Abstract
Sterol regulatory element-binding proteins (SREBPs) are a family of membrane-bound transcription factors that regulate the uptake and synthesis of cholesterol and fatty acids in mammalian cells. SREBP cleavage-activating protein (SCAP) is an endoplasmic reticulum (ER) protein that binds newly synthesized SREBP, retaining it in the ER where SREBP is inactive. SCAP binds cholesterol and functions as the cholesterol sensor in this regulatory system. Under low cholesterol conditions, SCAP escorts SREBP from the ER to the Golgi apparatus where two proteases sequentially cleave and activate SREBP. Given their central importance in maintaining cellular lipid homeostasis, other mechanisms exist to regulate SREBP activity, such as control of protein synthesis and degradation. Here, we describe methods to assay ER-to-Golgi transport of SCAP in vitro using immunofluorescence microscopy and two different cell systems, Chinese hamster ovary (CHO) cells stably expressing hamster GFP-SCAP and human HeLa cells transiently expressing human GFP-SCAP. These methods will permit investigators to determine if cellular perturbations act by affecting the ER-to-Golgi transport of SCAP.
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Acknowledgments
We thank Drs. Michael Brown and Joseph Goldstein (University of Texas-Southwestern) for providing CHO/GFP-SCAP cells.
Financial Support
This work was supported by the National Institutes of Health (HL077588 and GM126088, PJE).
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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
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Ishida, C.T., Shao, W., Espenshade, P.J. (2023). Assaying Sterol-Regulated ER-to-Golgi Transport of SREBP Cleavage-Activating Protein Using Immunofluorescence Microscopy. In: Wang, Y., Lupashin, V.V., Graham, T.R. (eds) Golgi. Methods in Molecular Biology, vol 2557. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2639-9_45
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DOI: https://doi.org/10.1007/978-1-0716-2639-9_45
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