©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel Isoform of Syntaxin-binding Protein Homologous to Yeast Sec1 Expressed Ubiquitously in Mammalian Cells (*)

(Received for publication, November 15, 1994; and in revised form, December 28, 1994)

Hideki Katagiri Jungo Terasaki Tomiyasu Murata Hisamitsu Ishihara (2) Takehide Ogihara Kouichi Inukai (2) Yasushi Fukushima Motonobu Anai Masatoshi Kikuchi (2) Jun-ichi Miyazaki (1) Yoshio Yazaki Yoshitomo Oka (§)

From the  (1)Third Department of Internal Medicine and Department of Disease-related Gene Regulation Research (Sandoz), Faculty of Medicine, University of Tokyo, Hongo, Tokyo 113, Japan and the (2)Institute for Adult Disease, Asahi Life Foundation, Nishishinjuku, Shinjuku, Tokyo 160, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

munc-18/n-Sec1/rbSec1, a brain homologue of the yeast Sec1p protein, is thought to participate in regulating the docking and fusion of synaptic vesicles. We have screened the mouse cDNA library of an MIN6 cell line, derived from pancreatic beta cells, for its novel isoform and have identified a cDNA encoding a 593-amino acid protein having 63, 53, and 30% identity with munc-18/n-Sec1/rbSec1, Caenorhabditis elegans unc18, and Saccharomycescerevisiae Sec1p, respectively. While munc-18/n-Sec1/rbSec1 expression has been reported to be neural-specific, RNA blot analysis has revealed that the novel isoform, which we refer to as muSec1 (mammalian ubiquitous Sec1), is expressed ubiquitously. We have also identified mouse munc-18/n-Sec1/rbSec1 from the MIN6 cDNA library, indicating that different isoforms of a protein participating in vesicular transport exist in a single cell. muSec1 bound to glutathione S-transferase-syntaxin 1A and, although with lower affinity, to glutathione S-transferase-syntaxin 4 fusion protein. These findings suggest that muSec1 is, via its binding to the syntaxin family, involved in the protein trafficking from the Golgi apparatus to the plasma membrane and that the fundamental mechanisms of protein trafficking have been conserved from yeast through virtually all mammalian cells.


INTRODUCTION

The molecular mechanisms of vesicular transport have been intensively investigated, especially in neurotransmitter-containing synaptic vesicles(1, 2) . In neurons, a multi-subunit complex can be extracted as a 20 S particle containing synaptobrevin, syntaxin, and SNAP-25 suggesting that the membrane fusion machinery is based primarily on the interactions between soluble fusion factors, such as N-ethylmaleimide-sensitive factor and soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAPs),^1 and their receptors (SNAP receptors; SNAREs)(3) . According to the SNARE hypothesis, vesicle docking is proceeded by the interaction of synaptic vesicle proteins (v-SNARE), such as synaptobrevin(4, 5) , with the target membrane proteins (t-SNARE), such as syntaxin (6) and SNAP-25(7) , prior to the actual membrane fusion. In the yeast secretory system, which is constitutive, it has recently been demonstrated that several proteins homologous to SNAREs participate in vesicular transport (for a recent review, see (8) ). Thus, these underlying mechanisms appear to be conserved among all eukaryotic cells and also between constitutive and regulatory pathways of secretion.

Recently, a 67-kDa neural-specific syntaxin-binding protein (munc-18/n-Sec1/rbSec1), a rat brain homologue of yeast Sec1p, has been identified by several laboratories(9, 10, 11, 12) . The yeast syntaxin homologues Sso1p and Sso2p were identified as multicopy suppressors of SEC1 mutation(13) . Furthermore, the Caenorhabditiselegans homologue of Sec1p, unc18, is a protein whose mutations lead to a paralytic phenotype and accumulation of acetylcholine(14, 15, 16) , which is consistent with an impairment of presynaptic function. These findings suggest that vesicle docking and/or fusion to the plasma membrane might be regulated via binding of the Sec1 homologue to the syntaxin family.

