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Published on: September 15, 2023
Human TMEM30a promotes uptake of antitumor and bioactive choline phospholipids into mammalian cells
Rui Chen1, Erin Brady, Thomas M McIntyre
1Department of Cell Biology, Cleveland Clinic, Cleveland, OH 44195, USA.
Abstract:
Antitumor alkylphospholipids initiate apoptosis in transformed HL-60 and Jurkat cells while sparing their progenitors. 1-O-Alkyl-2-carboxymethyl-sn-glycero-3-phosphocholine (Edelfosine) like other short-chained phospholipids--inflammatory platelet-activating factor (PAF) and apoptotic oxidatively truncated phospholipids--are proposed to have intracellular sites of action, yet a conduit for these choline phospholipids into mammalian cells is undefined. Edelfosine is also accumulated by Saccharomyces cerevisiae in a process requiring the membrane protein Lem3p, and the human genome contains a Lem3p homolog TMEM30a. We show that import of choline phospholipids into S. cerevisiae ΔLem3 is partially reconstituted by human TMEM30a and by Lem3p-TMEM30a chimeras, showing the proteins are orthologous. TMEM30a-GFP chimeras expressed in mammalian cells localized in plasma membranes, as well as internal organelles, and ectopic TMEM30a expression promoted uptake of exogenous choline and ethanolamine phospholipids. Short hairpin RNA knockdown of TMEM30a reduced fluorescent choline phospholipid and [(3)H]PAF import. This knockdown also reduced mitochondrial depolarization from exogenous Edelfosine or the mitotoxic oxidatively truncated phospholipid azelaoyl phosphatidylcholine, and the knockdown reduced apoptosis in response to these two phospholipids. These results show that extracellular choline phospholipids with short sn-2 residues can have intracellular roles and sites of metabolism because they are transport substrates for a TMEM30a phospholipid import system. Variation in this mechanism could limit sensitivity to short chain choline phospholipids such as Edelfosine, PAF, and proapoptotic phospholipids.
Insights
Antitumor alkylphospholipids like Edelfosine induce apoptosis. The TMEM30a protein facilitates the import of these choline phospholipids into cells, impacting their antitumor activity and cellular response.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Antitumor alkylphospholipids, such as Edelfosine, induce apoptosis in cancer cells but their cellular uptake mechanism is unclear.
- Platelet-activating factor (PAF) and other phospholipids also exhibit intracellular activity, necessitating a transport system for cellular entry.
Purpose of the Study:
- To identify the cellular mechanism responsible for the uptake of short-chained choline phospholipids into mammalian cells.
- To investigate the role of the TMEM30a protein in phospholipid import and its contribution to Edelfosine-induced apoptosis.
Main Methods:
- Functional analysis of TMEM30a and its homolog Lem3p in yeast (Saccharomyces cerevisiae) for choline phospholipid import.
- Expression of TMEM30a-GFP chimeras in mammalian cells to determine localization and function.
- Short hairpin RNA (shRNA) knockdown of TMEM30a to assess its impact on phospholipid uptake and apoptosis.
Main Results:
- Human TMEM30a and yeast Lem3p are orthologous proteins that facilitate choline phospholipid import.
- TMEM30a localizes to plasma membranes and internal organelles in mammalian cells, promoting the uptake of exogenous phospholipids.
- TMEM30a knockdown reduced the uptake of choline phospholipids and [(3)H]PAF, decreased mitochondrial depolarization, and inhibited Edelfosine- and azelaoyl phosphatidylcholine-induced apoptosis.
Conclusions:
- Extracellular choline phospholipids with short sn-2 residues are transported into cells via the TMEM30a phospholipid import system, enabling intracellular functions.
- TMEM30a-mediated transport is crucial for the cytotoxic effects of short-chained phospholipids like Edelfosine and PAF.
- Variations in TMEM30a function may influence cellular sensitivity to these important bioactive phospholipids.
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