Orm1 and Orm2 are conserved endoplasmic reticulum membrane proteins regulating lipid homeostasis and protein quality control

Abstract

Yeast members of the ORMDL family of endoplasmic reticulum (ER) membrane proteins play a central role in lipid homeostasis and protein quality control. In the absence of yeast Orm1 and Orm2, accumulation of long chain base, a sphingolipid precursor, suggests dysregulation of sphingolipid synthesis. Physical interaction between Orm1 and Orm2 and serine palmitoyltransferase, responsible for the first committed step in sphingolipid synthesis, further supports a role for the Orm proteins in regulating sphingolipid synthesis. Phospholipid homeostasis is also affected in orm1Delta orm2Delta cells: the cells are inositol auxotrophs with impaired transcriptional regulation of genes encoding phospholipid biosynthesis enzymes. Strikingly, impaired growth of orm1Delta orm2Delta cells is associated with constitutive unfolded protein response, sensitivity to stress, and slow ER-to-Golgi transport. Inhibition of sphingolipid synthesis suppresses orm1Delta orm2Delta phenotypes, including ER stress, suggesting that disrupted sphingolipid homeostasis accounts for pleiotropic phenotypes. Thus, the yeast Orm proteins control membrane biogenesis by coordinating lipid homeostasis with protein quality control.

Fig. 1.

Stress response upon loss of Orm1 and Orm2. (A) Loss of Orm1 and Orm2 causes sensitivity to stress. Cells were normalized to 0.1 OD₆₀₀/mL and spotted onto plates containing 1 μg/mL tunicamycin in SC medium with 2% glucose, and incubated for 2 to 3 days at 30 °C. Tunicamycin sensitivity of orm1Δ orm2Δ cells is complemented by ORM2 expressed from a centromeric plasmid (pSH15) and suppressed by high-copy ORM1 (pSH16). For overexpression of CPY*, cells bearing vector or pGAL-CPY* (pES67) were spotted onto plates with 2% glucose or 2% galactose. (B) UPR is induced constitutively in orm1Δ orm2Δ cells. Wild-type (HXX1-7C) and orm1Δ orm2Δ (HXX1-7D) cells bearing a UPRE-lacZ reporter (pJC104) were incubated with or without 5 mM DTT for 1 h at 30 °C. Cell lysate was prepared and β-galactosidase assays were performed as previously described (45). Assays were performed in duplicate on at least three independent colonies. (C) Orm2 is increased in response to stress. Exponentially growing cells were incubated with or without tunicamycin (1 μg/mL), DTT (2 mM), or diamide (2 mM) for 1 h and 2 h at 30 °C in SC medium. Samples were normalized to lysate protein, and TAP-tagged Orm2 was analyzed by Western blot.

Fig. 2.

Suppression of ormΔ1 orm2Δ phenotypes by myriocin. (A) Suppression of orm1Δ orm2Δ cells by low-dose myriocin. Serial dilutions of cells were spotted onto SC medium with myriocin (0.05 μg/mL). Plates with or without tunicamyin (1 μg/mL) and myriocin were incubated at 30 °C for 2 days. To test cold sensitivity, cells were incubated for 5 days at 14 °C. (B) Slow ER-to-Golgi transport is suppressed by myriocin. Myriocin (1 μg/mL) was added 3 h before pulse-labeling as described in Fig. S2 legend.

Fig. 3.

Perturbation in sphingolipid homeostasis in orm1Δ orm2Δ cells. (A) Long chain base was quantitated by mass spectrometry in wild-type and single orm mutants, orm1Δ orm2Δ, and lcb3Δ orm1Δ orm2Δ cells, as described in Methods. Myriocin (0.5 μg/mL) was added to orm1Δ orm2Δ cells for 2 h. (B) Hypersensitivity of orm1Δ orm2Δ cells to exogenous PHS and suppression by high-copy RSB1. Cells with vector or RSB1 on a 2-μ plasmid were serially diluted and spotted on plates with SC medium with or without PHS (10 μM). (C) Rsb1 expression is increased in the absence of Orm1 and Orm2. rsb1Δ (ACX161-4C) and rsb1Δ orm1Δ orm2Δ (ACX161-4D) cells bearing a centromeric plasmid with RSB1-HA were analyzed by Western blot with anti-HA antibody. Brackets indicate glycosylated Rsb1, as described previously (16).

Fig. 4.

Co-IP of Orm proteins and SPT subunits. Exponentially growing cells in SC medium were harvested, and lysate was prepared and incubated with IgG-Sepharose under nondenaturing conditions. IPs were analyzed by Western blot with anti-rabbit to detect Orm2-TAP, and anti-HA or anti-myc antibodies. (A) Lysate was prepared from wild-type (untagged ORMs) and cells with chromosomal TAP-tagged ORM1 (SHY53) and ORM2-TAP bearing a centromeric plasmid with HA-LCB1. Pull-downs were analyzed by Western blot with anti-HA antibody to detect Lcb1. (B) Lysate was prepared from wild-type and TAP-ORM2 cells bearing a centromeric plasmid with myc-ERG11. After Orm2-TAP pull-down, anti-myc antibody was used to detect Erg11 as a negative control, and Orm2 was detected by rabbit secondary antibody. (C) Lysate was prepared from wild-type and ORM2-TAP cells bearing two plasmids with myc-tagged LCB1 and HA-LCB2. After Orm2-TAP pull-down, Western blots with anti-HA and anti-myc were used to detect HA-Lcb2 and myc-Lcb1, respectively. Efficiency of co-IP is similar for HA-Lcb2 and myc-Lcb1. (D) Lysate was prepared from wild-type, ORM2-TAP, and ORM2-TAP lcb2Δ (SHY54) bearing a centromeric plasmid with HA-tagged LCB1. After Orm2-TAP pull-down, anti-HA antibody was used to detect HA-Lcb1.

Fig. 5.

Inositol auxotrophy of orm1Δ orm2Δ cells. (A) Ino^- phenotype of orm1Δ orm2Δ cells is suppressed by overexpression of INO1, DGK1, and SCS2. Cells were serially diluted and spotted onto plates with SC medium with or without inositol at 30 °C. (B) Induction of INO1-lacZ is impaired in orm1Δ orm2Δ cells. Wild-type (HXX1-2A), orm2Δ (HXX1-7A), and orm1Δ orm2Δ (HXX1- 7D) cells bearing an INO1-lacZ reporter were grown at 25 °C in SC-uracil medium. Cells were washed and resuspended in fresh medium without inositol with or without myriocin (0.5 μg/mL) for 3 h. Lysate was prepared for β-galactosidase activity measurement. Assays were performed in duplicate on at least three independent colonies.