Although the syntaxin isoforms (syntaxins 2, 3, and 4) (17) and cellubrevin, a protein homologous to synaptobrevin(18) , which are thought to form SNAREs, are expressed in mammalian non-neural cells, the expression of munc-18/n-Sec1/rbSec1 is restricted to brain tissue (9, 10, 11) and some endocrine tissues(11, 12) . Furthermore, it has been shown that munc-18/n-Sec1/rbSec1 can bind to several isoforms of the syntaxin family but not to syntaxin 4(10) . Altogether, these findings suggest that additional isoform(s) of Sec1-related protein remain to be identified. In the present study, we have identified a novel mammalian isoform of Sec1p that can bind to syntaxins. We have termed this protein muSec1 (mammalian ubiquitous Sec1), as it appears to be ubiquitous.


EXPERIMENTAL PROCEDURES

cDNA Cloning of a Novel Sec1 Isoform

Two degenerate oligonucleotide primers were synthesized for the polymerase chain reaction (PCR). These two primers were 5`-TGYGCNACNCTRAARGARTAYCC-3` and 5`-ARRTCRTANGMCATNGCYTG-3`, corresponding to amino acid residues 179-186 and 250-256 of munc-18/n-Sec1/rbSec1 (9) which are highly conserved among munc-18/n-Sec1/rbSec1, unc-18, and Sec1p. The PCR was performed by using mouse skeletal muscle c DNA or cDNA from an MIN6 cell line, derived from mouse pancreatic beta cells(19) , as a substrate. Samples were amplified for 40 cycles under the following conditions: denaturation for 1 min at 94 °C, annealing for 1 min at 50 °C, and extension for 1 min 72 °C. A DNA fragment of approximately 230 bp was then separated by electrophoresis on a 2% low temperature melting agarose gel, cloned into TA vector pCRII (Invitrogen), and sequenced using a DNA sequencer (Applied Biosystems Inc.). We obtained two independent sequences: one, mUR1, encoding a protein related to rat munc-18/n-Sec1/rbSec1 (58% amino acid identity); and the other, mUR2, which turned out to be completely homologous at the amino acid level to rat munc-18/n-Sec1/rbSec1. 1.0 times 10^6 plaques of an oligo(dT)-primed ZAP II MIN6 cDNA library produced by a standard method (Stratagene) were screened under standard hybridization conditions using a P-labeled mUR2 and mUR1 cDNA fragment as probes to obtain mouse munc-18/n-Sec1/rbSec1 and its possible novel isoform, respectively. Positive clones were excised into pBluescript and sequenced. The final sequence was determined from both strands.

Northern Blot Analysis

Total cellular RNA was prepared from various mouse tissues and MIN6 cells using Isogen (Nippon Gene). For RNA blots, 20 µg of total RNA was subjected to formaldehyde-agarose gels and transferred onto Hybond-N nylon membrane (Amersham Corp.). The blot was hybridized in Rapid Hybridization Buffer (Amersham Corp.) with [-P]dCTP random-primed muSec1 cDNA fragment (729 bp), digested by restriction enzyme PvuII, as a probe. High stringency washes were performed at 65 °C in 0.1 times SSC (1 times SSC = 150 mM NaCl, 15 mM sodium citrate), 0.1% SDS.

In Vitro Transcription and Translation

cDNAs coding for mouse munc-18/n-Sec1/rbSec1 and muSec1 were linearized from pBluescript, transcribed in vitro with T3 phage polymerase using an mRNA capping kit (Stratagene), and translated in a rabbit reticulocyte lysate (Stratagene) supplemented with [S]methionine (ICN).

