©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Identification of Two Novel Munc-18 Isoforms Expressed in Non-neuronal Tissues (*)

(Received for publication, November 29, 1994; and in revised form, January 12, 1995)

Judy T. Tellam (§) Shane McIntosh David E. James (¶)

From the Center for Molecular Biology and Biotechnology and Department of Physiology and Pharmacology, University of Queensland, St Lucia, 4072, QLD, Australia

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Munc-18, also known as n-Sec1 or rbSec1, is a syntaxin-binding protein thought to play a role in regulating synaptic vesicle exocytosis. Although a gene family of syntaxins has been identified, only a limited subset bind to Munc-18. This implicates the existence of other mammalian Munc-18 homologues that may be involved in a range of vesicle transport reactions. The purpose of the present study was to identify other members of the Munc-18 family by cDNA cloning. Three distinct Munc-18 isoforms, Munc-18a, previously identified in neuronal tissue, and two novel isoforms, Munc-18b and Munc-18c, were isolated from a 3T3-L1 adipocyte cDNA library by screening with a rat brain Munc-18 DNA probe. Munc-18a is identical to Munc-18 and by Northern analysis is expressed predominantly in brain and to a lesser extent in testis and 3T3-L1 cells. Munc-18b is 62% identical to Munc-18 at the amino acid level and is expressed in testis, intestine, kidney, rat adipose tissue, and 3T3-L1 cells. Munc-18c is 51% identical to Munc-18 and is ubiquitously expressed. It is likely, based on these findings, that unique Munc-18/syntaxin interactions may play an important role in generating a combinatorial mechanism for the regulation of vesicle transport in mammalian cells.


INTRODUCTION

Identifying the molecular factors that regulate vesicle transport and fusion in eukaryotic cells has been the subject of intense investigation(1, 2) , particularly in the mammalian synapse. Here it has been demonstrated that many of the proteins that regulate synaptic vesicle exocytosis are similar to those observed in other cell types, including yeast, thus enabling the formulation of unifying models to explain all vesicle transport reactions(3) .

Recent biochemical studies have identified a number of multiprotein intermediates (SNARES) (^1)that regulate the specificity of synaptic vesicle exocytosis (reviewed in (4) ). A fusion protein complex, consisting of N-ethylmaleimide-sensitive factor and soluble N-ethylmaleimide-sensitive factor attachment proteins, interact with three proteins isolated from brain: synaptobrevin or vesicle-associated membrane protein, a synaptic vesicle membrane protein; and syntaxin and the synaptosomal-associated protein-25 (SNAP-25), both found on the presynaptic plasma membrane. The identification of this protein complex supports the recent SNARE hypothesis(3) , which implies that the specificity of membrane fusion is regulated by the specific binding of the donor vesicle (via the V-SNARE or synaptobrevin protein) with the acceptor membrane (via a T-SNARE or syntaxin). This basic template for molecular recognition and regulation of membrane fusion appears to be a general mechanism, since both T-SNARE and V-SNARE components belong to large gene families. In mammalian cells six syntaxin homologues have been cloned which exhibit a broad tissue distribution as well as variations in subcellular location(5) . Two distinct synaptobrevins, 1 and 2(6) , have been identified in synaptic vesicles, whereas cellubrevin, a synaptobrevin homologue, is targeted to recycling endosomes(7) . Other members of these two gene families have also been described in yeast. The yeast syntaxin homologues, Sed5p(8) , Sso1p, Sso2p(9) , and Pep12p(10) , together with a variety of synaptobrevin homologues, including Bet1p (11, 12) , Bos1p(13) , SNC1, and SNC2 (14) are thought to coordinate specific membrane trafficking events throughout the secretory pathway.

Another family of proteins believed to participate in this complex show homology to Sec1p. The Sec1p family of proteins includes Sly1(15) , Slp1/Vps33(16) , and Sec1(17, 18) , which act at many different stages along the secretory pathway in yeast. Studies in mammalian cells, however, have so far only identified one Sec1p family member, Munc-18/n-Sec1/rbSec1(19, 20, 21) , giving rise to the question as to whether other Sec1p-like proteins exist in mammalian cells. Munc-18 itself was identified as a mammalian syntaxin-binding protein. Although there is no functional data concerning the role of Munc-18 in vesicle transport, a highly related protein from Caenorhabditis elegans unc-18, has been identified and mutations in this gene product result in accumulation of acetylcholine containing secretory vesicles as well as abnormalities in the development of the C. elegans nervous system(22, 23) . Thus, given the likelihood of additional mammalian Sec1p-like proteins and their potentially important role in membrane trafficking events, we have identified and characterized further members of this gene family to more closely understand the regulation of vesicle transport.


