(Received for publication, November 15, 1994; and in revised form, December 28, 1994)
From the
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 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.
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), 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.
[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).
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 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).
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 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D42068[GenBank].