The unc-13 gene has been mapped to linkage group I, 0.025 map unit to the right of unc-15 (5, 14), the structural gene for paramyosin, which was cloned by ...
Proc. Nati. Acad. Sci. USA Vol. 88, pp. 5729-5733, July 1991 Biochemistry
A phorbol ester/diacylglycerol-binding protein encoded by the unc-13 gene of Caenorhabditis elegans (protein kinase C/regulatory domain/nervous system/cDNA cloning)
ICHIRO N. MARUYAMA AND SYDNEY BRENNER Medical Research Council Molecular Genetics Unit, Hills Road, Cambridge CB2 2QH, United Kingdom
Contributed by Sydney Brenner, March 6, 1991
Mutations in the unc-13 gene cause diverse ABSTRACT defects in the nervous system of the nematode Caenorhabditis elegans. Molecular cloning of the gene and sequencing of the cDNA revealed that the product encodes a protein, 1734 amino acids in length, with a central domain with sequence similarity to the regulatory region of protein kinase C. The domain was expressed in Escherichia coli and shown to bind specifically to a phorbol ester in the presence of calcium; diacylglycerol inhibited the binding in a competitive manner. These findings confirm that the unc-13 gene product has binding sites similar to those of protein kinase C and may be a component of an alternative transduction pathway of the diacylglycerol signal to a different effector function in the nervous system. In Caenorhabditis elegans, unc-13 mutants have uncoordinated movement and slow irregular pharyngeal pumping. There is evidence that the gene affects the nervous system rather than the musculature, which shows no visible defects. Abnormal connections between major interneurons through gap junctions have been found by reconstruction of part of the ventral nerve cord by serial section electron microscopy (I.N.M., unpublished results). It has been found by staining the nervous system with specific antibodies that motor and sensory neurons are misplaced in the unc-13 mutants (S. Siddiqui, personal communication). The mutants are also resistant to acetylcholinesterase inhibitors, such as aldicarb, but the enzyme is not altered (1). They also accumulate abnormally high levels of acetylcholine without alterations in the levels of choline or choline acetyltransferase activity (2). In this report, we describe the isolation and molecular characterization of the unc-13 gene* and show that it encodes a protein with a phorbol ester/diacylglycerol binding activity and a domain homologous to the regulatory region of protein kinase C (PKC), a multifunctional kinase that plays a central regulatory role in one of the major signal transduction pathways (3).
MATERIALS AND METHODS Nematode. The following C. elegans strains were used in this study. N2 is a standard wild-type; strain eSJ/e2274 is a partial revertant of the eSI mutant, e2153 and e2312 are weaker mutants spontaneously isolated from the background of a mutator strain, TR679 (4), and eJO91 is an amber mutant isolated from N2 after ethyl methanesulfonate mutagenesis. The conditions for growth, maintenance, and genetic manipulation of the nematode have been described (5, 6). DNA Procedures. Restriction enzymes, T4 DNA ligase, and Escherichia coli DNA polymerase Klenow fragment were used under the standard conditions (7). DNA sequencing was carried out by the chain-termination method (8) after cloning relevant fragments into M13 vectors (9). Oligonucle-
otides were synthesized by a DNA synthesizer (model 380B, Applied Biosystems). A genomic A library was made with A2001 (10) from N2 genomic DNA by partial digestion with Sau3A1 restriction enzyme, followed by purification of 15- to 20-kilobase (kb) fragments in an agarose gel. For cloning of the unc-13 cDNA, two libraries, a AgtlO phage (11) library provided by J. Ahringer and J. Kimble (University of Wisconsin, Madison) and a library constructed with AMGU2, were screened. The cDNA was made according to the protocol defined by the supplier (Amersham) from 10 ,tg of mRNA preparation and was cloned into AMGU2 with synthetic adaptors. Construction of the vector will be described elsewhere. Phorbol Ester Binding Assay. The segment of unc-13 cDNA, BamHI-EcoRI, that encodes amino acid residues 389-1090 was cloned in frame into a plasmid expression vector (12). The construct was transformed into E. coli BL21(DE3) cells, and when the bacterial cultures reached an OD6w of 0.5, recombinant proteins were induced with 0.5 mM isopropyl 8-D-thiogalactopyranoside for 3 hr. The cell cultures (10 ml) were centrifuged and pellets were resuspended in 200 A.l of lysis buffer (20 mM Tris HCI, pH 7.4/0.25 M sucrose/2 mM dithiothreitol/2 mM phenylmethylsulfonyl fluoride with lysozyme at 0.6 mg/ml) and kept on ice for 30 min. The suspensions were sonicated for 1 min. The total proteins were used for the binding assay under the published conditions (13) with minor modifications; 10 1.L of total proteins from a culture was incubated in 200 ul of 50 mM Tris HCI, pH 7.4/30 nM [3H]phorbol 12,13-dibutyrate ([3H]PBt2, 44.8 Ci/mmol, Amersham; 1 Ci = 37 GBq) in the presence of 2 mM CaCI2 or 2 mM EGTA. Phosphatidylserine was not added to the reaction mixture, because it had no detectable effect on the binding under various conditions tested. After incubation at 30'C for 15 min, the bound [3H]PBt2 was separated from the free ligand by filtration through a Whatman GF/B glass fiber filter pretreated with 0.3% polyethyleneimine and was washed five times with 1-ml portions of ice-cold 20 mM Tris HCI, pH 7.4. Nonspecific binding was determined under the same conditions with addition of 30 1LM unlabeled PBt2.
RESULTS The unc-13 gene has been mapped to linkage group I, 0.025 map unit to the right of unc-15 (5, 14), the structural gene for paramyosin, which was cloned by screening an expression library with a paramyosin-specific antibody (15). Using the paramyosin clone as a probe, we started chromosomal walking by screening a genomic library made with A2001. Phage A clones identified by hybridization were "fingerprinted" by A. Coulson and J. Sulston and matched by computer to their set of ordered cosmids (16). To determine the orientation of Abbreviations: PBt2, phorbol 12,13-dibutyrate; PKC, protein kinase C. *The sequence reported in this paper has been deposited in the GenBank data base (accession no. M62830).