EXPERIMENTAL PROCEDURES

Materials

Restriction enzymes were obtained from New England Biolabs, Inc. (Beverly, MA). Radioactive nucleotides and nylon membranes (Hybond-N) were from Amersham (Aylesbury, United Kingdom). Oligonucleotides were synthesized on an Applied Biosystems 394 DNA synthesizer. All chemicals were high purity commercial grades. 3T3-L1 fibroblasts, obtained from the American Type Tissue Culture Center, were cultured and differentiated into adipocytes as described previously(24) .

cDNA Cloning and Sequence Analysis of Munc-18 Isoforms

Two oligonucleotide primers corresponding to the 5` (TGCTCTAGAAGAACGCCATGGCCCCCATTGG; sense primer) and 3` (TGCTCTAGATTAACTGCTTATTTCTTCGTCTGTTTTATTCAG; antisense primer) ends of the rat brain Munc-18 cDNA (19) were synthesized and used to obtain a full length Munc-18 DNA fragment by reverse transcriptase-polymerase chain reaction from rat brain RNA. Rat brain RNA was isolated by the guanidine isothiocyanate procedure(25) . The polymerase chain reaction cycling profile was 94 °C for 20 s, 55 °C for 20 s, 72 °C for 2 min, repeated for 35 cycles. The authenticity of the amplified DNA fragment was verified by restriction mapping. The polymerase chain reaction product was isolated from a 1% agarose gel, radiolabeled with random hexamer primers (Promega Corp., Madison, WI) and used to screen a random-primed 3T3-L1 adipocyte cDNA library constructed in ZAP II, kindly provided by Dr. F. Fiedorek, University of North Carolina. A total of 250,000 plaques were screened. From 35 positives, isolated after sequential purifications, three distinct cDNA classes were identified. Clones were subcloned into pBluescript II SK- (Stratagene, La Jolla, CA) and sequenced manually using Sequenase version 2.0 (U. S. Biochemical Corp.) or by automated DNA sequencing (Applied Biosystems Inc., model 373A). Both strands of DNA for the entire Munc-18c cDNA and 97% of the Munc-18b cDNA were sequenced utilizing T3, T7, and gene-specific oligonucleotide primers as well as the Erase-a-base nested deletion kit (Promega). The remaining 5` sequence of Munc-18b was obtained using the 5`-Amplifinder rapid amplification of cDNA ends kit (Clontech, Palo Alto, CA), as per manufacturer's instructions. 3T3-L1 adipocyte poly(A) RNA (2 µg) was reverse-transcribed and then primed with oligonucleotide P1 (GCTGCTTTGTAGGTGAAGGTTGGTGTTCCC). A nested gene-specific primer P2 (CGCGGATCCCGTGGGACTCAGCAAATAAATTGCCTCC) was used in conjunction with this procedure. The deduced amino acid sequences of Munc-18b and Munc-18c were aligned with rat brain Munc-18a (19, 20, 21) and the C. elegans gene product unc-18 (23) using the computer program Clustal V(26) .

RNA Blot Analysis

Total RNA was isolated from rat tissues, 3T3-L1 fibroblasts, and adipocytes by the guanidine isothiocyanate procedure(25) . 3T3-L1 fibroblast and adipocyte poly(A) was obtained using the Pharmacia mRNA purification kit (Pharmacia, Uppsala, Sweden). RNA was electrophoresed using a 1% formaldehyde-agarose resolving gel and transferred to a nylon membrane. The blot was sequentially probed with Munc-18a, Munc-18b, and Munc-18c DNA fragments labeled with [alpha-P]dCTP by random priming. The hybridization conditions were: 50% formamide, 5 times SSPE (1 times SSPE = 0.15 M NaCl, 10 mM NaH(2)PO(4), 1 mM EDTA, pH 7.4), 5 times Denhardt's solution, 1% SDS, and 100 µg/ml denatured herring sperm DNA at 42 °C for 16 h. The blot was washed with 1 times SSC and 0.1% SDS at 50 °C. The RNA blots were also probed with DNA coding for glyceraldehyde-3-phosphate dehydrogenase for normalization of results. The conditions for hybridization of the RNA blot for each of the three Munc-18 DNA probes was individually determined such that they did not cross-hybridize.


