Palmitoylation controls trafficking of the intracellular Ca 2+ channel MCOLN3/TRPML3 to regulate autophagy

Autophagy. 2019 Feb;15(2):327-340. doi: 10.1080/15548627.2018.1518671. Epub 2018 Sep 14.

Abstract

MCOLN3/TRPML3 is a Ca2+-permeable cation channel that is expressed in multiple subcellular compartments with dynamic localization. Our previous studies suggest that upon macroautophagy/autophagy induction MCOLN3/TRPML3 is recruited and provides Ca2+ for the fusion process in autophagosome biogenesis. However, how intracellular trafficking and the Ca2+ channel function of MCOLN3/TRPML3 are related to autophagy are not known. Here we report that MCOLN3/TRPML3 undergoes palmitoylation at its C-terminal region, which is required for dynamic trafficking and cellular function of MCOLN3/TRPML3 in autophagy. Palmitoylation regulated MCOLN3/TRPML3 surface expression and trafficking, but not channel properties or localization and function of intracellular MCOLN3/TRPML3. Activation of intracellular MCOLN3/TRPML3 induced robust Ca2+ release, which solely increased autophagy in Ca2+- and palmitoylation-dependent manners. Palmitoylation regulated not only intracellular MCOLN3/TRPML3 trafficking to autophagic structures but also autophagic flux in induced autophagy. Importantly, nutrient starvation activated MCOLN3/TRPML3 to release Ca2+ and increased the level of MCOLN3/TRPML3 palmitoylation. Disruption of MCOLN3/TRPML3 palmitoylation, however, abolished the starvation-induced MCOLN3/TRPML3 activation without affecting channel activity. These results suggest that trafficking and channel function of MCOLN3/TRPML3 are regulated in the context of autophagy, and palmitoylation is a prerequisite for the function of MCOLN3/TRPML3 as a Ca2+ channel in autophagosome formation by controlling its trafficking between subcellular compartments. Abbreviations: 17-ODYA, 17-octadecynoic acid; 2-BP, 2-bromopalmitate; BFA, brefeldin A; DN, dominant-negative; GPN, glycyl-L-phenylalanine-beta-naphthylamide; HN, hydroxylamine; KD, knockdown; MCOLN3/TRPML3, mucolipin 3; MS, mass spectrometry; PAT, palmitoyl acyltransferase; PM, plasma membrane; WT, wild type; ZDHHC, a zinc-finger motif and an Asp-His-His-Cys sequence.

Keywords: Autophagy; Ca channel; MCOLN3/TRPML3; membrane trafficking; palmitoylation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Autophagy*
  • Calcium / metabolism
  • Calcium Channels / chemistry
  • Calcium Channels / metabolism*
  • Endocytosis
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Intracellular Space / metabolism*
  • Lipoylation*
  • Protein Transport
  • Transient Receptor Potential Channels / chemistry
  • Transient Receptor Potential Channels / metabolism*

Substances

  • Calcium Channels
  • MCOLN3 protein, human
  • Transient Receptor Potential Channels
  • Calcium

Grant support

This work was supported by the National Research Foundation of Korea [NRF-2014R1A1A2057830] and [NRF-2018R1A4A1023822].