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 5729
5730
Biochemistry: Maruyama and Brenner
Proc. NatL Acad. Sci. USA 88
the overlapping clones to the genetic map, we looked for genomic polymorphisms due to the sDf6 deletion with a left break point between the unc-15 and unc-13 mutations (14). One of the cosmids, C05B12, hybridized to a polymorphic band on Southern blots of genomic DNAs prepared from the sDff6 heterozygotes (Fig. 1 A and B). Then, polymorphisms due to unc-13 mutations were sought by hybridization of the serial cosmid clones to genomic Southern blots of unc-13 mutant DNAs. Polymorphisms were found in all four unc-13 mutants isolated from a TR679 background, which is a transposon high hopper strain (4), in one partial revertant of the eSl allele, but in none of 20 ethyl methanesulfonateinduced alleles. All of the polymorphisms were detected by the cosmids C44E1 and ZK516, but not by any others. The results with ZK516 are shown in Fig. 2A; a deletion of about 2.0 kb was detected in the e2312 allele, and complex patterns, possibly involving DNA rearrangements, were found in e2153 and in a partial revertant, eSJ/e2274, of the eSI mutant. These alterations were located within a 9.2-kb HindIII genomic DNA fragment, which was identified by testing subclones of the cosmid C44E1. To identify the unc-13 product, the 9.2-kb HindIII fragment was used as a probe to select clones from cDNA libraries prepared from mRNA from mixed-stage populations. After screening about 106 phage plaques, 11 positive clones were isolated. From the cDNA clone (2C1) with the largest insert, a 3.3-kb EcoRI fragment was subcloned into a pUC12 vector (9). With this plasmid clone as a probe, the unc-13 mRNA was identified in a Northern blot (Fig. 2B) as a 5.9-kb mRNA both in wild type and in the e1091 amber mutant. However, the 5.9-kb mRNA was reduced in size to 5.1 kb in the e2153 A
unc-15
(1991)
mutant, proving that it is coded for by the region associated with unc-13 mutations. By sequencing a set of cDNAs and part of genomic DNA from the A clones, we determined a single open reading frame surrounded by in-frame stop codons at the 5' and the 3' end, suggesting that the cDNAs encode the entire coding region (Fig. 3). The genomic DNA sequences provide us the transcription direction of the gene on the genetic map, shown in Fig. 1D. From the DNA sequences and a restriction map of the mutant e2312 genomic DNA, it was determined that the DNA in the 2.0-kb deletion of e2312 covers an exon coding for 65 amino acids, from 110 to 174. Based on this result and the genetic map positions, we searched for the sequence changes of the amber mutant el091 and of the eSI allele in their 9.2-kb HindIII genomic fragments, using as primers a set of synthetic oligonucleotides chosen from the wild-type cDNA sequence. The results are shown in Figs. 1C and 3. The mutation in eSI is a C-to-T transition converting the CGA codon of Arg-390 into a TGA stop. The mutation of el091 is a G-to-A transition converting the TGG codon ofTrp-431 into a TAG amber stop. These results are consistent with the genetic map positions of the mutants and with previous evidence from suppression that el091 has an amber mutation. Thus, the unc-13 gene is on a chromosomal region associated with the polymorphisms described above and codes for 5.9-kb mRNA. However, we have not identified the 5' and 3' ends of the mRNA, and the chromosomal region must therefore extend beyond the 35 kb characterized. A C'
unc-13 (e5l) Z
aW
ID
a
kb
S_-
0.01 m.u.
B
C05B12
ZK516 C15A11
bLPM5
X53
C44E1 -
C 0) ~0)
? I
-Ii
i
II I
IIIi
IIEI11iI
10 kb
/.
III
II
_0
.,
4m ,$
..
B
_~~~~
m _
aa
oD
z
- 36 ,-E
..4s W ..
I
HindIIII
"
_0
4.7
-.4
2 6
Amo
low.
-2. 1
-
26S
EI 4 kb
X18
X24
-
1.5
1SS
X38 X41 D E
B
E
HE
E
B
E 1 kb
2C1 -Cl
C3
2C4 2C5
FIG. 1. (A) Genetic map of the unc-lS-unc-13 interval. Region of the sDf6 deletion is indicated by an arrow. m.u., Map unit. (B) Cosmid and A clones spanning the genetic map interval. APM5 encodes the unc-lS (paramyosin) gene. (C) Restriction map of HindIII cleavage of genomic region covered by cosmids C44E1 and ZK516. The map was constructed using the cosmid clones and A clones depicted by lines under the map. Closed and open boxes under the map are exons identified from the genomic sequence and restriction fragments hybridized to the cDNA probes, respectively. Arrowheads above the map indicate the positions of the mutant alleles. (D) Restriction map of the unc-13 transcript deduced from the sequence of genomic and cDNA clones. The protein-coding region is defined by a thicker line. The transcription direction is indicated by an arrow. The lines under the map are the representatives of cDNA employed for sequencing. H, HindIII; E, EcoRI; B, BamHI.
FIG. 2. (A) Southern blots of genomic DNAs hybridized with the cosmid clone ZK516. Genomic DNA (1 ug) from mixed-stage population was digested with Xba I, electrophoresed in 0.7% agarose, and transferred by capillary blotting to a nylon membrane with 10x standard saline/citrate (SSC). The membrane was hybridized at 680C in 6x SSC with 32P-labeled cosmid ZK516, followed by final washing at 68°C in 0.2x SSC for 30 min. (B) Northern blot of total RNA from mutants and wild-type. Total RNA was prepared from mixed-stage culture according to the published procedure (7). RNA, 30 ,ug per lane except for e1091 (100 ,ug), was electrophoresed in a 1.2% agarose/formaldehyde gel and blotted onto a nylon membrane by standard methods. The membrane was hybridized at 42°C in 5 x standard saline/phosphate/EDTA (SSPE)/50% formamide with the 32P-labeled cDNA clones (p2Cl). rRNA size markers are indicated.