RESULTS

To examine the hypothesis that there is a large gene family of mammalian Munc-18 isoforms, a 3T3-L1 adipocyte cDNA library was screened with a 1.8-kb rat brain Munc-18 DNA fragment. Thirty-five positive clones were isolated, and 30 were characterized by DNA sequencing. Three distinct cDNA classes were identified, referred to here as Munc-18a, Munc-18b, and Munc-18c. Munc-18a was identical to Munc-18/n-Sec1/rbSec1 (19, 20, 21) as determined by sequencing the 5` 340 nucleotides of a full-length clone (M5A) and by restriction mapping. Three of the 30 characterized clones were assigned to this group. Munc-18b was the most abundant isoform isolated from the library (22 out of 30 clones), whereas the remaining five clones fell into the third class, Munc-18c. Both Munc-18b and Munc-18c represent novel Munc-18 isoforms. The inserts from two separate Munc-18b clones (M1B and M3A) were completely sequenced on both strands and were both found to be missing 5` ends, as was the case for all the remaining clones in this class. The 5`-coding region was obtained using a 5` rapid amplification of cDNA ends procedure (see ``Experimental Procedures''). A 270-bp DNA fragment was amplified by this technique and then subcloned into Bluescript, sequenced, and found to contain a start codon with a consensus Kozak sequence (27) followed by 18 bp of novel sequence and 200 bp that were identical to that of the 5` end of M1B. The entire open reading frame of Munc-18b is 1,779 bp encoding a protein of 593 amino acids with a predicted M(r) of 66,357 and pI of 6.72 (Fig. 1). A 2.5-kb Munc-18c full-length clone (M2I) was sequenced in both directions and found to contain a 1,776-bp open reading frame encoding a protein of 592 amino acids with a calculated M(r) of 67,942 and a pI of 7.96 (Fig. 2). None of these deduced amino acid sequences showed any evidence of a transmembrane region. The percentage identity between the three Munc-18 isoforms at the nucleotide level is 54-64%.


Figure 1: Complete nucleotide sequence of 3T3-L1 Munc-18b cDNA and deduced amino acid sequence of the protein. Capital letters indicate the coding sequence, whereas lowercase letters signify the 5`- and 3`-noncoding sequences. Amino acid residues are denoted by the standard three-letter code below the nucleotide sequence, and the sequences are numbered on the right.




Figure 2: Complete nucleotide sequence of 3T3-L1 Munc-18c cDNA and deduced amino acid sequence of the protein. Capital letters indicate the coding sequence, whereas lowercase letters signify the 5`- and 3`-noncoding sequences. Amino acid residues are denoted by the standard three-letter code below the nucleotide sequence, and the sequences are numbered on the right.



The amino acid sequences of the three Munc-18 3T3-L1 isoforms exhibit substantial similarity along their entire length to the C. elegans gene product, unc-18 (Fig. 3). Munc-18b and Munc-18c showed 62 and 51% amino acid identity, respectively, compared with Munc-18/n-Sec1/rbSec1, referred to here as Munc-18a (Fig. 3, Table 1). We have adopted a similar nomenclature to Hata et al. (19) to refer to these different isoforms rather than that used by others(20, 21) , since all of the clones isolated from mammalian cells showed a higher degree of amino acid identity to the C. elegans gene product, unc-18, than to the yeast homologue, Sec1p (Table 1). As shown in Table 1, Munc-18a is most similar to the Drosophila homologue, Ropp (65%) and C. elegans unc-18 (59%), whereas Munc-18b is increasingly less similar to Ropp (54%) and unc-18 (53%) with Munc-18c being the least identical to Ropp (44%) and unc-18 (43%). All three adipocyte Munc-18 homologues displayed much lower identities (17-27%) to Sec1p, Sly1p, and Slp1p (Table 1). The predicted secondary structures of Munc-18a, Munc-18b, and Munc-18c were all very similar (results not shown).