In Vitro Binding Assay

Two sets of degenerate oligonucleotide primers (S1S and S1A, S4S and S4A) were synthesized corresponding to amino acids 1-7 and 264-270 of rat syntaxin 1A (6) and 1-7 and 272-278 of rat syntaxin 4(17) : S1S, 5`-ATGAARGAYMGNACNCARGA-3`; S1A, 5`-CADATDATDATCATDATYTTYTT-3`; S4S, 5`-ATGMGNGAYMGNACNCAYGA-3`; S4A 5`-ATNGCDATCATNACYTTYTT-3`. PCRs were performed using S1S and S1A as primers and mouse brain cDNA as a substrate, and using S4S and S4A as primers and mouse skeletal muscle cDNA as a substrate, to amplify cDNA fragments of syntaxin 1A and syntaxin 4, respectively. The amplified cDNA fragments were sequenced directly and cloned into a TA vector followed by sequencing. We selected the clones whose sequences were identical to the results of the direct sequencing experiments, indicating that they encode mouse syntaxin 1A and mouse syntaxin 4 without PCR errors. These clones were subcloned into the bacterial expression vector pGEX-5T (Pharmacia) producing an in-frame recombination of proteins composed of glutathione S-transferase (GST) fused to amino acids 4-267 of syntaxin 1A and amino acids 4-275 of syntaxin 4.

[S]Methionine-labeled munc-18/n-Sec1/rbSec1 or muSec1 (15 µl) was incubated for 1 h with 20 µl of glutathione-Sepharose 4B beads (Pharmacia Biotech Inc.) to which the GST alone, GST fusion syntaxin 1A, or GST fusion syntaxin 4 protein had been coupled. Samples were thoroughly washed in phosphate-buffered saline intensively and subjected to SDS-gel electrophoresis. Following electrophoresis, the gel was treated with Enlightning (Amersham), dried, and subjected to autoradiography. The radioactivity of signals was measured using a BAS 2000 image analyzer (Fujix).


RESULTS

To obtain a cDNA fragment corresponding to a novel mammalian isoform of Sec1p, PCR was performed using degenerate oligonucleotides as primers and mouse skeletal muscle cDNA as a substrate, since, in skeletal muscle, syntaxin 4 is expressed predominantly as compared with other syntaxin isoforms(17) . PCR products 233 bp in size were obtained, subcloned, and sequenced. All sequenced clones, designated mUR1, encoded a sequence related to rat munc-18/n-Sec1/rbSec1 (58% amino acid identity). We also performed the PCR using cDNA from an MIN6 cell line, derived from mouse pancreatic beta cells(19) , as a substrate under the same conditions, and subcloned the amplified DNA. Three out of nine sequenced clones had the same sequence as mUR1, whereas the other six clones, mUR2, encoded a protein that was completely homologous at the amino acid level to rat munc-18/n-Sec1/rbSec1(9) . These results indicate that the MIN6 cell line expresses both munc-18/n-Sec1/rbSec1 and its novel isoform.

To isolate cDNAs corresponding to these two isoforms, an oligo(dT)-primed ZAP II MIN6 cDNA library was screened under standard hybridization conditions using the cloned mUR1 or mUR2 cDNA fragment as a probe. The mUR1 probe yielded three overlapping clones, which were subsequently subcloned and sequenced in their entirety. The initiation codon of the cDNA shown in Fig. 1is consistent with the consensus sequence defined by Kozak for initiation codons(20) . Alignment of the predicted amino acid sequence for the protein with the full-length sequence of munc-18/n-Sec1/rbSec1 (Fig. 2) reveals amino termini of similar sequences, supporting the notion that the postulated amino terminus is correct. This methionine is followed by a single 1779-bp open reading frame encoding a 593-residue protein with no apparent transmembrane region (molecular mass of 67 kDa) (Fig. 1), which we have termed muSec1 (mammalian ubiquitous Sec1) since, as is described below, it appears to be ubiquitous. On the other hand, using mUR2 as a probe, three additional positive clones were isolated which encoded a protein completely homologous, at the amino acid level, to rat munc-18/n-Sec1/rbSec1. These data indicate that these clones correspond to mouse munc-18/n-Sec1/rbSec1. muSec1 protein has 63, 56, 53, 30, 25, and 21% identity with munc-18/n-Sec1/rbSec1, rop (Drosophila melanogaster), unc-18 (C. elegans), Sec1p (yeast), Sly1p (yeast), and Slp1p (yeast), respectively. Of all the proteins present in the GenBank data bank, muSec1 shares the highest homology with rat munc-18/n-Sec1/rbSec1, confirming that muSec1 is a novel mammalian isoform of Sec1p (yeast).


