From the Phenotypes of Caenorhabditis elegans
unc-18 and unc-64 gene mutations are similar. While
unc-18 is known to be essential for normal synaptic
transmission (Hosono, R., Hekimi, S., Kamiya, Y., Sassa, T., Murakami,
S., Nishiwaki, S., Miwa, J., Taketo, A., and Kodaira, K.-I. (1992)
J. Neurochem. 58, 1517-1525), the function of
unc-64 remains unclear. Here we describe the cloning, and
the molecular and genetic characterization of the unc-64
gene, especially in relation to unc-18. unc-64 encodes a
protein (C. elegans syntaxin) showing sequence and
structural similarities to mammalian syntaxin 1A. From
unc-64, at least three types of poly(A)+ RNA
are transcribed, which encode two types of syntaxin that differ in the
deduced transmembrane domain. In gene expression, unc-64
closely resembles unc-18, that is, both are expressed in neural cells, especially in motor neurons and neurons constituting head
ganglions. C. elegans syntaxin binds to UNC-18 with high affinity. The unc-64 (e246) mutation producing
a mild phenotype causes an Ala Synaptic transmission is regulated by neurochemical secretion from
the presynaptic terminus. The process is accomplished by the docking of
synaptic vesicles to the plasma membrane and their signal-dependent fusion. At the interaction, the proteins
associated with synaptic vesicles and plasma membrane execute important
roles (1). A number of membrane proteins functioning in the vesicles mediate transport from synaptic vesicles to the plasma membrane. Syntaxin is one such protein functioning in several steps of the secretory process (2-5). The mammalian syntaxin has been found in a 20 S complex thought to be responsible for the docking of synaptic
vesicles at the presynaptic plasma membrane (6, 7). Synaptobrevin/vesicle-associated membrane protein-like proteins residing on transport vesicles are donated as vesicle-associated soluble N-ethylmaleimide-sensitive factor
(NSF)1 attachment
protein-SNAP receptors (v-SNAREs), and their target membrane proteins
including syntaxin as target membrane-associated SNAP receptors
(t-SNAREs) (8). Although the binding of synaptic vesicles to the plasma
membrane is critical to the synaptic transmission, its regulation
remains unknown. Recently syntaxin 1A has been shown to bind the
C. elegans UNC-18 protein homolog, n-sec-1 or Munc-18 (9-11). Since the UNC-18 homolog does not copurify with stoichiometric amounts of other proteins in the 20 S fusion complex, it
is suspected that it functions at a different step in this process (9).
We have been studying the C. elegans UNC-18 protein, which
is thought to play an important role in mediating synaptic vesicle interaction with plasma membrane (12-15). UNC-18 is rich in charged and hydrophilic amino acids. The protein is expressed exclusively in
neurons; a large amount is insoluble but a significant amount is in
bound form.2 In
unc-18 gene mutants, the release of neurotransmitters is
defective, probably due to blocked presynaptic function (12, 13,
16).
We have sought genetic and biochemical evidence for interaction between
UNC-18 and C. elegans syntaxin. To this end, we cloned the
genome and cDNAs encoding C. elegans syntaxin. Genetic
and biochemical analyses show that the unc-64 gene encodes
C. elegans syntaxin and is functionally related to
unc-18. In a preliminary study we showed that UNC-18 binds
to GST-C. elegans syntaxin fusion protein (14). We report
here the kinetics of the binding between UNC-18 and C. elegans syntaxin. During the study, a preliminary report that
unc-64 encodes C. elegans syntaxin was published
(17).
Strains--
General methods for C. elegans culture
and genetic manipulation were as described previously (18). The
following mutations were used: LGIII, unc-64
(e246), dpy-18 (e364); LGX,
unc-18 (e81, cn347, md1094,
md1294), lon-2 (e678). Double mutants
with unc-64 and unc-18 were generated from the
progeny derived from unc-18/lon-2; unc-64/dpy-18.
Homozygous unc-18/unc-18; unc-64/unc-64 genotype was determined by crossing with +/unc-64 and +/+ males.
