Doc2 has two C2 domains that interact with Ca2؉ and phospholipid. Munc13 has two C2 domains and one C1 domain that interacts with phorbol ester or diacylglycerol (DAG) and phospholipid. Both Doc2 and Munc13 are implicated in Ca 2؉ -dependent neurotransmitter release, but their modes of action still remain unclear. We show here that Doc2 interacts with Munc13 both in a cell-free system and in intact PC12 cells during the high K We have isolated Doc2 as a novel protein having two C2 domains that interact with Ca 2ϩ and PL 1 (1). Doc2 consists of two isoforms, Doc2␣ and Doc2 (1, 2). Doc2␣ is specifically expressed in neuronal cells, whereas Doc2 is ubiquitously expressed (1-3). Both isoforms have at least the N-terminal Doc2-specific region and C-terminal two C2 domains. We have moreover shown that overexpression of the N-terminal fragment of Doc2␣ or its C-terminal fragment including the C2 domains in PC12 cells inhibits Ca 2ϩ -dependent exocytosis (4).These results suggest that Doc2␣ is involved in Ca 2ϩ -dependent exocytosis and interacts with another component of Ca 2ϩ -dependent exocytotic machinery. To clarify the mode of action of Doc2␣ in Ca 2ϩ -dependent exocytosis, it is important to isolate its interacting protein(s). We have attempted here to isolate a Doc2␣-interacting protein from a rat brain cDNA library by use of the yeast two-hybrid system and isolated Munc13 as a Doc2␣-interacting protein.Munc13 has been isolated as a mammalian homologue of Caenorhabditis elegans unc-13, which is implicated in Ca 2ϩ -dependent neurotransmitter release (5, 6). Munc13 has three isoforms, Munc13-1, -2, and -3. All the isoforms have two C2 domains and Munc13-1 has another atypical C2 domain. They have moreover one C1 domain that interacts with PE or DAG and PL (5-7). Munc13 is specifically expressed in neuronal cells, and Munc13-1 is localized at the presynaptic plasma membrane (6).We describe here that Doc2␣ directly interacts with Munc13-1 in a DAG-dependent manner and that the Doc2␣-Munc13-1 interactions play an important role in Ca 2ϩ -dependent exocytotic machinery. EXPERIMENTAL PROCEDURESTwo-hybrid Assay-The N-terminal fragment (1-90 aa) of human Doc2␣ cDNA (1) was inserted into the pBTM116 (pLexA-Doc2␣N). The yeast reporter strain L40 was transformed with pLexA-Doc2␣N and a rat brain cDNA library constructed in pGAD10 (CLONTECH). Library plasmids from positive clones were analyzed by transformation tests and DNA sequencing. Overlapping clones containing the full-length coding region of Munc13-1 were isolated by screening the rat brain cDNA library. The cDNA fragments encoding several Munc13-1 deletion mutants were constructed from the overlapping clones and inserted into pGAD424. The cDNA fragments encoding several Doc2␣ deletion mutants were inserted into pBTM116. After co-transformation into yeast strain L40, -galactosidase activity was assayed by liquid and filter assays (8,9).Preparation of Recombinant Proteins-The cDNA fragments encoding the N-terminal fragment (1-90 aa) of human Doc2␣ (1) and Munc13-1-...
The let-60 ras gene acts in a signal transduction pathway to control vulval differentiation in Caenorhabditis elegans. By screening suppressors of a dominant negative let-60 ras allele, we isolated three loss-of-function mutations in the sur-5 gene which appear to act as negative regulators of let-60 ras during vulval induction. sur-5 mutations do not cause an obvious mutant phenotype of their own, and they appear to specifically suppress only one of the two groups of let-60 ras dominant negative mutations, suggesting that the gene may be involved in a specific aspect of Ras activation. Consistent with its negative function, overexpressing sur-5 from an extragenic array partially suppresses the Multivulva phenotype of an activated let-60 ras mutation and causes synergistic phenotypes with a lin-45 raf mutation. We have cloned sur-5 and shown that it encodes a novel protein. We have also identified a potential mammalian SUR-5 homolog that is about 35% identical to the worm protein. SUR-5 also has some sequence similarity to acetyl coenzyme A synthetases and is predicted to contain ATP/GTP and AMP binding sites. Our results suggest that sur-5 gene function may be conserved through evolution.The Ras-mediated signal transduction pathway plays important roles in specifying cell fates in a number of developmental events in the nematode Caenorhabditis elegans including vulval cell differentiation (17, 31), male spicule cell differentiation (4), germ nucleus exit from pachytene (5), sex-myoblast migration (32), and excretory duct cell differentiation (38). Vulval differentiation has been the main system used in a number of laboratories to identify new components of this pathway and study its regulation. Vulval differentiation in C. elegans hermaphrodites is controlled by the combination of several cellcell signaling events ( Fig. 1A) (17,31). In particular, an inductive signal from the anchor cell induces three of the six vulval precursor cells (VPCs, P3.p to P.8p) to differentiate into vulval cells (Fig. 1A). The let-60 ras gene acts in a conserved signal transduction cascade to transduce the anchor cell signal encoded by the lin-3 gene (Fig. 1C). Previous genetic screens in several laboratories have identified many components that are either key factors acting in the main backbone of the signaling cascade (Fig. 1C) or factors that regulate the activity of these key players in the pathway (17, 31). The functions of several genes as regulators of the signaling pathway (e.g., unc-101, sli-1, and ksr-1) have been identified only by the genetic suppressor phenotypes and the synergistic effects of their mutations in other mutant backgrounds, since mutants with loss-offunction mutations in these genes show few or no abnormalities in vulval development (14,18,19,30).To identify negative factors that down regulate the let-60 ras activity, we screened for mutations that suppress the Vulvaless phenotype caused by a let-60 ras dominant negative (dn) mutation, let-60(K16N). In this paper, we describe the gene sur-5, defined by thr...
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