Predominant localization of phosphatidylserine at the cytoplasmic leaflet of the ER, and its TMEM16K-dependent redistribution

Abstract

TMEM16K, a membrane protein carrying 10 transmembrane regions, has phospholipid scramblase activity. TMEM16K is localized to intracellular membranes, but whether it actually scrambles phospholipids inside cells has not been demonstrated, due to technical difficulties in studying intracellular lipid distributions. Here, we developed a freeze-fracture electron microscopy method that enabled us to determine the phosphatidylserine (PtdSer) distribution in the individual leaflets of cellular membranes. Using this method, we found that the endoplasmic reticulum (ER) of mammalian cells harbored abundant PtdSer in its cytoplasmic leaflet and much less in the luminal leaflet, whereas the outer and inner nuclear membranes (NMs) had equivalent amounts of PtdSer in both leaflets. The ER and NMs of budding yeast also harbored PtdSer in their cytoplasmic leaflet, but asymmetrical distribution in the ER was not observed. Treating mouse embryonic fibroblasts with the Ca2+ ionophore A23187 compromised the cytoplasmic leaflet-dominant PtdSer asymmetry in the ER and increased PtdSer in the NMs, especially in the nucleoplasmic leaflet of the inner NM. This Ca2+-induced PtdSer redistribution was not observed in TMEM16K-null fibroblasts, but was recovered in these cells by reexpressing TMEM16K. These results indicate that, similar to the plasma membrane, PtdSer in the ER of mammalian cells is predominantly localized to the cytoplasmic leaflet, and that TMEM16K directly or indirectly mediates Ca2+-dependent phospholipid scrambling in the ER.

Osh6 requires Ist2 for localization to ER–PM contacts and efficient phosphatidylserine transport in budding yeast

“…Finally, it is intriguing that the membrane-embedded portion of Ist2 bears structural homology to the mammalian TMEM16 family of proteins, many of which have been shown to function as Ca 2+ -activated lipid scramblases and/or ion channels in the PM or possibly in the ER (Bushell et al, 2019;Falzone et al, 2018;Jha et al, 2019). PS is synthesized exclusively on the cytosolic side of the ER (Chauhan et al, 2016), but was proposed to be enriched in the luminal leaflet at steady state, although this was recently put under question (Fairn et al, 2011;Tsuji et al, 2019). Lipid scrambling would be another way to regulate the size of the PS pool available for transport by a cytosolic LTP.…”

“TMEM16K is an interorganelle regulator of endosomal sorting”

“…Our results are consistent with a model in which the TMEM16K phospholipid scrambling function could, upon TMEM16K activation by binding of the specific phosphatidylinositols and calcium 45 , selectively modulate the local lipid environment in the endoplasmic reticulum at these sites of contact. Distribution of phospholipids across the ER membrane is thought to be largely symmetrical, with the exception of phosphatidylserine 54,57 , and TMEM16K was shown to be required for its calcium-induced leaflet redistribution 54 Our study further raises the possibility that mammalian TMEM16 family proteins could have functional roles as interorganelle regulators. Indeed, mammalian TMEM16H was recently shown to regulate Ca 2+ signaling at the ER and plasma membrane MCS 63 Immunocytochemistry to evaluate subcellular localization of TMEM16K mutants to endoplasmic reticulum.…”

Phosphatidylserine biosynthesis pathways in lipid homeostasis: Toward resolution of the pending central issue for decades

“…In addition, the PS transport from the ER at the ER-TGN contact sites by ORP10 discussed in the previous section likely contributes to the PS population on the cytosolic side of the TGN. A recent report on the PS transbilayer distribution measured by freeze-fracture electron microscopy indicated different results 129 ; preferential enrichment of PS on the cytosolic side of the ER and Golgi was observed in mammalian cells. In the endocytic pathways from the plasma membrane, where the TGN is also involved, significant levels of PS exposure to the cytosolic side were observed for early endosomes, recycling endosomes, late endosomes, and lysosomes.…”