Isolation and Analysis of cDNA Clones--
cDNAs
encoding C. elegans syntaxins were screened from a C. elegans Germline Transformation and unc-64 Rescue--
Germline
transformation was performed using standard techniques (19). Cosmid
clones located within the unc-64 gene and the genomic clones
were microinjected into the mitotic germline of hermaphrodites,
according to the method developed by Mello et al. (19). For
rescue experiments, the relevant DNA was injected at concentration of
5-100 µg/ml. pRF4, a plasmid containing the rol-6
(su1006) allele, was coinjected as a marker to identify transgenic animals. Since unc-64 mutants are abnormal in
locomotion, a line was considered to be rescued if the transgenic
homozygous unc-64 animals could generate a normal rolling
phenotype.
GFP Constructs--
Expression constructs were made using the
6.0-kbp HindIII fragment including the putative
unc-64 5 GST-Syntaxin Fusion Proteins--
Vectors encoding GST fusion
proteins with the C. elegans syntaxin were constructed using
PCR products and the pGEX vector essentially as described previously
(14).
Molecular Biology--
Standard molecular biology protocols (20)
were followed except where noted. Total RNA was extracted by the
guanidinium thiocyanate/cesium chloride centrifugation method, and
poly(A)+ RNA was isolated with Oligotex-dT30 (Takara).
Northern blots were performed using approximately 5 µg of
poly(A)+ RNA per lane and transferred to Hybond
N+ (Amersham, Buckinghamshire, UK) for hybridization. The
Northern blot was probed with the full-length C. elegans syntaxin A cDNA, which had been labeled
according to the manufacture's directions (Strategenes). DNA sequence
analysis was performed by an Applied Biosystems 377 sequencer.
Sequencing reaction was performed according to dye primer protocol
(Applied Biosystems Inc.).
Identification of unc-64 Mutation--
The unc-64
(e246) cDNA was prepared by RT-PCR using four different
primers: STX-1 (5 In Situ Hybridization--
The antisense riboprobe was labeled
with digoxigenin-UTP. The mean length of the in vitro
transcribed RNA was reduced to 100-200 bp by alkaline hydrolysis.
In situ hybridization was performed as described by Mitani
et al. (21), with slight modifications. The collected worms
were fixed in 3.7% formaldehyde. After washing three times with
methanol for 5 min, specimens were treated twice for 5 min each time
with 100 mM triethanolamine (pH 8.0) and then with 100 mM triethanolamine containing 0.5% acetic anhydride for 5 min. Hybridization was done in hybridization buffer containing 50%
formamide, 5 × SSC, 100 µg/ml salmon sperm DNA, 100 µg/ml yeast RNA, 0.1% SDS, and 1 µg/ml RNA probe at 60 °C overnight. For detecting the hybridized signal, specimens were incubated with
anti-digoxigenin antibody tagged on alkaline phosphatase. Visualization
of bound alkaline phosphatase was performed with 5-bromo-4-chloro-3-indolyl phosphate and 4-nitro blue tetrazolium chloride as substrates.
Phenotypes of the unc-64 Mutation and Genetic Interactions with
unc-18--
Genetic and biochemical analyses revealed that the
unc-64 and unc-18 genes are functionally related
(16, 22). That is, both gene mutations result in uncoordinated
locomotion, abnormal accumulation of acetylcholine, resistance to
acetylcholinesterase inhibitors, and developmental retardation (18, 22,
24). The phenotype is recessive and is probably produced by a defect in
neurotransmitter release. To elucidate further the relationship between
the two genes, we constructed double mutant strains carrying unc-64 and unc-18 mutations (Table
I). A double mutation of
unc-18 allele, e81 or cn347 and
unc-64 (e246) was lethal. Double mutants showing
mild phenotype were more severely defective in locomotion and
development than the single mutants. ACh levels were elevated by
unc-64 and unc-18 mutations. ACh levels in the
double mutants were further elevated, although not higher than the
additive values of both mutations. To further reveal the functional
relationship of both genes, we cloned the unc-64 gene.