Biochemistry: Maruyama and Brenner
Proc. Natl. Acad. Sci. USA 88 (1991)
The predicted protein from the DNA sequence contains 1734 amino acid residues. A database search revealed significant similarity between a domain of the unc-13 product G
M
Q
V
R
L
W
D
K
L
and the regulatory domain of PKC, the central 300 amino acids of unc-13 corresponding to the N-terminal half of PKC. This region of PKC is involved in regulation of the enzyme I
G
V
H
Y
M
P
L
S
I
E
R
Y
S
24 D 120 64 240 104
GMATTCATTTTTGAACAMAArCGACCTGACGACGGAA GGTAC TCGAATTA TGGGcc.AGGG TTTTATGGGACAMTrTAATAGG TGTTCATTATATGCCACTTrCGC.AGA TTAGATACA N
A
A
G
S
G
0
N
L
Q
M
D
H
E
L
S
G
L
S
E
D
S
D
Y
T
S
D
V
S
V
E
T
R
N
P
V
N
H
G 0 T V G T R G P T G H N L L T D V R F E GCAATGCAGCAGG TTCGGGTCAATGGC-rTCAAA'rGGA TCATGAAC T;AAACAAGAAATGG TCMAC TGTAGGMCACG TGGTCCMACAGGTCATAACTTG TTGACTGATG TTCGAT TTG L P F D V Q G H D E D V Q S R L L A L N G L I E H D Q L G P N N H H R A P F N H AGC TTCC TTTTGATG TAC-AACGCCACGACGAAGATrGT~kG rCcAGATTGCITCGCGTTGAACGGACTCA TCGAACACGATAGCTGGGCCCCAACAACCATCACAGGGCGCCG TTTAAC C
AT=rGGG TTTGAG rAAGACAGTGATTATACATCCGACG TG rCcG R
L
Q
L
H
T
E
R
G
A
A
S
Y
F
H
H
L
Q
P
S
N
S
H
A
Y
Q
E
H
S
L
P
H
H
3360
T
R
144 4 80 184
CCAGTCAACCATCATICArTACATCCAACAGTTCGGCACATCMATACGAATCACAT TACATCCACAT'CGAA
D
E
E
D
A
Y
H
A
R
H
Q
E
P
S
D
D
Y
H
N
T
D
Q
0
D
Y
H
CACGTC.AATTG.TTACACACTCGTGAAGGCGCAIGCGAG TTAs GASGACGAAGAGACGCC TAICACGC TAGACAT'CMCCCGATCCGACGACTATACCACCAAGACACCTACGATCAAC S
S
Y
Y
N
D
E
Y
A
P
G
S
S
S
S
Q
Y
R
Q
G
Q
0
Y
D
Q
D
Q
Q
H
N
Q
Y
I
D
E
T
V
P
T
6600
I 224 720 264
V
A ~rc TCGTATTATAACGACGMATACGCGCCMAGTGGTAGTAGCAGTCMTATCAACGGCAAGGATATCAAQATCAAGA TCACAACATCAGAATATATACGAGGACACGGTGAC TCCGG R E F G E S A V P P A A S S S R R Q F D Q V Y G Y A S S S E E R Y D T P M S S G
TTCGAGAATTTGGCGAGTCGGCCGTTCCGCCGGCTGC
5731
TCATICGTCTAGACGTCAATTCGATCAGGTTTACGGGTACGCATCTAGCAGTGAAGAACGATATGACACACCGATGTCAAGTG 8840
R L P R D E P I L E H S E P E Y V Y D Q N G Y P E E D N Y G I N P T Y S E D H F G TCGACTGCCAAGAGATGMACCAATTCTTGAGCACAG TGAACCAGATACGTiG TCGATCAGAATGG E G Q T N D Y S T T H 0 E P N D F R N D Y N S S Y Q R E Y N N E S E P L S Y N S T T'GMGG TCAMKAAATGACTAT TC TACGACACATCAAGAACC AATGATTTTAGGAATGATTATAATTC TA^GTTATCAAAGAGAATATTGGAATGAGAGTGAGCCGC TTTCT TATAATA R P P N G H I R T G A N T W R E P S T S S R P T S S 0 A W N Y Q D D T H Q Y D E G TAGGCC TCCAAACGGACATATTAGAACAGGTGCAMACACTTGGCGCGAACCATC TAlCGTCATCICGcCCAACATCTTcCCAAGCATGGAATTATICAAATGACACTCACCAATArGATG V D R G S\R V S F T R T P S V D R T D R P S E S G G G F Y D E M S E S G R P G R AAG TTGATCGTGGATCCCGAG TATC TTTCACAAGAKCACCATCAiGTAGArAGAACAGATAGACCAAG TAAAGTGGTGGAiGGATTTTATGArGAAATGTCAGAAAGTGGAAGACCAiGGGC P D S H H N WNR Y D S I Q E E D N E K D N N K Q H V E G Y E E G 0 E E K Q K D N G TCCAGATTCTCACCATMATTGGAGATATGATTCAATTCMAGAAGMAGATAMTAAAMAGACAAC TGAAACAACATGTGGAAGGCTACGAAGAAGGACAGGAAGAGAAACAAAAGGACA Q K P N D H S A A S P 0 D H Y H R S D S T A Q Q D F G N N I V R Q T I Q E E E E A K R N Y Q E L W H N A Y K R V C A D L G I K S T V L D G N G S S A A N A F Y K S AMMAAGGAATTATCAGGAAC TTTGGCATAATr&CTTACAAGAGAG TTTG TCTGA~TCTCGGMATTAAGAGTAC TGTGCTTGKCGGGAACGGGAGTTCGGCTGCCAACGCCTTC TACAMAT I D A A P N M N V A R T K T S I P L V S E L V L K T M A T K R A Q A G L A N A A
304 960 344
TTATCCCGAAGAAGACAACTATGGAATTAMCCAACATATT'CGGAAGATCATT
rAAAAACCTAMTATCATAGTGCAGCTrcACCTCAAGATCACTATCA~rGATICAGATTCAACAGCGCAACAAGATTTCGGAAACAATATCGTTCGICAGAMTCAAGM"GAAGAGG
1080 384 12 00 424 13 20 464 14 4 0 504 15 60 544 16 80 584
