Munc13 proteins are essential in neurotransmitter release, controlling the priming of synaptic vesicles to a release-ready state. The sequences responsible for this priming activity are unknown. Here we identify a large alpha-helical domain of mammalian Munc13-1 that is autonomously folded and is sufficient to rescue the total arrest in neurotransmitter release observed in hippocampal neurons lacking Munc13s.

Three sterol regulatory element-binding proteins (SREBP-1a, -1c, and -2) stimulate transcription of genes involved in synthesis and receptor-mediated uptake of cholesterol and fatty acids. Here, we explore the individual roles of each SREBP by preparing lines of Chinese hamster ovary (CHO) cells that express graded amounts of nuclear forms of each SREBP (designated nSREBPs) under control of a muristerone-inducible nuclear receptor system. The parental hamster cell line (M19 cells) lacks its own nSREBPs, owing to a deletion in the gene encoding the Site-2 protease, which releases nSREBPs from cell membranes. By varying the concentration of muristerone, we obtained graded expression of individual nSREBPs in the range that restored lipid synthesis to near physiologic levels. The results show that nSREBP-2 produces a higher ratio of synthesis of cholesterol over fatty acids than does nSREBP-1a. This is due in part to a selective ability of low levels of nSREBP-2, but not nSREBP-1a, to activate the promoter for squalene synthase. nSREBP-1a and -2 both activate transcription of the genes encoding stearoyl-CoA desaturase-1 and -2, thereby markedly enhancing the production of monounsaturated fatty acids. nSREBP-1c was inactive in stimulating any transcription at the concentrations achieved in these studies. The current data support the emerging view that the nSREBPs act in complementary ways to modulate the lipid composition of cell membranes.Cholesterol and fatty acids, the building blocks of cell membranes, are synthesized by regulated pathways in animal cells. Both pathways are influenced by a single family of transcription factors designated sterol regulatory element binding proteins (SREBPs), 1 whose concerted actions optimize the lipid content of membranes (reviewed in Brown and Goldstein (1)). Appropriate to their role in modulating membrane composition, the SREBPs are bound intrinsically to cell membranes.They are synthesized as long precursors of ϳ1150 amino acids with three domains. The NH 2 -terminal domain of ϳ480 amino acids is a classic basic helix-loop-helix-leucine zipper transcription factor. This is followed by a membrane attachment domain of ϳ80 amino acids and a COOH-terminal domain of ϳ590 amino acids that performs a regulatory function. The SREBPs are attached to membranes of the nuclear envelope and endoplasmic reticulum in a hairpin fashion with their NH 2 -terminal and COOH-terminal domains projecting into the cytosol and the membrane attachment domain projecting into the lumen (2).In order to reach the nucleus, the NH 2

Metabolites of vitamin D3 (D3) (cholecalciferol) are recognized as enzymatically formed chemicals in humans that can influence a wide variety of reactions that regulate a large number of cellular functions. The metabolism of D3 has been extensively studied, and a role for three different mitochondrial cytochrome P450s (CYP24A, CYP27A, and CYP27B1) has been described that catalyze the formation of the 24(OH), 25(OH), and 1(OH) metabolites of D3, respectively. The hormone 1,25-dihydroxyvitamin D3 has been most extensively studied and is widely recognized as a regulator of calcium and phosphorous metabolism. Hydroxylated metabolites of D3 interact with the nuclear receptor and thereby influence growth, cellular differentiation, and proliferation. In this article, we describe in vitro experiments using purified mitochondrial cytochrome P450scc (CYP11A1) reconstituted with the iron-sulfer protein, adrenodoxin, and the flavoprotein, adrenodoxin reductase, and show the NADPH and time-dependent formation of two major metabolites of D3 (i.e., 20-hydroxyvitamin D3 and 20,22-dihydroxyvitamin D3) plus two unknown minor metabolites. In addition, we describe the metabolism of 7-dehydrocholesterol by CYP11A1 to a single product identified as 7-dehydropregnenolone. Although the physiological importance of these hydroxylated metabolites of D3 and their in vivo formation and mode of action remain to be determined, the rate with which they are formed by CYP11A1 in vitro suggests an important role.C ytochrome P450scc (CYP11A1) is a mitochondrial hemeprotein oxygenase of great interest because of its identification as a key enzyme in the metabolism of cholesterol to pregnenolone (1). This reaction is the initial step in the pathway leading to the synthesis of a number of metabolically important steroid hormones. Like other mitochondrial P450s this enzyme requires a minielectron transport chain consisting of the iron-sulfur protein, adrenodoxin (Adr), and the FADcontaining NADPH-Adr reductase (AdrR) for the transfer of reducing equivalents from NADPH to the P450. To date, CYP11A1 has no known natural substrates other then cholesterol. The conversion of cholesterol to pregnenolone (P5) is reported to occur by forming in sequence the monohydroxylated product [22R-(OH)cholesterol] followed by the formation of the dihydroxylated intermediate [20␣, 2 cholesterol] with subsequent carbon-carbon bond cleavage at the C20-C22 position to form the C19 steroid pregnenolone (1-5). It has been difficult to identify these hydroxylated intermediates of cholesterol metabolism during catalysis by CYP11A1 because they are not released from the active site of CYP11A1 during metabolism (1-5). Of interest is the recent report that the oxysterol 22R-(OH)cholesterol plays an important role as a ligand for the liver X receptor, a transcription factor (6).In mammalian tissues 7-dehydrocholesterol (7-DHC) is the direct precursor of cholesterol in the Kandutsch-Russell cholesterol biosynthetic pathway. A deficiency of ⌬7-DHC reductase, the enzyme responsible fo...