Figure 3: Comparison of the deduced amino acid sequences of Munc isoforms. Three different Munc-18 clones were isolated from a 3T3-L1 adipocyte cDNA expression library. Munc-18a was partially sequenced (see underlined region) and was found to be identical to a rat brain isoform previously referred to as Munc-18/nSec1/rbSec1(19, 20, 21) . Munc-18b and Munc-18c are novel cDNAs. Also included in the alignment is a C. elegans gene product, unc-18 (GenBank accession number S66176). Amino acids are shown in the single-letter code and numbered on the right. Amino acids that are identical between all four homologues are denoted by an asterisk, whereas conserved substitutions are indicated with a dot. Gaps introduced to generate this alignment are represented by dashes. Sequences were aligned using the computer program Clustal V.





The tissue distribution of Munc-18a, Munc-18b, and Munc-18c was studied by Northern blot analysis and was found to be unique for each isoform (Fig. 4A). In agreement with previous studies (19, 20, 21) , Munc-18a had a transcript size of 3.8 kb and was expressed predominantly in rat brain, but lower levels were also detected in testis. Munc-18b was expressed at highest levels in rat testis, and lower levels of expression were detected in intestine, kidney, and epididymal fat pad. A major Munc-18b transcript of 2.3 kb and a minor transcript of 3.2 kb were detected in each of these tissues (Fig. 4A). The molecular basis and significance of the two transcripts remains to be determined. Munc-18c (transcript size: 3.0 kb) was expressed ubiquitously in liver, kidney, intestine, testis, heart, skeletal muscle, brain, and epididymal fat. A second Munc-18c transcript of 1.7 kb was also detected in rat testis. Each of the Munc-18 isoforms was expressed in 3T3-L1 fibroblasts and adipocytes, consistent with the fact that these clones were isolated from a 3T3-L1 adipocyte cDNA library. The expression of Munc-18a decreased following differentiation into adipocytes, whereas increased mRNA levels of both Munc-18b and Munc-18c were observed in 3T3-L1 adipocytes compared with the undifferentiated fibroblasts (Fig. 4B).


Figure 4: RNA blot analysis of 3T3-L1 Munc-18 isoform mRNAs in various rat tissues and 3T3-L1 fibroblasts and adipocytes. Total RNA from eight different rat tissues and poly(A) RNA from 3T3-L1 fibroblasts and adipocytes were hybridized with alpha-P-labeled probes derived from the three different Munc-18 isoforms (Munc-18a, Munc-18b, and Munc-18c) as well as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as described under ``Experimental Procedures.'' A, expression of Munc-18a, Munc-18b, Munc-18c, and glyceraldehyde-3-phosphate dehydrogenase mRNAs in rat tissues. B, expression of Munc-18a, Munc-18b, and Munc-18c mRNAs in 3T3-L1 fibroblasts (F) and adipocytes (A). The size (in kilobases) of the major transcripts are indicated at the left.




DISCUSSION

In this study we have identified two novel Munc-18 isoforms which exhibit broad and distinct tissue distributions and which are highly homologous to the previously described neural isoform, Munc-18/n-Sec1/rbSec1(19, 20, 21) . We have referred to these gene products as: Munc-18a, which is the neural-specific protein; Munc-18b, found in testis, kidney, intestine, and adipose tissue; and the ubiquitously expressed Munc-18c. Previous studies in a variety of organisms have ascribed an important role to the Munc-18/Sec1p gene product in the regulation of vesicle transport. In the mammalian synapse Munc-18a has been shown to bind to the presynaptic membrane protein syntaxin (19, 20, 21) . Furthermore, a genetic interaction between Sec1p and two syntaxin homologues, Sso1 and Sso2, has been found in yeast(9) . In view of the putative role of syntaxin in the formation of a vesicle docking complex, it has been suggested that members of the Munc-18/Sec1p family may play a proofreading function in vesicle docking/fusion reactions(20) .

