Summary Water-soluble Niemann-Pick C2 (NPC2) and membrane-bound NPC1 are cholesterol-binding lysosomal proteins required for export of lipoprotein-derived cholesterol from lysosomes. The binding site in NPC1 is located in its N-terminal domain (NTD), which projects into the lysosomal lumen. Here, we perform alanine-scanning mutagenesis to identify residues in NPC2 that are essential for transfer of cholesterol to NPC1(NTD). Transfer requires three residues that form a patch on the surface of NPC2. We previously identified a patch of residues on the surface of NPC1(NTD) that are required for transfer. We present a model in which these two surface patches on NPC2 and NPC1(NTD) interact, thereby opening an entry pore on NPC1(NTD) and allowing cholesterol to transfer without passing through the water phase. We refer to this transfer as a hydrophobic handoff and hypothesize that this handoff is essential for cholesterol export from lysosomes.

The endoplasmic reticulum (ER)4 protein Scap is unique in nature because it serves as a cholesterol sensor that ensures the proper amount of cholesterol in membranes of animal cells (1, 2). The function of Scap derives from its ability to mediate the regulated transport of sterol regulatory element-binding proteins (SREBPs) from ER to Golgi. SREBPs are a family of three transcription factors that activate all of the genes necessary to produce cholesterol, fatty acids, and triglycerides (3). The SREBPs are synthesized as intrinsic transmembrane proteins of the ER. Immediately after their synthesis, the SREBPs bind to Scap, which serves as the nidus for incorporation into COPIIcoated vesicles, which bud from the ER and travel to the Golgi. There the SREBPs are processed sequentially by two proteases, thereby releasing the active transcriptional fragments that travel to the nucleus. When cholesterol accumulates in ER membranes, Scap binds the cholesterol and undergoes a conformational change that causes it to bind to Insig, an ER-resident protein (4). As a result of the conformational change and its stabilization by Insig (5), the Scap-SREBP complex is no longer incorporated into budding vesicles, and the active fragment cannot reach the nucleus. As a result, synthesis of cholesterol and fatty acids declines.The 1276 amino acids of Scap can be divided into two functional regions (see Fig. 1). The COOH-terminal domain of ϳ540 amino acids extends into the cytosol. It contains at least four WD repeat sequences that mediate its binding to SREBPs. The NH 2 -terminal region of ϳ735 amino acids is the membrane attachment domain. It contains eight ␣-helices separated by hydrophilic loops (6). Three of the loops (Loops 1, 6, and 7) are long enough to have significant structure. Helices 2-6 contain the Insig binding site (7,8). Loop 6, which faces the cytosol, contains the hexapeptide sequence MELADL, which serves as the binding site for the COPII proteins that cluster the Scap-SREBP complex into COPII-coated vesicles that bud from ER membranes (2, 9). When the cholesterol content of ER membranes exceeds a sharp threshold of 4 -5% of total lipids, the cholesterol binds to the membrane region of Scap (10), and this elicits a conformational change in Loop 6 that can be monitored by a protease protection assay (11). The change is reflected by the exposure of a novel arginine (Arg 505 ) to cleavage by trypsin (Fig. 1).The cholesterol-induced conformational change in Loop 6 causes the MELADL sequence to become inaccessible to COPII proteins, thereby precluding transport to the Golgi (2). Although the conformational change does not require Insig, binding to Insig stabilizes the inactive conformation, thereby lowering the threshold for cholesterol (10).Our previous cholesterol-binding studies were performed with a recombinant form of Scap that contained the entire membrane attachment domain (TM1-8) (1,12). Within this domain, the precise site of cholesterol binding was not established. In the current study, we localize the choleste...

Background: Scap controls cholesterol by transporting SREBPs from ER to Golgi in a sterol-sensitive fashion. Two luminal loops of Scap (Loops 1 and 7) may play a regulatory role. Results: A point mutation in Scap Loop 7 prevents interaction with Loop 1 and abolishes ER exit. Conclusion: Scap movement and activation of SREBPs require interaction between Loops 1 and 7. Significance: This work provides insight into how cells control cholesterol.

Insulin resistance and type 2 diabetes mellitus (T2DM) result from an inability to efficiently store and catabolize surplus energy in adipose tissue. Subcutaneous adipocytes protect against insulin resistance and T2DM by coupling differentiation with the induction of brown fat gene programs for efficient energy metabolism. Mechanisms that disrupt these programs in adipocytes are currently poorly defined, but represent therapeutic targets for the treatment of T2DM. To gain insight into these mechanisms, we performed a high-throughput microscopy screen that identified ubiquitin carrier protein 9 (Ubc9) as a negative regulator of energy storage in human sc adipocytes. Ubc9 depletion enhanced energy storage and induced the brown fat gene program in human sc adipocytes. Induction of adipocyte differentiation resulted in decreased Ubc9 expression commensurate with increased brown fat gene expression. Thiazolidinedione treatment reduced the interaction between Ubc9 and peroxisome proliferator-activated receptor (PPAR)γ, suggesting a mechanism by which Ubc9 represses PPARγ activity. In support of this hypothesis, Ubc9 overexpression remodeled energy metabolism in human sc adipocytes by selectively inhibiting brown adipocyte-specific function. Further, Ubc9 overexpression decreased uncoupling protein 1 expression by disrupting PPARγ binding at a critical uncoupling protein 1 enhancer region. Last, Ubc9 is significantly elevated in sc adipose tissue isolated from mouse models of insulin resistance as well as diabetic and insulin-resistant humans. Taken together, our findings demonstrate a critical role for Ubc9 in the regulation of sc adipocyte energy homeostasis.

Steroid receptor coactivator 1 (SRC-1) drives diverse gene expression programs necessary for the dynamic regulation of cancer metastasis, inflammation and gluconeogenesis, pointing to its overlapping roles as an oncoprotein and integrator of cell metabolic programs. Nutrient utilization has been intensely studied with regard to cellular adaptation in both cancer and noncancerous cells. Nonproliferating cells consume glucose through the citric acid cycle to generate NADH to fuel ATP generation via mitochondrial oxidative phosphorylation. In contrast, cancer cells undergo metabolic reprogramming to support rapid proliferation. To generate lipids, nucleotides, and proteins necessary for cell division, most tumors switch from oxidative phosphorylation to glycolysis, a phenomenon known as the Warburg Effect. Because SRC-1 is a key coactivator responsible for driving a hepatic gluconeogenic program under fasting conditions, we asked whether SRC-1 responds to alterations in nutrient availability to allow for adaptive metabolism. Here we show SRC-1 is stabilized by the 26S proteasome in the absence of glucose. RNA profiling was used to examine the effects of SRC-1 perturbation on gene expression in the absence or presence of glucose, revealing that SRC-1 affects the expression of complex I of the mitochondrial electron transport chain, a set of enzymes responsible for the conversion of NADH to NAD(+). NAD(+) and NADH were subsequently identified as metabolites that underlie SRC-1's response to glucose deprivation. Knockdown of SRC-1 in glycolytic cancer cells abrogated their ability to grow in the absence of glucose consistent with SRC-1's role in promoting cellular adaptation to reduced glucose availability.