Department of Biology,
Department of Nutrition,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Val conversion in the conserved
COOH-terminal region in mammalian syntaxin 1A or Drosophila
syntaxin-1A whose site is included in three types of transcripts. The
binding of the mutant C. elegans syntaxin to UNC-18 is
greatly reduced, indicating the mutation site contributes to the
binding.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
ZAP cDNA library (a gift from R. Barstead and R. Waterston) using the RT-PCR product and the cm04d2 C. elegans syntaxin cDNA (provided by C. Martin and R. Waterston)
as probes. Longer clones (10 in all) were characterized by restriction
analysis and sequenced after subcloning into M13 vectors using standard
procedures. SwissProt data bases were searched for other C. elegans syntaxin family proteins using the BLAST and/or FASTA
programs.
-regulatory region to direct expression of the GFP
expression plasmid pPD
95.75.3 This comprised
nucleotides of genomic DNA immediately upstream of the initiator
methionine codon in exon 1 plus 3.5 kb of additional down stream
genomic sequence including exon 1 through exon 5.
-GACAGTCCAACAATAGGAAC-3
,
58 to
39), STX-2
(5
-ATTAGGCCAGTGATGAGTAT-3
, 842-823), STX-3
(5
-ACACAGACTGATTATCGAG-3
, 412-430), and STX-4
(5
-TTTCCTCCAAATCCTCATCT-3
, 505-486). These primers cover the entire
C. elegans syntaxin A cDNA. After 30 cycles of
amplification using LA Taq polymerase (Takara), the products
were cloned into the pCRTMII cloning vector
(Invitrogen).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Phenotypes of double mutants of unc-18 and unc-64
Cloning of the unc-64 Gene-- All members of the syntaxin family contain a highly conserved sequence located in the cytoplasmic domain. C. elegans syntaxin homologs were amplified by RT-PCR using degenerated primers derived from two short regions in the carboxyl-terminal portion of mouse syntaxin 1A. A C. elegans cDNA library was screened with the 0.5-kb PCR product as probe, and 58 cDNA clones were obtained. The cDNAs were mapped at the region close to unc-64 on the right of chromosome III (Fig. 1A). We tested cosmids B0502 and C26A4 in germline transformation experiments with unc-64 gene allele e246, and an 8.0-kb XbaI genomic DNA fragment was also injected. Wild-type locomotion was restored for the unc-64 allele following the injection of the two cosmids and the genomic clone, indicating that unc-64 encodes C. elegans syntaxin.
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Molecular Identification of unc-64 Mutation-- We analyzed the unc-64 mRNA from e246 animals in the hope of identifying the defect. The mutant RNA is unchanged in content and in size. We generated cDNA from e246 by RT-PCR and sequenced it by linear amplification PCR. The e246 mutation occurs in the coding region of the mRNA (Figs. 2A and 3C), in which conserved alanine at position 248 is converted to valine.
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Protein Structure of C. elegans Syntaxin-- We compared the sequence of the predicted syntaxin with human and Drosophila syntaxins (Fig. 3A). C. elegans syntaxin shares extensive overall sequence homology with other syntaxins: 63% identical to human, and 66% identical to Drosophila syntaxin (25).
C. elegans syntaxin is hydrophilic at the N terminus (Fig. 3B). The predicted secondary structure is an amphipathicThe unc-64 Gene Transcripts-- To examine the expression of the unc-64 gene transcript, Northern blot analyses were performed. As shown in Fig. 4A, at least four bands were detected using the C. elegans syntaxin A cDNA as a probe. The developmental profile of the expression pattern of the major transcripts, 1.0- and 3.0-kb RNAs, are similar (Fig. 4B). That is, two of the transcripts are most abundant at the embryonic stage and decrease with the progress of development. The 1.0-kb transcript is identical to the C. elegans syntaxin cDNAs in size. To identify the source of the 3.0-kb RNA, Northern blotting using various DNA fragments from the unc-64 genome as probe was carried out. However, we failed to find the region of the genome corresponding to the large transcript.