The SNARE hypothesis (3) proposes that the specificity of different vesicle fusion reactions is determined by specific proteins in both the donor and acceptor membrane compartments. In the case of the mammalian synapse, these include syntaxin, synaptobrevin, synaptosomal-associated protein-25 (SNAP-25), and small molecular weight GTP-binding proteins of the Rab family (reviewed in (4) ). Each of these proteins belongs to a large gene family, the individual members of which display differences in tissue expression and intracellular location. Another difference is that both genetic studies in yeast and biochemical studies in the mammalian synapse have revealed a particular specificity in the interaction between different members of these gene families. For instance, synaptobrevin or vesicle-associated membrane protein 1 and 2 bind to syntaxin 1A and 4 but not to syntaxin 2 and 3(28) . On the basis of such studies it has been suggested that the specificity of vesicle transport may be regulated by a combinatorial mechanism. We propose on the basis of the findings in the present study that different Munc-18 isoforms are also involved in regulating distinct vesicular transport steps, thus elaborating the fidelity of a combinatorial mechanism of sorting.

Two lines of evidence predicted the existence of multiple homologues of Munc-18 in mammalian cells. First, three distinct yeast genes have been identified that are involved in discrete vesicle transport events. These include Sec1(17) , which is involved in post-Golgi secretion, Sly1(15) , in endoplasmic reticulum to Golgi transport, and Slp1(16) , that has a function in vacuolar trafficking. Second, the neural homologue of Sec1, referred to here as Munc-18a, binds to syntaxin 1A, 2, and 3 but not to syntaxin 4(20) . Furthermore, the expression of Munc-18a is primarily confined to neural tissue (19-21; Fig. 4A), whereas members of the syntaxin family display a broad tissue distribution(5) .

Based on the amino acid homology between Munc-18a, Munc-18b, and Munc-18c as well as their tissue distributions, it is likely that they may be involved in a wide array of vesicle transport steps. Munc-18b showed the highest amino acid sequence identity to Munc-18a (62%, Table 1). The relatively limited tissue distribution of Munc-18b implies that it may be involved in more specialized vesicle trafficking events common to testis, intestine, and kidney. The tissue distribution of Munc-18b does not correlate with that of any of the known mammalian syntaxin isoforms(5) . This raises the possibility that a Munc-18b-specific syntaxin remains to be identified. However, the neural-specific isoform, Munc-18a, interacts with multiple syntaxin's (20) , both neural and non-neural, and so it is conceivable that different Munc-18/syntaxin combinations may occur in vivo, depending upon the native expression of these proteins in a particular cell type. Munc-18c exhibited a broad tissue distribution implicating its involvement in a more constitutive vesicle transport event that is common to all cells. Consistent with this, Munc-18c showed the least identity to Munc-18a, Ropp, and unc-18 (43-51%, Table 1), which have all been implicated in regulated exocytosis in more specialized secretory cells. In order to define the role of these new Munc-18 isoforms in vesicle transport, it will be necessary to determine the intracellular location of Munc-18b and Munc-18c using isoform-specific antibodies and to study the specificity of the interaction between different members of the syntaxin and Munc-18 gene families.

The identification of a mammalian Munc-18 gene family provides further insight into our understanding of vesicle transport regulation. These proteins are likely to play an important role in vesicle docking and/or fusion and should be considered as an integral component of the SNARE complex. Future studies will be required to map the specific interactions between different syntaxin's and Munc-18 isoforms in order to define their role in vesicle transport. It is also possible that other members of this gene family exist, which are either not expressed at a significant level in 3T3-L1 cells or are less homologous than the three Munc-18 proteins described here.


FOOTNOTES

*
This work was supported by the National Health and Medical Research Council of Australia. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U19520 [GenBank](munc-18b gene) and U19521 [GenBank](munc-18c gene).

§
National Health and Medical Research Council (Dora Lush) postgraduate scholar.

Wellcome Research Fellow. To whom correspondence should be addressed. Tel.: 61-7-365-4986; Fax: 61-7-365-4388.

(^1)
The abbreviations used are: SNAREs, soluble N-ethylmaleimide-sensitive factor attachment protein receptors; V-SNARE, vesicle membrane SNARE; T-SNARE, target membrane SNARE; Munc-18, mammalian homologue of unc-18; kb, kilobase(s); bp, base pair(s).


ACKNOWLEDGEMENTS

We thank Professor John Shine, Dr. Richard Alm, and Dr. Robert Piper for providing invaluable advice and support during these studies. We also thank Shane Rea and Kirsten Blake for technical help. We are indebted to Dr. Amanda Carozzi for providing 3T3-L1 fibroblasts and adipocytes enabling the production of RNA.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.