1800 CM1T0CGATGCAGC0TCCAMCAMTG TGGCCCGAACGMGACAAGTATTCCACTT0GTTrCGAACTTGTTCTAAAGACGATGGCCACAAAACGTGCGCAAGCAGGTCTGGCGAATCGG
R T T F S D T E L K T H V Y K K T L Q A L I Y P I S A T T P H N F A T T T F Q T 624 CAAGAACGACG TrTCTAGATACAGAATTAzAAAACTCArG TCTACAAGAAACTCTACAAGCATTGATCTAiCCCGATCAGlTGCAACAACTCC TCACAACTTCGCAACTAiCAACATTCCAAA 1 920 P T F E G L L W G L A R 0 G L R Y E 0Q V K V H D X Q R E L L S A 664 CACCCACGTTTTGTTACGAATGCGAAGGATTATTGTGGGGArTGCTAGACAAGGATTGCGATGTACTCAiGTTCAMGTGAAAGT~TACATAAMTGTCGTG.AATTGCTCAGCGC TGATT 204 0 L Q R A A E K S T K H G E A D R T Q S L V N V I R D R M K I Q E Q N K P E V F Q 704
0
G
D0Q
2160 2C1TTCAGAGCAGCCGAGAAGTCGACGAAACATGGAGAAGCTGATA0GACAGTCACTTGTAArGTTATCAGAGA0TGAATrAGAT7CAGGAACAGAATAAGCCTGAAGTGTTTC M I R T V F V D
E
D
I
N
K
Q
E
L
T
K
K
V
T
S
A
I
L
E
G
N
K
S
S
S
A
K
I
T
T
L
L
V
744 22 80 784 24 00 N E K F H F E C H N S T D R I K V R V W D E D N D L K S K L R Q K L T R E S D D 824 GGAATGAGAAATTTCATTTTIGAMTGCCACAACTCCACGGACAGAATTAAAGTTCAGTTTGGAGACGGATAATrATrrGAAMTCCAAGTTAAGGCAAAAATT'GACTCG TMTAC TATG 25 20 F L G Q T V I E V R T L S G E M D V N Y N L E K R T D K S A V s G A I R L H I N 864 ATTTC 2 64 0 V E I K G E E K L A P Y H V 0 Y T C L H E H L F A A H C V D E E V K L P K V R G 904 ATGTTGAMTCMAGGGAGAAGAGMAAC TTGCACCGTATCATGTTCAG TATACATGTC TTCATMACATC TTTTTGCTGCT'CATTGTGTAGATMAGAAGTTMAAC TTCCAAMAGTTCGAG 2 760 E D S W K V C F Q E T G Q E I A E E F A M R Y G I E S I Y Q A N T H F A C L C T 944 GCGMAGATAGTTGGAMAGTTTGC TTCCAGGAAACTGG 7TCMGAGATTGC TGMAGAATTTGCGATGCGGTAT(GGMTTGAMAGTATTTACCAGGCCATGACTCATTT'CGCATGCCTATGTA 28 80 R Y M C A G V P A V L S T L L A N I N A Y Y A H T T A T S A V S A P D R F A A S 984 C TAGATACATG TCGCAGGAGTACC s-vCAGTCTTGTCMACACTTTTGGCCAATATMAATGCGTAC TATGCTCATACAACr TCACGTCAGCAGlTC TCTCTCC l'ATAGATTTG TCAT 30 00 N F 0 K E R F V K L L D Q L H N S L R I D L S A Y R N H F P S S S P A K L 0 D L 1024 CCAAC TGCCAAAC TTCAAGATC 3120 K S T V D L L T S I T F F R M K V L E L A S P P R A S T V V R E C A K A C M Q Q 1064 TTAAATCMACCGTTGATCTTC TTAC TTCAATTACATTCTTTCGCATGAAAG TTCTCGAG TTGGCGAGCCCACCGCGAGCATCAACAG TTGTTCGCGAATGCGCAAAMGCCTGCATGACGC 324 0 T Y Q L N F E S C A E Q F P I L D T S V 0 F N Y E F I D Y I M R V I E E D Q K N 1104 AGACC TATCAACTGATGTrTGMATATGCGC TIGACAATTCCCAATTTTGGACAc TTCAGTTCAATTCTCGGTATGAATTCATCGACTATATAATGcGAG TAATT'GAAGAAGAtCAAA 3 360 Y T P A L N 0 F P Q E L N V G N L S A E T L W S M Y K N D L K M A L E E H A 0 K 1144 34 80 K R C K 0 P E Y N N L Y F K V K G F Y F K Y V A D L S T Y K S S I P E F P A N F 1184 AGAMACGATGCAAGACGCC T 3 600 I P F V N 0 N L N E N D E H S M D I L R N A Y N C R 0 A D T S H K H Q N T Q I L 1224 'rTArrCCATTCGTCATGGATTGGCTCAATGAAAATGAcGAACATTCGATGGATATr-I CAGAAAT'GCATACAACTGTAGACAGC TACAC TTCCCACAAACATCAGMACACACAMATTC 3 720 EH V V 0 V F T 0 L N A A L K L L K Q N 0 C P N P E V A A 0 N N K KR F S K 0 L N 1264
AGATGATTAGMACAG
TTTTMATGTGGATGAGAATAT~TAAAAGAiGACT-TTGMAACTGTMAGGCGTCTATTTTIGAAGGAAGTAWAAATGGAG TGCAMAGATTACATTGACAGTGC C A Q G L I A K D K T G K S D P Y V T A Q V G K TIK R R TJ R T I H Q E L N P V N TTTGTGCGCAGGGAC TTATTGCCMAGGACAAMACCGGAAAAAG TATCCATATGTGACAGCTCAAGTTGGAAMGACCAAACGAAGGACACGAACCATACAiCCAAGAGCTCAATrCCAGTTT
TTGGGACAAACAGTAATTGAAGTTCGAACTTTATCTGGTGAAATGGATGTCTGGTATAATCTTGAAAAGAGAACTGATAAATCTGCTGTATCCGiG~cCATTCGATTGCATATCA
TTCGGAAAAGAACGATTTGTGAAACTTCTTGATCAACT~TACACAGTTTGAGAATTGACTTGTCAGCATATAz;GAATCATTTTCCCTCrTCGTCGCC
ACTATACACCAGCATTGMATCAATTCCCTCAAGAACTGAATGTrtGGAATTTA~TCGCTGAAACACTTTGGAGTATGTACAAGAMTATTTGAAAATGGCATTAGAAGAACATGCACAAA
