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.
Subject(s)
Dictyostelium , Animals , Dictyostelium/genetics , Endosomes , Humans , Mammals , Nutrients , Oligopeptides , PinocytosisABSTRACT
Macropinocytosis, an evolutionarily conserved endocytic pathway, mediates nonselective bulk uptake of extracellular fluid. It is the primary route for axenic Dictyostelium cells to obtain nutrients and has also emerged as a nutrient-scavenging pathway for mammalian cells. How cells adjust macropinocytic activity in various physiological or developmental contexts remains to be elucidated. We discovered that, in Dictyostelium cells, the transcription factors Hbx5 and MybG form a functional complex in the nucleus to maintain macropinocytic activity during the growth stage. In contrast, during starvation-induced multicellular development, the transcription factor complex undergoes nucleocytoplasmic shuttling in response to oscillatory cyclic adenosine 3',5'-monophosphate (cAMP) signals, which leads to increased cytoplasmic retention of the complex and progressive downregulation of macropinocytosis. Therefore, by coupling macropinocytosis-related gene expression to the cAMP oscillation system, which facilitates long-range cell-cell communication, the dynamic translocation of the Hbx5-MybG complex orchestrates a population-level adjustment of macropinocytic activity to adapt to changing environmental conditions.
Subject(s)
Dictyostelium , Animals , Dictyostelium/metabolism , Pinocytosis/physiology , Cytoplasm , Cell Nucleus , Transcription Factors/metabolism , MammalsABSTRACT
Phospholipid hydroperoxide glutathione peroxidase (PHGPx or GPx4; EC1.11.1.12) is a selenoperoxidase that can directly reduce phospholipid and cholesterol hydroperoxides. The mature cytoplasmic GPx4 is a monomeric protein with molecular weight of 19.5 kDa. In this study, human GPx4 (hGPx4) gene was amplified from the complementary DNA library of human hepatoma cell line. Eukaryotic expression plasmid pSelExpress1-leader-GPx4 was constructed and transfected into the eukaryotic cells HEK293T. Expression of hGPx4 was detected by Western blotting, and the target protein was purified by immobilized metal affinity chromatography. The results of the activity and kinetics of the purified protein show that the obtained protein follows a "ping-pong" mechanism, which is similar to that of native cytosolic glutathione peroxidase (GPx1; EC1.11.1.9). This is the first time that hGPx4 could be expressed and purified from HEK293T cells, and this work will provide an important resource of hGPx4 for its functional study in vitro and in vivo.