Figure 1: Nucleotide sequence of mouse muSec1 cDNA and predicted amino acid sequence of mouse muSec1. The amino acid sequence is shown in single-letter code below the nucleotide sequence, and the sequences are numbered on the right.




Figure 2: Comparison of amino acid sequences of mouse muSec1, munc-18/n-Sec1/rbSec1, and yeast Sec1p. Identical amino acids at a given position are boxed.



RNA blotting study (Fig. 3) has revealed that an approximately 2.7-kilobase transcript of muSec1 is expressed in all tissues examined, although it was detected at a very low level in the heart and the additional band of more kilobases was detected in the brain and MIN6. The expression of muSec1 in heart and brain tissue was also confirmed by direct sequencing of reverse transcription-PCR products (data not shown).


Figure 3: Northern blot analysis of muSec1 mRNA in various mouse tissues and the MIN6 cell line. A, 20 µg of total RNA from the tissues and cells were denatured and electrophoresed as described in the text. For autoradiography, the nylon membrane was exposed to x-ray film with an intensifying screen at -80 °C for 14 days. B, the gel was stained with ethidium bromide before transfer. 28 S and 18 S ribosomal RNAs are indicated.



To characterize the interaction of muSec1 with syntaxins, an in vitro binding assay (10) was performed. [S]Methionine-labeled munc-18/n-Sec1/rbSec1 and muSec1 proteins were incubated with GST-syntaxin fusion protein coupled to Sepharose beads. The bound radioactive proteins were eluted, electrophoresed, and visualized by autoradiography. It has been reported that rat munc-18/n-Sec1/rbSec1 binds to GST-syntaxin 1A fusion protein, which contains most of the cytoplasmic domain (amino acid residues 4-267) of syntaxin 1A, but does not bind to GST-syntaxin 4 fusion protein(10) . In the present study, we obtained the same results; mouse munc-18/n-Sec1/rbSec1 was found to bind to GST-syntaxin 1A, but not to GST-syntaxin 4 or GST alone (Fig. 4, lanes 4-6). Approximately 3.3% of the labeled munc-18/n-Sec1/rbSec1 protein bound to GST-syntaxin 1A, based on the radioactivity values obtained from computer image analysis. On the other hand, muSec1 also bound to GST-syntaxin 1A and, although with a lower affinity, to GST-syntaxin 4 fusion protein, which contains most of the cytoplasmic domain (amino acid residues 4-275) of syntaxin 4 (Fig. 4, lanes 7-9). Approximately 4.7% of the labeled muSec1 protein bound to GST-syntaxin 1A, while 0.8% bound to GST-syntaxin 4. In all binding experiments, approximately equal amounts of fusion protein were employed as demonstrated by Coomassie Blue staining of the proteins in SDS-polyacrylamide gels (data not shown).


Figure 4: Binding of mouse muSec1 and munc-18/n-Sec1/rbSec1 to syntaxin 1A and syntaxin 4 in vitro. Autoradiograms of S-labeled muSec1 and munc-18/n-Sec1/rbSec1 binding to GST-syntaxin fusion proteins coupled to Sepharose beads. Lane1, RNA free translation sample; lane2, 1 µl of S-labeled munc-18/n-Sec1/rbSec1 probe; lane3, 1 µl of S-labeled muSec1 probe; lane4, munc-18/nSec-1/rbSec-1 bound to the GST protein; lane5, munc-18/n-Sec1/rbSec1 bound to GST-syntaxin 1A(4-267); lane6, munc-18/n-Sec1/rbSec1 bound to GST-syntaxin 4(4-275); lane7, muSec1 bound to the GST protein; lane8, muSec1 bound to GST-syntaxin 1A(4-267); lane9, muSec1 bound to GST-syntaxin 4(4-275).