FIG. 3. Nucleotide sequence of the unc-13 cDNA and
TGAATATATGAATTTATATTTCAAGGTTAAAGGTTTCTATTTCAAATAMTGTGCTGATCTTTCAACATKCAAATCAlTCMTTCCCGAGTTTCCTIGCATGG
TCGMCATGTTGTTGATGTGTTCACCCAACTAAACGC CCAAATCAGAAGTTGC CTGATATGATGAAGAGATTAGTAAAACATTA 3840 3840TTGAAGCTCCITCMKAAATGGKCG K V L L A Y A 0 N V 0 K 0 F P K F A H D E K L A C I L M N N V 0 L K 1304 Q L R V
TACGAAACAATGGGCGGAGCAGAACTTGACGAACATATTGGACAAGITGCTTACAGTCC71r-X
TTaCAAACACAAGTICAACCGGAAGCCGATGCAGTTCTAGAACCGTTGA
AGATrGTTACGAAGTTAATGTCAACAAACAT'CMAAGTATAAAGGGAATGAAT~TCGTTAAAGATA TGACGCCATTAAAGACAGTlbrrArklrrr
FKE T T CATTCTGAAAACACCTAACTCCAATC TCCAAAAATGCACATCAGTTTATATACACAGACTAC L
K
S
P
L
E
V
Q
G
E
V
V
S
0
K
L
S
Y
0
V
D
L
F
S
P
H
G
E
QL
I
K
-.
TF I T S R P D L S1544 4G6CAAC80TCA CGTTTATTACA80CA CGAC GATCTCCAA 4680
K
A
0
V
K
I
L
A
A
0
N
0
0
L 480G0GCACCAGTTGGAGGTTAGTGTTCMGTTGACTTATTCAG TAATCCAGGCAC ;GGGAACAGMGGCTACCGTTAAAATCTTCCGCAAACGATCTTCGATGGCAAACC T
G
E
Q
T
D
R
W
T
1584
S
4800
F
K
P
predicted amino acid
se-
from
E Q ATAAAGTTC TTCTTG.CATACGCTGA rATGTTCAAAAAGAMTTCCGAAATTTGCACATGACGAGAAGCTTGCGTGTATTC TAATGAACAATGTTCAGCAATTACGTGTGCAACTCGAAA 3 960 I Y E 0 N 0 0 A K L 0 K H I G Q V L T V L Q K K L N S V L D R L S A E F V T T L 1344 AGATC MAFGAGTTGAATTCTGTTC TAGACAGACTTTCAGC TATrTG TACCACTT 4 080 E P H I H E Q TSI K L GNM L L V K I K G P Q L Q K T Q V Q P E A D A V L E P L M 1384 TGGAACCCCATATTCATGAACAAACAAT'CAAATTGGGTAMtCTCC TCGTTAAAATAAAGGG TCCACAAC 4 200 0 L L K 0 5 L R 8 Y A 0 0 C K K 0 V L K Y I L K E L N K I T I V N M E K R V V L 1424 TGGATCTrTCTTGAAGGA TCATTGAGAAGATACGCGGA~TAG TGTGAGAJAAACGGTTC TGAAGTATAT~TCTCAAAIGMCTTT(GGATCATTATTGAAAATGTC432 C4320 P P L 5 0 K A L L K 0 L P N A K I 0 D V T K L M S T N N AATAArGTAAGGAAAAT Q S I K G M N S V K D M 1464 TACCACCATTATCAGATAAGGCTCTATTGAAACAMcTwGCCGMTGccAA-TT 4 4 40O N 0 N A R K S E K S L 0 P K 0 C T V L 0 C A L D A I K D S F H A S G K G L K K S 1504 -GA GATAlGCAAGAGArCTGAAAAAC TC TAACACCCAGACAAGTACAGTTTTGATTGT-GCAC F
the
quence of its product. The entire coding sequence was obtained
overlapping cDNA clones by sequencing both strands. The first methionine immediately after the stop codon in the longest reading frame was taken as the first amino acid and the stop codons in the same reading frame are indicated by asterisks. The segments having significant similarity to PKC are underlined. Potential serine or threonine phosphorylation sites (17) are boxed. Typical cysteine recommon in both unc-13 peats 1 41and PKC are circled. The posi-
A P K F N F 1624 KK 4GTCAGC9T20AAACCATTTGGTNAAG TGCTCGTCGGACCACATCTTTCAGACAAGAGAMATGGTCGACAMGACCGCAGGCAACTGGGCTCCAA ACGAACTT HFF L G N E G E P E H Y E L M F Q V K D Y C F A R D D R V V G V G V L L S S 1664 Itions of the mutations, C to T TT'CATTTiC CTCCTTGGAAATGMGGAIGAACC TGMCA TTACGAGCG TTATGTTTCAAG TCMGGATTACTGTTTTTGCACGGAGGATACGAGTTTGT.TGAGTTGGAG TTCTGCAAGTTATCAT 5040 iand G to A, that occurred in the L I L L I K
K
K
A
N
920
4
Q
0
C
A
V
L
T
R
T
V
L
H
I
D
E
T
G
A
S
S
L
S
Q
R
0
T
D
E
V
A 1704
516T0ATCAAGCGGGATCC TGCGCCATGTGGGT TCAACT TACACCGGAC GCATATCGACGAGACTGWGCATTTTCTCAGGATTTTCACAGCG TAAScATGATGG 5110 F
CAAAGGAC A
VKR
L
K
T
E
C
R
Y
E
T
C0A rGC0CTAAACCAAA G
GAA
E
0AC0
M
A
A
S
ATGGCTGCATCAGCATCAAGTC
N
I
N
R
T
*
*
1734
CMCACA2GA0CATAGT TCAAGTGCCTTTTTTTGTTGG 5A3M76ATAAGT GTTCTTCATTCGATCATTTCATCTCCTCTACGAATACCTGTACATTTTGTGTTTTTTTTTTCTCGATTC 5
5
280
e5 and e1091 alleles are marked by arrows above cytosine at 1218 and guanine at 1342,
i t I
respectively.