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Expression of the unc-64 Gene-- To determine where and when the unc-64 gene is expressed during C. elegans development, we used in situ hybridization and transformation with the unc-64::GFP reporter gene. The gene is specifically expressed in neurons, and the transcript is exclusively localized also in neurons (Fig. 5). The pattern of unc-64 gene expression and transcriptional localization is very similar to that of the unc-18 gene. However, the unc-18 transcript presents abundantly in the hermaphrodite specific neurons, whereas the unc-64 transcript is not detectable in this type of neuron. Further, the unc-64 gene is expressed in almost all neurons constituting the head ganglion, while the unc-18 gene is expressed in only a limited number of neurons, including AVE, SIBD, and SIBV interneurons.4
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Interaction between C. elegans Syntaxin and UNC-18-- We already showed that in the cytoplasm C. elegans syntaxin binds to UNC-18 (14). To examine further the interaction of the two proteins, we assessed the binding of purified recombinant C. elegans syntaxin to UNC-18 (Fig. 6). Both the N-terminal and C-terminal halves of C. elegans syntaxin were devoid of the binding ability. Only the intact cytoplasmic domain has the binding ability (Fig. 6, A and B), indicating that a full-length of C. elegans syntaxin is necessary for the binding. We compared the binding of the wild-type and mutant C. elegans syntaxin to UNC-18 (Fig. 7). Half-maximal binding of the wild-type C. elegans syntaxin to UNC-18 occurred at 50 nM. The binding ability is approximately identical to the ability between n-sec-1 and syntaxin 1A (~80 nM) (10). However, the binding affinity between the mutant C. elegans syntaxin and UNC-18 was greatly reduced with half-maximal binding at 300 nM.
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DISCUSSION |
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unc-64 Encodes the Syntaxin 1A Homolog-- During the transport of synaptic vesicles, docking/fusion is regulated by synaptic proteins. A previous genetic and biochemical study suggested that the unc-64 gene is required for the synaptic transmission (16). Mutation in unc-64 results in resistance to inhibitors of acetylcholinesterase and in an abnormal accumulation of ACh, probably due to a defect in synaptic transmission (13). Here we demonstrate that the gene encodes a mammalian syntaxin 1A homolog.
By Northern blotting, we detected at least four positive bands (3.0- and 1.0-kb major bands, and 3.3- and 1.6-kb minor bands) using the C. elegans syntaxin A cDNA as probe. Yeast artificial chromosome grids covering almost all the C. elegans genome were constructed by Coulson et al. (28). By hybridization to the YAC grid filter, we detected three YAC clones, Y47C12, Y54C1, and Y21D2, other than Y43F4 covering unc-64. Y47C12 hybridized strongly and was located to the right of lev-10 on chromosome I (LGI). Y54C1 and Y21D2 hybridized weakly and were located to the left side of unc-64 (LGIII), and between gpa-3 and gpa-1 (LGV), respectively. These results show that C. elegans syntaxins constitute a family as to mammalian syntaxins. We are currently analyzing C. elegans genes encoding syntaxins other than C. elegans syntaxin. The C. elegans genome sequence project identifies several genes encoding syntaxins: SYN1, SYN2, SYN3, and SYN4. The predicted peptide sequences of these genes are not strikingly similar to C. elegans syntaxin: SYN1 (27.2% identity), SYN2 (24.2%), SYN3 (22.4%), and SYN4 (25.6%). The SYN1 coding peptide is more homologous to C. elegans syntaxin in amino acid sequence and size (299 amino acids) than the other three SYN genes. The SYN1 gene is located close to the region covered by Y21D2 on LGV, suggesting that the gene may encode a member of the C. elegans syntaxin family. To identify the source of the 3-kb RNA detected by the C. elegans syntaxin cDNA probe, we carried out Northern blotting with unc-64 genomic DNA containing the upstream 5unc-64 Gene Expression-- The pattern of unc-64 expression suggests the gene is directly involved in sensory transduction. unc-64::GFP gene fusions are expressed at high levels in the nerve ring and ventral nerve cord. It is not yet clear whether the three transcripts are differently expressed, either spatially or temporally. In situ hybridization and unc-64::GFP transgenic reporters revealed that unc-64 is specifically expressed in neurons. However, the two methods gave different weights to each expression pattern that is, the unc-64::GFP fusions were expressed around the nerve ring and ventral nerve cord, whereas in situ hybridization detected more unc-64 gene transcripts in motor neurons than in interneurons. These differences might reflect that in situ hybridization is quantitative, whereas GFP expression is qualitative.
