The function of the fatty acyl-CoA synthetase ACSL3 in the dynamic metabolism of lipid droplets
Final Report Abstract
ACSL3 is an efficient long chain fatty acyl-CoA synthetase isoenzyme, activating fatty acids for lipid metabolism. It appears unique among the larger enzyme family of fatty acid CoA ligases in that it shows a dual localization to the endoplasmic reticulum (ER) and to lipid droplets (LDs). Previously, we also showed that ACSL3 translocates rapidly from the ER to newly emerging LDs in response to neutral lipid synthesis. The enzyme activity of ACSL3 and related proteins may be precisely determined from minute amounts of material by an in vitro assay. A robust and broadly applicable protocol was developed. The N-terminus of ACSL3 contains all the necessary sorting information for targeting to LDs, and has served as a sensitive membrane marker for lipid droplets in several applications including live microscopy. Therefore, cDNA fusions with fluorescent proteins have been recently renamed to "lipotracker". The expression of the corresponding protein was also instrumental in establishing a novel LD quantification method based on iterative image preprocessing. Fatty acids activated by ACSL3 may be reduced by peroxisomal fatty acyl-CoA reductases (Far enzymes). We found that human Far1 features a dual membrane topology enabling it to also localize to lipid droplets. This was a very surprising finding, suggesting a close functional relationship between peroxisomes and LDs. ACSL3 is the dominantly expressed acyl-CoA synthase in several mammalian tissue culture cell models like A431 or COS-7. Depletion of ACSL3 reduced fatty acid uptake and metabolism, suggesting that ACSL3 regulates cellular fatty acid transport indirectly by metabolic trapping. The molecular stoichiometry of ACSL3 in A431 cells was analyzed in detail to determine molecule numbers/ cell, synthesis of fatty acyl-CoA molecules/ s, triglyceride synthesis over time, LD number and volume gain after fatty acid supplementation. ACSL3 was nine-fold concentrated on LDs compared to the ER, but the majority of ACSL3 was nevertheless still localized to the ER. Taken together, there was not sufficient ACSL3 on LDs to account for the observed synthesis of triglycerides and the corresponding growth of LDs. In conclusion, LD growth requires substantial transport of neutral lipids from the ER, and cannot be explained solely by the localized biosynthesis at the surface of LDs. This study therefore contributes to a paradigm shift regarding LD growth.
Publications
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(2016). Measurement of long chain fatty acyl-CoA synthetase activity. Meth Mol Biol 1376, 43-53
Füllekrug, J. and M. Poppelreuther
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(2018). The metabolic capacity of lipid droplet localized acyl-CoA synthetase 3 is not sufficient to support local triglyceride synthesis independent of the endoplasmic reticulum in A431 cells. BBA Mol Biol Cell Lipids 1863(6), 614-624
Poppelreuther, M., Sander, S., Minden, F., Dietz, M.S., Exner, T., Du, C., Zhang, I., Ehehalt, F., Knüppel, L., Domschke, S., Badenhop, A., Staudacher, S., Ehehalt, R., Stremmel, W., Thiele, C., Heilemann, M. and J. Füllekrug
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(2019). An alternative membrane topology permits lipid droplet localization of peroxisomal fatty acyl-CoA reductase 1. J Cell Sci 132(6)
Exner, T., Romero-Brey, I., Yifrach, E., Rivera-Monroy, J., Schrul, B., Zouboulis, C.C., Stremmel, W., Honsho, M., Bartenschlager, R., Zalckvar, E., Poppelreuther, M. and J. Füllekrug
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(2019). Lipid droplet quantification based on iterative image processing. J Lipid Res.60(7), 1333-1344
Exner, T., Beretta, C., Gao, Q., Afting, C., Romero-Brey, I., Bartenschlager, R., Fehring, L., Poppelreuther and J. Füllekrug