376 r
Biochemistry: Maruyama and Brenner
5732
Proc. Natl. Acad. Sci. USA 88 (1991)
and includes the highly conserved CI and CII subdomains, which are responsible for the diacylglycerol (or phorbol ester), phospholipid, and Ca2' dependence of the kinase activity of this protein (3). The CI subdomain of PKC typically contains two repeats of a cysteine-rich sequence,
T
4-
Cys-Xaa2-Cys-Xaal3-Cys-Xaa2-Cys-Xaa7-Cys-Xaa7-Cys,
3.
0
but only one is found in the unc-13 product (Fig. 4A). However, a single repeat has been found in a new PKC enzyme, rat ; type (24), in the human rafoncogene product (25), and also in n-chimaerin (26), a sequence with unknown function that is specifically expressed in the human brain; furthermore, one repeat is sufficient to bind phorbol ester (13). The unc-13 product has also a region homologous to the CII subdomain of PKC. This subdomain is responsible for Ca2" dependency of this enzyme activity. Some PKCs, rat 6 and E and rabbit novel type, do not have the CII subdomain, and this correlates with Ca2+ independence of the kinase activity (23, 29). A similar sequence is also found in the phosphatidylinositol-specific phospholipase C (30) and a synaptic vesicle-specific protein (28) (Fig. 4B). The putative ATP-binding site, Gly-Xaa-Gly-Xaa2-Gly-Xaa16-Lys, which is observed not only in PKC but also in many other protein kinases, is absent in the unc-13 product, as are other sequences characteristic of the kinase domain. This indicates that the product is unlikely to be a protein kinase. Except for the central domain, the unc-13 product has no significant similarity to any protein in the databases available, including GenBank, Swiss-Prot, and the Protein Identification Resource (Release 28.0). The sequence analysis suggests that the unc-13 product may interact with phorbol esters and Ca2". To investigate these possible activities, a recombinant protein containing amino acid residues 389-1090 was expressed in E. coli by subcloning a 2.1-kb BamHI-EcoRI fragment of the cDNA into a plasmid expression vector (12). The construct has a segment homologous to the CI and CII subdomains of the PKC regulatory region. The total protein prepared from bacteria harboring the construct was analyzed by in vitro binding to PBt2 in the presence or absence of Ca2+. Fig. 5 shows that the protein specifically binds to PBt2 in a Ca2+_ dependent manner as found in PKCs which have the CII subdomain. The binding to phorbol ester was inhibited competitively by the diacylglycerol diolein, with 50%6 inhibition at
U x 4J
E:
2-
77 4
1-
0 II
--Al.
2
1
FIG. 5. Binding of phorbol ester to the recombinant unc-13 proteins. [3H]PBt2 was reacted with total proteins from bacteria harboring plasmids in the presence (+) or absence (-) of Ca2+. Bars 1 and 2 correspond to proteins from bacteria harboring the plasmids without inserts, and with the fragments encoding amino acid residues 389-1090, respectively. The rightmost bar 2 shows the [3H]PBt2 binding in the presence of diolein (10 pug/ml). The specific phorbol ester binding activity was expressed as cpm per 10 ml of culture (OD600 = 0.5). The results represent the mean ± SEM of three to six independent experiments using separately prepared samples.
=10,g/ml, which is roughly the same as found with PKC. However, unlike PKCs, the protein does not require phosphatidylserine for binding under the conditions tested.
