Summary: In Arabidopsis, copper (Cu) transport to the ethylene receptor ETR1 mediated using RAN1, a Cu transporter located at the endoplasmic reticulum (ER), and Cu homeostasis mediated using SPL7, the key Cu‐responsive transcription factor, are two deeply conserved vital processes. However, whether and how the two processes interact to regulate plant development remain elusive. We found that its C‐terminal transmembrane domain (TMD) anchors SPL7 to the ER, resulting in dual compartmentalisation of the transcription factor. Immunoprecipitation coupled mass spectrometry, yeast‐two‐hybrid assay, luciferase complementation imaging and subcellular co‐localisation analyses indicate that SPL7 interacts with RAN1 at the ER via the TMD. Genetic analysis revealed that the ethylene‐induced triple response was significantly compromised in the spl7 mutant, a phenotype rescuable by RAN1 overexpression but not by SPL7 without the TMD. The genetic interaction was corroborated by molecular analysis showing that SPL7 modulates RAN1 abundance in a TMD‐dependent manner. Moreover, SPL7 is feedback regulated by ethylene signalling via EIN3, which binds the SPL7 promoter and represses its transcription. These results demonstrate that ER‐anchored SPL7 constitutes a cellular mechanism to regulate RAN1 in ethylene signalling and lay the foundation for investigating how Cu homeostasis conditions ethylene sensitivity in the developmental context.

ABSTRACT: Macropinocytosis mediates non-selective bulk uptake of extracellular fluid. It is the major route by which axenic Dictyostelium cells obtain nutrients and has emerged as a nutrient-scavenging pathway in mammalian cells. How environmental and cellular nutrient status modulates macropinocytic activity is not well understood. By developing a high-content imaging-based genetic screen in Dictyostelium discoideum we identified Slc15A, an oligopeptide transporter located at the plasma membrane and early macropinosome, as a novel macropinocytosis regulator. We show that deletion of slc15A but not two other related slc15 genes, leads to reduced macropinocytosis, reduced cell growth and aberrantly increased autophagy in cells grown in nutrient-rich medium. Expression of Slc15A protein or supplying cells with free amino acids rescues these defects. In contrast, expression of transport-defective Slc15A or supplying cells with amino acids in their di-peptide forms fails to rescue these defects. Therefore, Slc15A modulates the level of macropinocytosis by maintaining the intracellular availability of key amino acids through extraction of oligopeptides from the early macropinocytic pathway. We propose that Slc15A constitutes part of a positive feedback mechanism coupling cellular nutrient status and macropinocytosis. This article has an associated First Person interview with the first authors of the paper.