ABSTRACT
The dipeptide glycyl-l-phenylalanine 2-naphthylamide (GPN) is widely used to perturb lysosomes because its cleavage by the lysosomal enzyme cathepsin C is proposed to rupture lysosomal membranes. We show that GPN evokes a sustained increase in lysosomal pH (pHly), and transient increases in cytosolic pH (pHcyt) and Ca2+ concentration ([Ca2+]c). None of these effects require cathepsin C, nor are they accompanied by rupture of lysosomes, but they are mimicked by structurally unrelated weak bases. GPN-evoked increases in [Ca2+]c require Ca2+ within the endoplasmic reticulum (ER), but they are not mediated by ER Ca2+ channels amplifying Ca2+ release from lysosomes. GPN increases [Ca2+]c by increasing pHcyt, which then directly stimulates Ca2+ release from the ER. We conclude that physiologically relevant increases in pHcyt stimulate Ca2+ release from the ER in a manner that is independent of IP3 and ryanodine receptors, and that GPN does not selectively target lysosomes.
Subject(s)
Calcium Signaling/drug effects , Calcium/metabolism , Cytosol/drug effects , Dipeptides/pharmacology , Endoplasmic Reticulum/drug effects , Biological Transport , CRISPR-Cas Systems , Calcium Channels/genetics , Calcium Channels/metabolism , Cathepsin C/genetics , Cathepsin C/metabolism , Cell Line, Tumor , Cytosol/metabolism , Endoplasmic Reticulum/metabolism , Gene Editing , Gene Expression , Gene Knockdown Techniques , HEK293 Cells , HeLa Cells , Humans , Hydrogen-Ion Concentration/drug effects , Inositol 1,4,5-Trisphosphate Receptors/genetics , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Leukocytes/cytology , Leukocytes/drug effects , Leukocytes/metabolism , Lysosomal Membrane Proteins/genetics , Lysosomal Membrane Proteins/metabolism , Lysosomes/drug effects , Lysosomes/metabolism , Ploidies , Ryanodine Receptor Calcium Release Channel/genetics , Ryanodine Receptor Calcium Release Channel/metabolismABSTRACT
Newly synthesised histones are thought to dimerise in the cytosol and undergo nuclear import in complex with histone chaperones. Here, we provide evidence that human H3.1 and H4 are imported into the nucleus as monomers. Using a tether-and-release system to study the import dynamics of newly synthesised histones, we find that cytosolic H3.1 and H4 can be maintained as stable monomeric units. Cytosolically tethered histones are bound to importin-alpha proteins (predominantly IPO4), but not to histone-specific chaperones NASP, ASF1a, RbAp46 (RBBP7) or HAT1, which reside in the nucleus in interphase cells. Release of monomeric histones from their cytosolic tether results in rapid nuclear translocation, IPO4 dissociation and incorporation into chromatin at sites of replication. Quantitative analysis of histones bound to individual chaperones reveals an excess of H3 specifically associated with sNASP, suggesting that NASP maintains a soluble, monomeric pool of H3 within the nucleus and may act as a nuclear receptor for newly imported histone. In summary, we propose that histones H3 and H4 are rapidly imported as monomeric units, forming heterodimers in the nucleus rather than the cytosol.