DISCUSSION In C. elegans, most genes are localized in clusters on the five autosomal linkage groups (LG). On LGI, of about 100 genes positioned, 35 are located within the 2-map-unit interval dpy-5-in-10, and 10 genes are found in an interval of 0.2 map unit. This clustering might reflect nonuniform recombination
A U N C - 13
6 1 °
TPA- 1 d PI bP K h PX r PK r PK r PK r PK
C C a C b Cg C d C e C z
h R A F n C H
86
0A TPHN F
IVD Ii
TT TFQ T P T F C
jFICE]YjNJFKSPTFC
126
P F TY A G P T F C H NFE H F I HT Y G S P T F C RS I H T Y S S P T FC RS K H IF R N H K r R L H S Y S S P T FC D P HIRIFKVYNYOSPTFC VNIPHIX FGIHNY V PTFC YRAN GHJLFQAKR NR AYC
134
VPLETHFHN
1 0 6 9 7 9 7 9 5 226
230*
Ii
41
0P
D P D P D P NI
KYEXI
RK TFILELA
TV V H F
Y
C D I
RrGEHWC
GLARQGLRC T
E CEGLL
Q
rm
n
H CG S L Y G F Q G L RC E v H C GS LLYIGIY HQ GL C S A C D T H C G S L L Y G L I HIQ G H C G S L L Y G L I H Q GO HC D T H C G S L L LGC LL H Q G K C D H CGT LLWLG V QG LC LQCL C K v H CGSLLG G Q C S EIW G LAR GYR C I N D D D D D D D
BY
CQKF
--L--OFKC
:ANFM WLIA
OVEK
QT
AD
,V
L Q ROARKK GVNQEHS LIC HiECEIEMi]N
DEC
|N|D M NSH 9AA
A KC
E
L[A
L
KujN
V
DC
P SL C G C D H T
|D M N|VH M M N|V HI KRCVMNVP SLC E M N|V H RKCVKSVP SLC G M N|V H HlC EKVAN L C NNVHV RERCE TNVAP N C K K L L H| K RClH VEV P L TIC KQCVINVPSLC
G Y0Fi EHCSTKVP TNC G I NE KQJCSEKVPPND
EI
G MND H TIE.AJ G T D H T ER G V D H T E R
G I
NQK L L
RG I RRHNDSV G VD V D
W SN
I R
LK
HV
K PD
B U N C
-
1 3
dPKC
b P K Ca b
r P
K Cb C
7 3 6
EliL
K g bP LC
7
1 0
P C
8
r P 6 5
LTCVIR
[A
GIADKTKSDPY
V IKEGRNLIP L LIP 1 70 DBEKLH VERD 1 7 0 R E VP L I V VRDAK P I4 VG ARNLP 1 7 0 MD 179
2 8 6
A
flH
AiC I EGAR
G[-VV
E
- - -
TEV- -TRR|TRTI|H-QELNPVWNEK-H
EC HNT --K-RW D E D|
DPNGLSDPYV KVKLIPDDIKIDQSKKKTRTIK-ACLNPVWNET-LTYDLKPEDKIDKRIRL-IEVWDVD -TFKLKPSDKDRRLS-EEW DWD DPNGLSDPYVKLKLIPDPKNESKQK D L P D PN|GL| S P YT YVK LK I D PSS S|LN TIK- C KDRgR PE VNET KE D L S EI VDD OK -|FIFIL D
TTIR-STLlNPKWQES
-nV
RNlLTKQKTKTK-ATLlNPVWNET-F -F PV AMP FlNLKPGDVVURRLS-EjEVWD HL-P0NGDPlNGLSDPY GIVCP8 VIEVGAEYDSIKTE VV|YEED QNUR VPFEQIQKVQVV[DTVL
KNLKKMIVGGLDPY
KLKLIPDP
FVVD NG|LN
I SP E F AF
P
-
LR
HF
K
IH L
L K K KT-
K K N Tk L
Y
YD
YES-F
FIG. 4. (A) Alignment of the sequence of the unc-13 product with the various kinds of PKC CI subdomains. dPKC stands for Drosophila PKC, likewise b for bovine, h for human, r for rat. PKCa stands for PKC type b for ., g for y, d for 8, e for E, and z for The amino acid sequences of PKCs used in the alignment are TPA-1 (tpa-J product; ref. 18), dPKC (19), bPKCa (20), bPKCb (21), hPKCb (21), rPKCg (22), rPKCd (23), rPKCe (23), and rPKCz (24). All PKCs except for rat C type (rPKCz) have two repeats of six cysteine residues at the conserved positions, but only the second segments are employed for alignment. Also shown are the sequences of the human raf gene product (hRAF; ref. 25) and n-chimaerin (nCHIM; ref. 26). Identical amino acid residues in the unc-13 sequence and others are boxed. (B) Alignment of the sequence of the unc-13 product with the CII subdomains of PKC, part of a bovine phosphatidylinositol-specific phospholipase C (bPLC) (27), and one of the repeating units, homologous to the CII subdomain of PKC, of a rat synaptic vesicle-specific protein (rP65) (28). a,
?.
Biochemistry: Maruyama and Brenner frequency along the autosomes or a real nonrandom distribution of genes on the physical map. In the lin-12 cluster on LGIII, it has been suggested that clustering results at least in part from local decreased recombination frequency by comparison of physical distance and genetic map length (31). We can estimate the ratio of physical distance to map length for the unc-15-unc-13 genes, located in the heart of the cluster on the LGI and separated by 0.025 map unit. The physical distance between the two genes is =80 kb as measured from restriction maps of the cosmid clones spanning this chromosomal region. This gives 3200 kb/map unit, which is 10 times greater than the average distance, 330 kb/map unit, estimated for the whole genome (i.e., 105 kb/300 map units), and -3 times greater than the distance in the cluster on LGIII (900 kb/map unit), suggesting that the apparent clustering results from decreased recombination frequency in this chromosomal region. The unc-13 product has binding sites in common with PKC and binds to a PKC ligand in vitro. It may be part of a signal transduction pathway transducing the signal from diacylglycerol to effector functions specified by the novel N- or C-terminal domains. Mutation in unc-13 causes diverse defects in the nervous system in C. elegans. Phenotypes of the mutant, such as resistance to acetylcholinesterase inhibitor and accumulation of acetylcholine at an abnormally high level, may be explained by a defect in acetylcholine release, storage, or uptake (2). In fact, phorbol esters and Ca2l synergistically enhance neurotransmitter release by neurons (32-34). These findings, along with the present results, suggest a primary role for the unc-13 product in neurotransmitter release from neurons. Since neurotransmitters also play important roles in neuronal development, including outgrowth and establishment of the circuitry (35-37), the abnormal neuronal development in unc-13 mutants may be the secondary consequence of the altered function. Alternatively, the abnormal neuronal development (misplaced axons and abnormal gap junctions) may have produced a decreased rate of neurotransmitter release. Further analysis of function of the gene product should provide clues for understanding the phenotypes. We thank P. Cuello for assistance in genomic DNA sequencing; H. Kagawa for the unc-15 clone; A. Coulson and J. Sulston for cosmid clones; R. Hoskins, J. Mancillas, and J. Hodgkin for their generous gifts of mutants; and J. White and D. Albertson for critically reading the manuscript.
