Gene Structure and Promoter Function of Murine Munc18-2, a Nonneuronal Exocytic Sec1 Homolog Anurag Agrawal,* ,1 Roberto Adachi,* Michael Tuvim,* Xiao-Tian Yan,* Abigail H. Teich,* and Burton F. Dickey† , ‡ *Department of Internal Medicine, †Department of Molecular Physiology and Biophysics, and ‡Department of Molecular and Cellular Biology, Baylor College of Medicine and Houston VA Medical Center, Houston, Texas 77030 Received August 14, 2000 Sec1 family proteins are regulators of diverse exo- cytic processes, from yeast to man. Three mammalian homologues, Munc18-1, -2, and -3 have been described. We have studied the structure and expression of the mouse Munc18-2 gene. The Munc18-2 gene comprises 19 exons whose sizes range from 50 to 158 bp, with a total gene size of approximately 11 kb. A single tran- script of 2.1 kb is expressed in multiple non-neuronal murine tissues. Munc18-2 has a striking resemblance to Munc18-1 in structure despite only 60% sequence identity, suggesting a recent gene duplication event. Analysis of the region upstream of the transcription start site shows that Munc18-2 has a TATA-less pro- moter, with a consensus initiator (Inr) sequence at the start of transcription, several Sp1 binding sites, and strong promoter activity in RBL-2H3 mast cells. The region from 5 to 430 is more active than 5 to 800, suggesting upstream repressor elements. © 2000 Academic Press Key Words: Sec1; Munc18; exocytosis; vesicle traf- ficking; secretion; syntaxin; SNARE. Recent evidence from a variety of biochemical and genetic approaches has revealed a conserved mecha- nism of vesicular transport from yeast to man. The central components are SNARE proteins that are lo- calized both on transport vesicles (v-SNAREs, such as VAMP) and target membranes (t-SNAREs, such as Syntaxin and SNAP-25) (1, 2). These come together in a parallel four-helix bundle called the core complex. Initial formation of this complex appears to mediate tight docking between transport vesicle and target membrane, while completion of the energetically fa- vored coiling process appears to drive lipid bilayer fu- sion (3). Numerous trafficking proteins regulate func- tion of the core complex (4). Among these are Sec1 family proteins that interact with high affinity with Syntaxin family proteins (5– 8). Syntaxin is the most important trafficking protein since absence of Syntaxin in D. melanogaster causes a complete loss of neurose- cretion, whereas absence of other exocytic SNAREs causes severe but incomplete defects (9 –11). Syntaxin is the only SNARE whose structure is ordered in iso- lation, whereas the others only become ordered through interaction with Syntaxin. In isolation, Syn- taxin exists in a closed conformation in which the coil it contributes to the core complex is instead complexed with three other internal coils (12, 13). In metazoan systems (the situation appears to differ in yeast (14)), Sec1 opens the structure of Syntaxin, and then must dissociate to allow Syntaxin to participate in core com- plex formation (8, 13). Experimentally, these sequen- tial interactions have been detected as positive and negative roles in transport, whereby absence of Sec1 causes a complete loss of neurosecretion, and overex- pression also impairs secretion (15). In Saccharomyces cerevesiae, four Sec1-related pro- teins are present (16 –19). Sec1p is required for vesicle transport from the Golgi complex to the plasma mem- brane, Sly1p for transport from endoplasmic reticulum to the Golgi complex, and Slp1p/VPS33 and VPS45 for vacuolar transport. In higher organisms, proteins clos- est in structure to Sec1p have been described that similarly function in secretion (20). A single exocytic Sec1 isoform exists in C. elegans (Unc18) and in D. melanogaster (Rop), while three have been described in mammals (Munc18-1, -2, and -3). Munc18-1 was de- scribed as a neuron-specific Sec1 ortholog, and regula- tion of its interaction with Syntaxin-1A has been stud- ied in detail (5, 8). Munc18-2 and Munc18-3 were identified in non-neuronal tissues and have not been The nucleotide sequence data reported in this paper will appear in the DDBJ/GenBank/EBI nucleotide sequence database with the Ac- cession Nos. AF263345 (Munc18-2 cDNA sequence) and AF263346 (Munc18-2 gene sequence). Release date: May 22, 2000. 1 To whom correspondence should be addressed at Baylor College Medicine and the Houston VA Medical Center, 2002 Holcombe Blvd, MS-151B, Houston, TX 77030. Fax: 713-794-7853. E-mail: aagrawal@bcm.tmc.edu. Biochemical and Biophysical Research Communications 276, 817– 822 (2000) doi:10.1006/bbrc.2000.3513, available online at http://www.idealibrary.com on 817 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.