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Sterol-regulated transport of SREBPs from endoplasmic reticulum to Golgi: Insig renders sorting signal in Scap inaccessible to COPII proteins.

ABSTRACT: Two classes of sterols, cholesterol and oxysterols, block export of sterol regulatory element-binding proteins (SREBPs) from the endoplasmic reticulum (ER) to the Golgi by preventing the binding of COPII-coated proteins to a hexapeptide sorting signal (MELADL) in Scap, the SREBP-escort protein. Here, we show that anti-MELADL blocks COPII binding in vitro, and microinjection of Fab anti-MELADL blocks Scap.SREBP movement in cells. Cholesterol and oxysterols block COPII binding to MELADL by binding to different intracellular receptors, cholesterol to Scap and oxysterols to Insig. Cysteine labeling shows that both binding events produce a conformational change near the MELADL sequence, abrogating COPII binding but not anti-MELADL binding. Mutagenesis experiments raise the possibility that the distance of MELADL from the ER membrane is crucial for COPII binding, and we speculate that sterols and Insig block SREBP transport by altering the location of MELADL with respect to the membrane, rendering it inaccessible to COPII proteins.

SUBMITTER: Sun LP

PROVIDER: S-EPMC1851663 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Sterol-regulated transport of SREBPs from endoplasmic reticulum to Golgi: oxysterols block transport by binding to Insig.

Project description:Cholesterol synthesis in animals is controlled by the regulated transport of sterol regulatory element-binding proteins (SREBPs) from the endoplasmic reticulum to the Golgi, where the transcription factors are processed proteolytically to release active fragments. Transport is inhibited by either cholesterol or oxysterols, blocking cholesterol synthesis. Cholesterol acts by binding to the SREBP-escort protein Scap, thereby causing Scap to bind to anchor proteins called Insigs. Here, we show that oxysterols act by binding to Insigs, causing Insigs to bind to Scap. Mutational analysis of the six transmembrane helices of Insigs reveals that the third and fourth are important for Insig's binding to oxysterols and to Scap. These studies define Insigs as oxysterol-binding proteins, explaining the long-known ability of oxysterols to inhibit cholesterol synthesis in animal cells.

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Insig-2, a second endoplasmic reticulum protein that binds SCAP and blocks export of sterol regulatory element-binding proteins.

Project description:This paper describes insig-2, a second protein of the endoplasmic reticulum that blocks the processing of sterol regulatory element-binding proteins (SREBPs) by binding to SCAP (SREBP cleavage-activating protein) in a sterol-regulated fashion, thus preventing it from escorting SREBPs to the Golgi. By blocking this movement, insig-2, like the previously described insig-1, prevents the proteolytic processing of SREBPs by Golgi enzymes, thereby blocking cholesterol synthesis. The sequences of human insig-1 and -2 are 59% identical. Both proteins are predicted to contain six transmembrane helices. The proteins differ functionally in two respects: (i) production of insig-1, but not insig-2, in cultured mammalian cells requires nuclear SREBPs; and (ii) at high levels of expression, insig-1, but not insig-2, can block SCAP movement in the absence of exogenous sterols. The combined actions of insig-1 and -2 permit feedback regulation of cholesterol synthesis over a wide range of sterol concentrations.

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Sterols block binding of COPII proteins to SCAP, thereby controlling SCAP sorting in ER.

Project description:Sterols inhibit their own synthesis in mammalian cells by blocking the vesicular endoplasmic reticulum-to-Golgi transport of sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP), a sterol-sensing protein that escorts SREBPs. Unable to reach the Golgi, SREBPs are not processed by Golgi-resident proteases, and they fail to activate genes required for cholesterol synthesis. The current studies were designed to reveal whether sterols block SCAP movement by inhibiting synthesis of special vesicles dedicated to SCAP, or whether sterols block SCAP incorporation into common coat protein (COP)II-coated vesicles. Through immunoisolation, we show that SCAP-containing vesicles, formed in vitro, also contain vesicular stomatitis virus glycoprotein (VSVG) protein, a classic marker of COPII-coated vesicles. Sterols selectively block incorporation of SCAP into these vesicles without blocking incorporation of VSVG protein. We show that the mammalian vesicular budding reaction can be reconstituted by recombinant yeast COPII proteins that support incorporation of SCAP as well as VSVG into vesicles. Sterols block SCAP incorporation into vesicles by blocking Sar1-dependent binding of the COPII proteins Sec 23/24 to SCAP. These studies demonstrate feedback control of a biosynthetic pathway by the regulated binding of COPII proteins to an endoplasmic reticulum-to-Golgi transport protein.