1. Chalfie, M. & White, J. G. (1988) in The Nematode Caenorhabditis elegans, ed. Wood, W. B. (Cold Spring Harbor Lab., Cold Spring Harbor, NY), pp. 337-391. 2. Hosono, R., Sassa, T. & Kuno, S. (1989) Zool. Sci. 6, 697-708. 3. Nishizuka, Y. (1988) Nature (London) 334, 661-665. 4. Collins, J., Saari, B. & Anderson, P. (1987) Nature (London) 328, 726-728. 5. Brenner, S. (1974) Genetics 77, 71-94. 6. Sulston, J. E. & Brenner, S. (1974) Genetics 77, 95-104. 7. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold Spring Harbor, NY).
Proc. Natl. Acad. Sci. USA 88 (1991)
5733
8. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 9. Vieira, J. & Messing, J. (1982) Gene 19, 259-268. 10. Kahn, J., Matthes, H. W. P., Gait, M. T. & Brenner, S. (1984) Gene 32, 217-224. 11. Huynh, T. V., Young, R. A. & Davis, R. W. (1984) in DNA Cloning Techniques: A Practical Approach, ed. Glover, D. (IRL, Oxford), pp. 49-78. 12. Studier, F. W. & Moffatt, B. A. (1986) J. Mol. Biol. 189, 113-130. 13. Ono, Y., Fujii, T., Igarashi, K., Kuno, T., Tanaka, C., Kikkawa, U. & Nishizuka, Y. (1989) Proc. Natl. Acad. Sci. USA 86, 4868-4871. 14. Rose, A. M. & Baillie, D. L. (1980) Genetics 96, 639-648. 15. Kagawa, H., Gengyo, K., McLachlan, A. D., Brenner, S. & Karn, J. (1989) J. Mol. Biol. 207, 311-333. 16. Coulson, A. R., Sulston, J. E., Brenner, S. & Karn, J. (1986) Proc. Natl. Acad. Sci. USA 83, 7821-7825. 17. Krebs, E. G. & Beavo, J. A. (1979) Annu. Rev. Biochem. 48, 923-959. 18. Tabuse, Y., Nishiwaki, K. & Miwa, J. (1989) Science 243, 1713-1716. 19. Rosenthal, A., Rhee, L., Yadegari, R., Paro, R., Ullrich, A. & Goeddel, D. V. (1987) EMBO J. 6, 433-441. 20. Parker, P. J., Coussens, L., Totty, N., Rhee, L., Young, S., Chen, E., Stabel, S., Waterfield, M. D. & Ullrich, A. (1986) Science 233, 853-859. 21. Coussens, L., Parker, P. J., Rhee, L., Yang-Feng, T. L., Chen, E., Waterfield, M. D., Francke, U. & Ullrich, A. (1986) Science 233, 859-866. 22. Knopf, J. L., Lee, M., Sultzman, L., Kriz, R., Loomis, C., Hewick, R. M. & Bell, R. M. (1986) Cell 46, 491-502. 23. Ono, Y., Fujii, T., Ogita, K., Kikkawa, U., Igarashi, K. &
Nishizuka, Y. (1988) J. Biol. Chem. 263, 6927-6932. 24. Ono, Y., Fujii, T., Ogita, K., Kikkawa, U., Igarashi, K. &
Nishizuka, Y. (1989) Proc. Natl. Acad. Sci. USA 86, 30993103. 25. Bonner, T. I., Oppermann, H., Seeburg, P., Kerby, S. B., Gunnell, M. A., Young, A. C. & Rapp, U. R. (1986) Nucleic Acids Res. 14, 1009-1015. 26. Hall, C., Monfries, C., Smith, P., Lim, H. H., Kozma, R., Ahmed, S., Vanniasingham, V., Leung, T. & Lim, L. (1990) J. Mol. Biol. 211, 11-16. 27. Stahl, M. L., Ferenz, C. R., Kelleher, K. L., Kriz, R. W. & Knopf, J. L. (1988) Nature (London) 332, 269-272. 28. Perin, M. S., Fried, V. A., Mignery, G. A., Jahn, R. & Sudhof, T. C. (1990) Nature (London) 345, 260-263. 29. Ohno, S., Akita, Y., Konno, Y., Imajoh, S. & Suzuki, K. (1988) Cell 53, 731-741. 30. Baker, M. E. (1989) Mol. Cell. Endocrinol. 61, 129-131. 31. Greenwald, I., Coulson, A., Suiston, J. & Priess, J. (1987) Nucleic Acids Res. 15, 2295-2307. 32. Tanaka, C., Taniyama, K. & Kusunoki, M. (1984) FEBS Lett. 175, 165-169. 33. Zurgil, N. & Zisapel, N. (1985) FEBS Lett. 185, 257-261. 34. Matthies, H. J. G., Palfrey, H. C., Hirning, L. D. & Miller, R. J. (1987) J. Neurosci. 7, 1198-1206. 35. McCobb, D. P., Cohan, C. S., Conner, J. A. & Kater, S. B. (1988) Neuron 1, 377-385. 36. Parnavelas, J. G. & Cavanagh, M. E. (1988) Trends Neurosci. 11, 92-93. 37. Lipton, S. A. & Kater, S. B. (1989) Trends Neurosci. 12, 265-270.