DISCUSSION

Research on vesicular transport from the Golgi apparatus to the plasma membrane has focused mainly on the constitutive secretion in yeast and the regulatory secretion of neurotransmitters at synapses. Structurally related proteins may participate in these two processes, and it is thought that specific proteins such as synaptotagmin, which has domains for Ca regulation(21) , confer the regulatory properties on the fundamental mechanism which is conserved from yeast to neuron. The previously identified mammalian homologue of Sec1p, munc-18/n-Sec1/rbSec1, is expressed predominantly in the brain and, although only at barely detectable levels, in some endocrine tissues (11, 12) , but not in most non-neural tissues.

In non-neural tissues, however, it is hypothesized that protein trafficking from Golgi to the plasma membrane is carried out utilizing a similar mechanism. The findings that muSec1, a Sec1p homologue, is expressed ubiquitously and is capable of binding to syntaxins are consistent with the concept that non-neural cells also shares the fundamental mechanisms. We cannot rule out the possibility that muSec1 is involved in the other vesicular transport steps, such as endoplasmic reticulum to Golgi and Golgi to vacuole. However, muSec1 has greater homology with Sec1p, which participates in vesicular transport from Golgi to the plasma membrane in yeast, than with Sly1p (endoplasmic reticulum to Golgi) or Slp1p (Golgi to vacuole). In addition, muSec1 can bind to syntaxins which are localized at the plasma membrane. These findings suggest that muSec1 plays a role in vesicular transport from the Golgi apparatus to the plasma membrane. Further studies using a specific antibody against muSec1 are required to elucidate the intracellular localization and function of muSec1 in greater detail.

In the present study, we found that two types of Sec1p-related protein, munc-18/n-Sec1/rbSec1 and muSec1, were present in an MIN6 cell line which exhibits characteristics similar to those of isolated islets(19, 22) . These results demonstrate that different isoforms of a protein involved in the vesicular transport exist in a single cell, suggesting that the specificity of vesicular transport is guaranteed by a specific interplay among proteins constituting the fundamental machinery of vesicle docking and/or fusion. Which (or whether both) protein(s) is responsible for insulin-containing vesicle fusion, i.e. insulin secretion in pancreatic beta cells, should be determined.

Since mutations of unc18 in C. elegans lead to the accumulation of acetylcholine(14, 15, 16) , it is possible that dysfunction of muSec1 may lead to disordered protein translocation to the plasma membrane. For instance, in muscle and adipocytes, GLUT4 glucose transporters translocate from low density microsomes to the plasma membrane in response to the insulin stimulation (23, 24, 25) and it has been reported that synaptobrevin is localized on vesicles containing GLUT4(26) . These results may suggest that the fundamental mechanism of such protein trafficking is based on the interactions among proteins such as synaptobrevin, syntaxin, and muSec1. Thus, dysfunction of these molecules may result in cellular disorders such as insulin resistance. Several potential phosphorylation sites for protein kinase A, as well as casein kinase II, are conserved between munc-18/n-Sec1/rbSec1 and muSec1. Phosphorylation by these kinases may regulate protein trafficking from the Golgi apparatus to the plasma membrane.


FOOTNOTES

*
This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture and also by a grant for diabetes research from the Ministry of Health and Welfare of Japan. 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.

§
To whom correspondence should be addressed. Present address: Third Dept. of Internal Medicine, Yamaguchi University School of Medicine, Kogushi 1144, Ube, Yamaguchi 755, Japan. Fax: 81-836-22-2342.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D42068[GenBank].

(^1)
The abbreviations used are: SNAP, soluble N-ethylmaleimide-sensitive factor attachment protein; SNARE; SNAP receptor; PCR, polymerase chain reaction; bp, base pair(s).


ACKNOWLEDGEMENTS

We thank Dr. Eric van Breda for critical reading of this manuscript.