Interaction between unc-18 and unc-64-- We provide direct evidence that unc-18 and unc-64 genetically interact. First, the double mutation results in a more severe defect than do single mutations. Second, both gene products have high binding affinity. Third, the binding ability was reduced by the unc-64 mutation. C. elegans syntaxin binds to UNC-18 with high affinity (Fig. 7). The cytoplasmic domain of C. elegans syntaxin has clustered, reactive hydrophobic amino acids consisting of helical wheels. To elucidate which domain contributes to the binding with UNC-18, we carried out a binding assay. The experiments assessing the ability of the binding domain (Fig. 6, A and B) suggest that the intact cytoplasmic domain of C. elegans syntaxin is essential for binding. Mutation at the C terminus of C. elegans syntaxin reduces the binding ability to UNC-18. However, it is unlikely that only the C-terminal region contributes to the binding. While the interaction could be mediated directly, the simplest model is that C. elegans syntaxin interacts with UNC-18 as receptor and ligand, respectively. Vertebrate syntaxin binds to synaptobrevin, a component of synaptic vesicles. UNC-18 has a regulatory function in the binding between syntaxin and synaptic vesicles.
Mutations in unc-18 (md1094) and unc-64 (e246) produce mildly defective phenotypes. However, double mutants show phenotypes severely defective in development and locomotion, presumably due to the great impediment of synaptic transmission. This is because the ACh levels are much higher in the double than single mutants. unc-18 (md1094) has a small deletion in the 3 ![]() |
ACKNOWLEDGEMENTS |
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We thank C. Martin and R. Waterston, who provided the cm04d2 clone and T. Ishihara, who identified cells expressing unc-18 and unc-64 gene. We are grateful to A. Coulson and J. Sulston for providing cosmids and S. Noji (University of Tokushima) for help in performing in situ hybridization. S. Matsudaira and R. Kitamura technically supported the work.
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FOOTNOTES |
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* This study was supported in part by a Grant-in-Aid for Scientific Research from the NEC Corp. from the Ministry of Education, Science and Culture of Japan and from "Research for the Future" Program of the Japan Society for the Promotion (RFTR) of Science (to R. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be 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 GenBankTM/EMBL Data Bank with accession number(s) AB008842, AB008843, and AB008844.
The C. elegans syntaxin family (SYN1, SYN2, SYN3, and SYN4) discussed in this paper has been submitted to the SwissProt Data Bank with accession number(s) Q20024, Q20574, Q20797, and P91409.
§ These authors contributed equally to this work.
To whom correspondence should be addressed: Dept. of Physical
Information, Faculty of Medicine, 13-1 Takara-machi, Kanazawa University, Kanazawa, Ishikawa 920, Japan. Tel.: 81-76-265-2183; Fax:
81-762-34-4202; E-mail: rhosono{at}kenroku.ipc.kanazawa-u.ac.jp.
1 The abbreviation used are: NSF, N-ethylmaleimide-sensitive factor; SNAP, soluble N-ethylmaleimide-sensitive factor attachment protein; v-SNARE, vesicle-associated soluble NSF attachment protein-SNAP receptor; SNAP-25, synaptosomal associated protein of 25 kDa; t-SNAREs, target membrane-associated SNAP receptors; ACh, acetylcholine; GST, glutathione S-transferase; YAC, yeast artificial chromosome; RT, reverse transcriptase; PCR, polymerase chain reaction; LG, linkage group; bp, base pair(s); kb, kilobase pair(s).
2 S. Harada and R. Hosono, unpublished results.
3 A. Fire, S. Xu, J. Ahnn, and G. Seydoux, personal communication.
4 T. Ishihara, personal communication.
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