Reconstitution Assay System for Ceramide Transport With Semi-Intact Cells

doi:10.1016/B978-0-12-386487-1.00006-7

Methods in Cell Biology

Volume 108, 2012, Pages 117-129

https://doi.org/10.1016/B978-0-12-386487-1.00006-7 Get rights and content

Abstract

The intracellular transport of lipids from the sites of their synthesis to their appropriate destination is a critical step for lipid metabolism. One well-defined inter-organelle lipid movement is the transport of ceramide by ceramide transport protein (CERT). Ceramide, a key intermediate for both sphingomyelin and glycosphingolipids, is synthesized at the endoplasmic reticulum and delivered to the Golgi apparatus to be converted to sphingomyelin. CERT delivers ceramide from the ER to the Golgi apparatus in a non-vesicular and ATP-dependent manner. This chapter describes a reconstitution assay system for ceramide transport with semi-intact cells, which is useful for the study of the CERT-mediated inter-organelle transport of ceramide.

Introduction

The intracellular transport of lipids from the sites of their synthesis to their appropriate destination is a critical step for lipid metabolism, because various steps of lipid biosynthesis occur in different intracellular compartments (van Meer et al., 2008). The cellular lipids are transported by vesicular and non-vesicular mechanisms, however, little is known about the molecular machinery for the inter-organelle movement of lipids. One well-defined inter-organelle lipid movement is the transport of ceramide in sphingomyelin (SM) biosynthesis. Ceramide, a key intermediate for both SM and glycosphingolipids, is synthesized at the cytosolic face of the endoplasmic reticulum (ER) (Futerman and Riezman, 2005, Mandon et al., 1992). It is then delivered to the luminal side of the Golgi apparatus to be converted to SM by SM synthase 1, which catalyzes the transfer of phosphocholine from phosphatidylcholine to ceramide (Huitema et al., 2004, Yamaoka et al., 2004). Ceramide transport protein (CERT) delivers ceramide from the ER to the Golgi apparatus in a non-vesicular and ATP-dependent manner (Hanada et al., 2009). In this chapter, we describe a reconstitution assay system for ceramide transport with semi-intact cells, which is useful for the study of the CERT-mediated inter-organelle transport of ceramide (Funakoshi et al., 2000, Hanada et al., 2009).

The reconstitution assay system is outlined in Fig. 1. LY-A, a CHO-K1-derived cell line, is deficient in ceramide transport activity due to a mutation converting glycine 67 to glutamic acid in CERT cDNA (Fukasawa et al., 1999, Hanada et al., 1998, Hanada et al., 2003). Perforated cells are prepared by scraping adhered LY-A cells under hypotonic conditions. Cytosolic factors leak from the small pores formed at the plasma membrane while other cellular structures and the localization of various membrane-bound enzymes are maintained (Beckers et al., 1987). When [³H]sphingosine is added to the perforated cells, [³H]ceramide is synthesized and accumulated in the ER membrane where most of the ceramide synthases exist (Mizutani et al., 2005, Riebeling et al., 2003, Venkataraman et al., 2002). It is important to keep the temperature below 15 °C at this step to suppress ceramide transport from the ER. Then, exogenous CERT diluted in the cytosol fraction of LY-A is added to the perforated cells. Because some yet to be identified cytosolic components seem necessary for efficient ATP-dependent ceramide transport, the cytosol fraction of LY-A, of which endogenous CERT is deficient, is used for the reconstitution system when the activity of exogenous CERT is examined (Fig. 2). When the perforated cells are added to the cytosol fraction including functional CERT, [³H]ceramide is transported from the ER to the Golgi apparatus to synthesize [³H]SM in an ATP-dependent manner (Fukasawa et al., 1999, Funakoshi et al., 2000). The cellular lipids are extracted and analyzed by thin layer chromatography (TLC) (Fig. 3). Ceramide transport activity is estimated by quantifying [³H]SM with an image analyzer.

The materials and methods for the assay of the activity of ER-to-Golgi transport of ceramide of purified recombinant CERT are described below.

Section snippets

Materials

All chemicals of reagent grade or better are used. Water purified with Milli-Q (Millipore) is used.

1.
ES medium (Nissui Pharmaceutical Co., Tokyo, Japan), prepared just before use (see Subheading III B 2 and Note 1).
2.
Ham's F-12 medium (Gibco), stored at 4 °C
3.
HEPES-NaOH (pH 7.4), sterilized by filtration (0.22 μm filter), aseptically stored at room temperature at a concentration of 1 M.
4.
Ten percent NaHCO₃, sterilized by filtration (0.22 μm filter), aseptically stored at room temperature.
5.
10 ×

Cell Culture

1.
CHO-K1 is a permanent cell line derived from Chinese hamster ovary. LY-A, a CHO-K1-derived mutant cell line, is deficient in ceramide transport activity due to a mutation converting glycine 67 to glutamic acid in CERT cDNA (Fukasawa et al., 1999, Hanada et al., 1998, Hanada et al., 2003). The LY-A cell line is available from RIKEN Cell Bank (http://www.brc.riken.jp/lab/cell/english/).
2.
LY-A cells are routinely maintained in F-12 culture medium (Ham's F-12 medium supplemented with 10% newborn

Notes

1.
ES medium is a modified autoclavable Eagle's minimum essential medium enriched with amino acids, sodium pyruvate, and vitamin B₁₂ to facilitate cell proliferation and to reduce serum consumption (Koyama and Kodama, 1982). ES medium of Nissui Pharmaceutical Co. contains kanamycin, an autoclavable anti-bacterial drug. The three lids of the spinner flask are capped with pieces of aluminum foil. Make sure that at least one of the lids keeps loosened during autoclaving, cooling, and cell culturing

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Chapter 6 - Reconstitution Assay System for Ceramide Transport With Semi-Intact Cells

Abstract

Introduction

Section snippets

Materials

Cell Culture

Notes

Arch. Biochem. Biophys.

Cell

Cell

J. Biol. Chem.

Trends Cell Biol.

Anal. Biochem.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.