Abstract 2229 BLTP2 is a Vps13-like pro-survival protein that controls plasma membrane fluidity in human cancer cells

Non-vesicular lipid transport at membrane contact sites (MCS) is conducted by Lipid Transport Proteins (LTPs). In eukaryotes, LTPs were thought to operate by shuttling lipids between two membranes. Only recently, a class of giant (∼3000 amino acids) proteins, with structural similarity to Vps13/Atg2 were discovered that are thought to act as conduits between organelle-membranes, facilitating bulk lipid transport. These have been termed Bridge-like Lipid Transport Proteins (BLTPs). Mutations in each BLTP are linked to specific human diseases. However, the mechanisms and functions of most BLTPs remain enigmatic. We are studying BLTP2, which is overexpressed in breast cancer. We find that the BLTP2 gene is expressed 2.5-fold higher in the aggressive triple negative epithelial breast cancer cell line MDA-MB-231 than non-cancerous breast epithelial cell line MCF10A . Acute knockdown of BLTP2 significantly decreases the proliferation of MDA-MB-231 but not MCF10A. Silencing BLTP2 also increases apoptosis of MDA-MB-231 cells and limits their ability to metastasize in an in vivo model system. Because BLTP2 is conserved in eukaryotes, we used budding yeast, a genetically tractable model organism, to study the function of BLTP2. Fmp27 is a yeast homolog of BLTP2. We observed that yeast cells lacking FMP27 have a growth defect at low temperature, which was surprisingly rescued by upregulating PE production through the Kennedy pathway. We find that C. elegans mutants of BLTP2 also depend on an intact Kennedy pathway for their survival and fertility. Upregulating PtdEtn (PE):PtdCho (PC) ratio is one way yeast adapt to cold. Since Fmp27 localizes to ER-plasma membrane (PM) contact sites, we hypothesized it might regulate the PE:PC ratio of the PM. Indeed, using highly pure organelle-isolation, we found that PE:PC ratio drops dramatically in plasma membrane from cells lacking Fmp27, while the ratio remained unchanged in the ER. Maintaining membrane fluidity is essential for cold adaptation in yeast. One method by which eukaryotes maintain membrane fluidity is by altering their PE:PC ratio. Consistent with that, we observed that fmp27Δ has significantly altered plasma membrane fluidity, which was restored by upregulating PE biosynthesis. Our results suggest that yeast BLTP2 maintains plasma membrane fluidity by regulating PE transport from the ER to the PM. We assessed whether BLTP2 has a similar role in human cells. We found that deletion of BLTP2 (sgBLTP2) reduces the proliferation rate of HeLa cells. Moreover, cellular level of PE but not PC, is significantly reduced in sgBLTP2 and these cells have significantly altered plasma membrane fluidity. Like yeast cells lacking Fmp27, both the proliferation and membrane fluidity defects of sgBLTP2 cells are complimented by upregulating PE biosynthesis. Additionally, using lipidomics, we found that both yeast and cancer cells lacking BLTP2 does not alter the acyl chain composition of their phospholipids. This suggests that BLTP2 regulates PM fluidity by altering PE:PC ratio instead of changing acyl chain length or saturation. Furthermore, using DepMap search and subsequent characterization we observed that TLCD1, a protein known to regulate PE composition and PM fluidity in cells, synergizes with BLTP2 to regulate PM fluidity and cell proliferation. This suggested that human cancer cells have multiple mechanisms to control PM fluidity by regulating the phospholipid composition of their PM. This research was supported by NIH/NIDDK Intramural funding and by the UT Southwestern Medical Center.