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HSPA8 dampens SCAP/INSIG split and SREBP activation by reducing PKR-mediated INSIG phosphorylation

Project description:Lipid accumulation in the renal tubules is a major determinant of diabetic kidney disease (DKD), and activation of SREBPs plays a central role in this process. Our study aimed to explore whether HSPA8, a molecular chaperone, is the master regulator of INSIG/SREBPs function in DKD. Here, we showed that tubular epithelial cell (TEC)-specific knockout of HSPA8 upregulated the phosphorylation of INSIG1 and INSIG2, which disrupted the interaction between INSIG proteins and SCAP, leading to the translocation of the SCAP-SREBP complex to the Golgi apparatus, and the activation of SREBPs. TEC-specific overexpression of HSPA8 restrained these changes. INSIG1 and INSIG2 can be phosphorylated by protein kinase R (PKR), while HSPA8 can recognize PKR and recruit the E3 ubiquitin ligase (CHIP) to promote PKR ubiquitination and degradation. SREBP1 transcriptionally activated HSPA8 expression under temporary hyperglycemic stimulation. However, persistent hyperglycemia reduced HSPA8 levels by promoting nuclear factor (NF)-κB-mediated transcriptional inhibition and reducing USP43-mediated deubiquitination of HSPA8. Collectively, these findings indicate that the molecular chaperone HSPA8 serves as a negative feedback regulator of SREBPs, lipogenesis, and DKD development.

2024-12-12 | GSE283754 | GEO

Epithelial SCAP/INSIG/SREBP signaling regulates multiple biological processes during perinatal lung maturation.

Project description:Pulmonary surfactant is required for lung function at birth and throughout postnatal life. Defects in the surfactant system are associated with common pulmonary disorders including neonatal respiratory distress syndrome and acute respiratory distress syndrome in children and adults. Lipogenesis is essential for the synthesis of pulmonary surfactant by type II epithelial cells lining the alveoli. This study sought to identify the role of pulmonary epithelial SREBP, a transcriptional regulator of cellular lipid homeostasis, during a critical time period of perinatal lung maturation in the mouse. Genome wide mRNA expression profiling of lung tissue from transgenic mice with epithelial-specific deletions of Scap (Scap(Δ/Δ), resulting in inactivation of SREBP signaling) or Insig1 and Insig2 (Insig1/2(Δ/Δ), resulting in activation of SREBP signaling) was assessed. Differentially expressed genes responding to SREBP perturbations were identified and subjected to functional enrichment analysis, pathway mapping and literature mining to predict upstream regulators and transcriptional networks regulating surfactant lipid homeostasis. Through comprehensive data analysis and integration, time dependent effects of epithelial SCAP/INSIG/SREBP deletion and defined SCAP/INSIG/SREBP-associated genes, bioprocesses and downstream pathways were identified. SREBP signaling influences epithelial development, cell death and cell proliferation at E17.5, while primarily influencing surfactant physiology, lipid/sterol synthesis, and phospholipid transport after birth. SREBP signaling integrated with the Wnt/β-catenin and glucocorticoid receptor signaling pathways during perinatal lung maturation. SREBP regulates perinatal lung lipogenesis and maturation through multiple mechanisms by interactions with distinct sets of regulatory partners.

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PAQR3 Regulates Endoplasmic Reticulum-to-Golgi Trafficking of COPII Vesicle via Interaction with Sec13/Sec31 Coat Proteins.

Project description:Endoplasmic reticulum (ER)-to-Golgi anterograde transport is driven by COPII vesicles mainly composed of a Sec23/Sec24 inner shell and a Sec13/Sec31 outer cage. How COPII vesicles are tethered to the Golgi is not completely understood. We demonstrated here that PAQR3 can facilitate tethering of COPII vesicles to the Golgi. Proximity labeling using PAQR3 fused with APEX2 identified that many proteins involved in intracellular transport are in close proximity to PAQR3. ER-to-Golgi trafficking of N-acetylgalactosaminyltransferase-2 on removal of brefeldin A is delayed by PAQR3 deletion. RUSH assay also revealed that ER-to-Golgi trafficking is affected by PAQR3. The N-terminal end of PAQR3 can interact with the WD domains of Sec13 and Sec31A. PAQR3 enhances Golgi localization of Sec13 and Sec31A. Furthermore, PAQR3 is localized in the ERGIC and cis-Golgi structures, the acceptor sites for COPII vesicles. Taken together, our study uncovers a role for PAQR3 as a player in regulating ER-to-Golgi transport of COPII vesicles.

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The role of cholesterol binding in the control of cholesterol by the Scap-Insig system.

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