AtNHX5 and AtNHX6 in protein transport. 5 They found that nhx5 nhx6 produced large, heavy seeds with a dark coat. Additionally, protein storage vacuoles (PSVs) were smaller but their number was increased in nhx5 nhx6.…”

“…[1][2][3] They are localized in the Golgi, trans-Golgi network (TGN), and prevacuolear compartment (PVC). [4][5][6] Studies have shown that AtNHX5 and AtNHX6 facilitate ion and pH homeostasis, and are crucial for growth and development. [4][5][6] Accumulating evidence indicates that AtNHX5 and AtNHX6 play an important role in protein transport.…”

“…[4][5][6] Studies have shown that AtNHX5 and AtNHX6 facilitate ion and pH homeostasis, and are crucial for growth and development. [4][5][6] Accumulating evidence indicates that AtNHX5 and AtNHX6 play an important role in protein transport. 5,[7][8][9] AtNHX5 and AtNHX6 have diversified roles in protein transport Reguera et al examined the role of AtNHX5 and AtNHX6 in protein transport.…”