Cholesterol is a major structural component of the plasma membrane (PM). The majority of PM cholesterol forms complexes with other PM lipids, making it inaccessible for intracellular transport. Transition of PM cholesterol between accessible and inaccessible pools maintains cellular homeostasis, but how cells monitor the accessibility of PM cholesterol remains unclear. We show that endoplasmic reticulum (ER)-anchored lipid transfer proteins, the GRAMD1s, sense and transport accessible PM cholesterol to the ER. GRAMD1s bind to one another and populate ER-PM contacts by sensing a transient expansion of the accessible pool of PM cholesterol via their GRAM domains. They then facilitate the transport of this cholesterol via their StART-like domains. Cells that lack all three GRAMD1s exhibit striking expansion of the accessible pool of PM cholesterol as a result of less efficient PM to ER transport of accessible cholesterol. Thus, GRAMD1s facilitate the movement of accessible PM cholesterol to the ER in order to counteract an acute increase of PM cholesterol, thereby activating non-vesicular cholesterol transport.

In Gram-negative bacteria, lipid asymmetry is critical for the function of the outer membrane (OM) as a selective permeability barrier, but how it is established and maintained is poorly understood. Here, we characterize a non-canonical ATP-binding cassette (ABC) transporter in Escherichia coli that provides energy for maintaining OM lipid asymmetry via the transport of aberrantly localized phospholipids (PLs) from the OM to the inner membrane (IM). We establish that the transporter comprises canonical components, MlaF and MlaE, and auxiliary proteins, MlaD and MlaB, of previously unknown functions. We further demonstrate that MlaD forms extremely stable hexamers within the complex, functions in substrate binding with strong affinity for PLs, and modulates ATP hydrolytic activity. In addition, MlaB plays critical roles in both the assembly and activity of the transporter. Our work provides mechanistic insights into how the MlaFEDB complex participates in ensuring active retrograde PL transport to maintain OM lipid asymmetry.DOI: http://dx.doi.org/10.7554/eLife.19042.001

The outer membrane (OM) of Gramnegative bacteria is a permeability barrier that impedes the entry of external insults, such as antibiotics and bile salts.

Cholesterol is essential for cell physiology. Transport of the "accessible" pool of cholesterol from the plasma membrane (PM) to the endoplasmic reticulum (ER) by ER-localized GRAMD1 proteins (GRAMD1a/1b/1c) contributes to cholesterol homeostasis. However, how cells detect accessible cholesterol within the PM remains unclear. We show that the GRAM domain of GRAMD1b, a coincidence detector for anionic lipids, including phosphatidylserine (PS), and cholesterol, possesses distinct but synergistic sites for sensing accessible cholesterol and anionic lipids. We find that a mutation within the GRAM domain of GRAMD1b that is associated with intellectual disability in humans specifically impairs cholesterol sensing. In addition, we identified another point mutation within this domain that enhances cholesterol sensitivity without altering its PS sensitivity. Cell-free reconstitution and cell-based assays revealed that the ability of the GRAM domain to sense accessible cholesterol regulates membrane tethering and determines the rate of cholesterol transport by GRAMD1b. Thus, cells detect the codistribution of accessible cholesterol and anionic lipids in the PM and fine-tune the non-vesicular transport of PM cholesterol to the ER via GRAMD1s.

The clinical effects, recovery characteristics, and costs of total intravenous anesthesia (TIVA), sevoflurane, and isoflurane anesthesia have been measured in various out-patient operations, but have not been evaluated in patients undergoing laminectomy or discectomy. In the current study, the authors assessed the hemodynamic characteristics, recovery, and cost analyzes after laminectomy and discectomy operations, comparing TIVA, sevoflurane, and isoflurane anesthesia. Sixty American Society of Anesthesiologists I and II patients were randomly divided into three groups, each consisting of 20 patients. Group I received propofol-alfentanil, Group 2 received sevoflurane-N2O, and Group 3 received isoflurane-N2O. At the end of surgery, the anesthetics were discontinued, and recovery from anesthesia was assessed by measuring the time until spontaneous eye opening and the time until response to verbal commands. The drug and delivery costs were calculated in United States dollars. No significant differences were found in the demographic data. Heart rate and mean arterial pressure decreased significantly after induction of anesthesia in the TIVA group, compared to the two other groups ( P < .05 for both comparisons). The fastest recovery was seen in the TIVA group. Incidences of postoperative nausea, vomiting, and pain were significantly reduced after TIVA ( P < .05 for both comparisons). Thus, TIVA patients required fewer additional drugs and showed the lowest additional costs in the post-anesthesia care unit. However, the total cost was significantly higher in the TIVA group than in the sevoflurane and isoflurane groups (52.73 dollars, 29.99 dollars, and 24.14 dollars, respectively) ( P < .05). Total intravenous anesthesia was associated with the highest intraoperative cost but provided the most rapid recovery from anesthesia, and the least frequent postoperative side effects.