The role of cholesterol in the association of endoplasmic reticulum membranes with mitochondria

Abstract

The unique endoplasmic reticulum (ER) subdomain termed the mitochondria-associated ER membrane (MAM) engages the physical connection between the ER and the mitochondrial outer membrane and plays a role in regulating IP(3) receptor-mediated Ca(2+) influx and the phospholipid transport between the two organelles. The MAM contains certain signaling and membrane-tethering proteins but also lipids including cholesterol. The biophysical role of lipids at the MAM, specifically in the physical interaction between the MAM of the ER and mitochondria, remains not totally clarified. Here we employed the in vitro membrane association assay to investigate the role of cholesterol in the association between MAMs and mitochondria. The purified MAMs and mitochondria were mixed in vitro in a test tube and then the physical association of the two subcellular organelles was quantified indirectly by measuring the presence of the MAM-specific protein sigma-1 receptors in the mitochondria fraction. Purified MAMs contained free cholesterol approximately 7 times higher than that in microsomes. We found that depletion of cholesterol in MAMs with methyl-β-cyclodextrin (MβC) significantly increases the association between MAMs and mitochondria, whereas MβC saturated with cholesterol does not change the association. (14)C-Serine pulse-labeling demonstrated that the treatment of living cells with MβC decreases the level of de novo synthesized (14)C-phosphatidylserine (PtSer) and concomitantly increases greatly the synthesis of (14)C-phosphatidylethanolamine (PtEt). Apparently, cholesterol depletion increased the PtSer transport from MAMs to mitochondria. Our findings suggest that cholesterol is an important substrate in regulating the association between MAMs of the ER and mitochondria.

Fig. 1. The in vitro MAM-mitochondria association assay

A. MAM and mitochondrial membranes from CHO cells. P1, nuclear; Mito, mitochondrial; P3, microsomal; Cyt, cytosolic fractions. Sig-1R, sigma-1 receptor; IP3R3, IP₃ receptor type-3; CRT, calreticulin. Levels of respective organelle markers were measured by immunoblotting. Note that ER chaperones involved in the ER-to-Golgi vesicle transport (i.e., BiP, CRT) are also detected in the cytoplasmic fraction. B. In vitro association of MAMs with purified mitochondria. MAMs and mitochondria membranes were prepared from CHO cells or rat livers. In the MAM(+)-Mito(+) sample, the two membranes were incubated together for 30 min. Pellets and supernatants (SNT) were obtained by a centrifugation at 6,300g. The level of MAM-enriched protein sigma-1 receptors (Sig-1R) was measured by immunoblotting.

Fig. 2. Depletion of cholesterol in MAMs with MβC

A. The level of free cholesterol in isolated mitochondria, MAMs, and microsomal membranes. N=3. Mean±SEM. B. Time-dependent effect of MβC in depleting membrane cholesterol. The crude mitochondrial fraction (containing both mitochondria and MAMs) were incubated with 5 mM of M®C at 37 or 4 °C for indicated periods of time.

Fig. 3. Effect of cholesterol depletion on the association between MAMs and mitochondria

MAMs were incubated with MβC or MβC-Chol at 37 °C for 30 min. The MAM-mitochondria association assay was then performed as described in Figure 1. Mito, mitochondria; SNT, supernatant. Sigma-1 receptors (Sig-1R) were measured by immunoblotting.

Fig. 4. Effect of cholesterol depletion on the de novo biosynthesis of PtSer and PtEt in CHO cells

CHO cells were treated with 5 mM of MβC or MβC-Chol for 2 h prior to ¹⁴C-serine pulse-labeling for 1 h. Total lipids were extracted and visualized by HPTLC followed by autoradiography. SM, sphingomyelin.