REFERENCES

  1. Südhof, T. C., De Camilli, P., Niemann, H., and Jahn, R. (1993) Cell 75, 1-4 [Medline] [Order article via Infotrieve]
  2. Bennet, M. K., and Sheller, R. H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2559-2563 [Abstract]
  3. Sollner, T., Whiteheart, S. W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst, P., and Rothman, J. E. (1993) Nature 362, 318-324 [CrossRef][Medline] [Order article via Infotrieve]
  4. Trimble, W. S., Cowan, D. M., and Sheller, R. H. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4538-4542 [Abstract]
  5. Baumert, M., Maycox, P. R., Navone, F., De Camilli, P., and Jahn, R. (1989) EMBO J. 8, 379-384 [Abstract]
  6. Bennet, M. K., Calakos, N., and Sheller, R. H. (1992) Science 257, 255-259 [Medline] [Order article via Infotrieve]
  7. Oyler, G. A., Higgins, G. A., Hart, R. A., Battenberg, E., Billingsley, M., Bloom, F. E., and Wilson, M. C. (1989) J. Cell Biol. 109, 3039-3052 [Abstract]
  8. Ferro-Novick, S., and Jahn, R. (1994) Nature 370, 191-193 [CrossRef][Medline] [Order article via Infotrieve]
  9. Hata, Y., Slaughter, C. A., and Südhof, T. C. (1993) Nature 366, 347-351 [CrossRef][Medline] [Order article via Infotrieve]
  10. Pevsner, J., Hsu, S.-C., and Sheller, R. H. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1445-1449 [Abstract]
  11. Garcia, E. P., Gatti, E., Butler, M., Burton, J., and De Camilli, P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2003-2007 [Abstract]
  12. Hodel, A., Schafer, T., Gerosa, D., and Burger, M. M. (1994) J. Biol. Chem. 269, 8623-8626 [Abstract/Free Full Text]
  13. Aalto, M. K., Ronne, H., and Keranen, S. (1993) EMBO J. 12, 4095-4104 [Abstract]
  14. Hosono, R., Hekimi, S., Kamiya, Y., Sassa, T., Murakami, S., Nishiwaki, K., Miwa, J., Taketo, A., and Kodaira, K. I. (1992) J. Neurochem. 58, 1517-1525 [Medline] [Order article via Infotrieve]
  15. Gengyo-ando, K., Kamiya, Y., Yamakawa, K., Kodaira, K., Nishiwaki, K., Miwa, J., Hori, I., and Hosono, R. (1993) Neuron 11, 703-711 [Medline] [Order article via Infotrieve]
  16. Pelham, H. R. B. (1993) Nature 364, 582 [Medline] [Order article via Infotrieve]
  17. Bennet, M. K., Garcia-Arraras, J. E., Elferink, L. A., Peterson, K., Fleming, A. M., Hazuka, C. D., and Scheller, R. H. (1993) Cell 74, 863-873 [Medline] [Order article via Infotrieve]
  18. McMahon, H. T., Ushkaryov, Y. A., Edelmann, L., Link, E., Bintz., T., Niemann, H., Jahn, R., and Südhof, T. C. (1993) Nature 364, 346-349 [CrossRef][Medline] [Order article via Infotrieve]
  19. Miyazaki, J., Araki, K., Yamato, E., Ikegami, H., Asano, T., Shibasaki, Y., Oka, Y., and Yamamura, K. (1990) Endocrinology 127, 126-132 [Abstract]
  20. Kozak, M. (1991) J. Cell Biol. 115, 887-903 [Abstract]
  21. Perin, M. S., Fried, V. A., Mignery, G. A., Jahn, R., and Südhof, T. C. (1990) Nature 345, 260-263 [CrossRef][Medline] [Order article via Infotrieve]
  22. Ishihara, H., Asano, T., Tsukuda, K., Katagiri, H., Inukai, K., Anai, M., Kikuchi, M., Yazaki, Y., Miyazaki, J., and Oka, Y. (1993) Diabetologia 36, 1139-1145 [Medline] [Order article via Infotrieve]
  23. Suzuki, K., and Kono, T. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 2542-2545 [Abstract]
  24. Cushman, S. W., and Wardzala, L. J. (1980) J. Biol. Chem. 255, 4758-4762 [Free Full Text]
  25. Oka, Y., and Czech, M. P. (1984) J. Biol. Chem. 259, 8125-8133 [Abstract/Free Full Text]
  26. Cain, C. C., Trimble, W. S., and Lienhard, G. E. (1991) J. Biol. Chem. 267, 11681-11684 [Abstract/Free Full Text]

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