(Received for publication, December 18, 1995; and in revised form, January 9, 1996)
From the
Cysteine string proteins (Csps) are synaptic vesicle proteins thought to be involved in calcium-dependent neurotransmitter release at nerve endings. Here, we report the cloning of two Csp variants, termed Csp1 and Csp2, from bovine adrenal medullary chromaffin cells. The bovine Csp1 appears to be the homologue of rat brain Csp, sharing 95% identity at the amino acid level. The nucleotide sequence of csp2 is identical with that of csp1 except for a 72-base insert which introduces a stop codon into the coding sequence, which would be predicted to result in a truncated protein 3.3 kDa smaller than Csp1. Furthermore, polymerase chain reaction analysis detected homologues of Csp1 and Csp2 in rat kidney, liver, pancreas, spleen, lung, and adrenal gland. Expression of Csps in non-neuronal tissues was confirmed by Northern blotting and by immunoblotting with anti-Csp1 antiserum which also demonstrated expression of both full-length and truncated Csps in spleen. The widespread tissue distribution is inconsistent with a role for Csps as specific regulators of presynaptic calcium channels as previously proposed. We suggest that Csps may have a more general role in membrane traffic in non-neuronal as well as neuronal cells.
Calcium influx stimulates the secretion of neurotransmitters and catecholamines from presynaptic nerve terminals and adrenal chromaffin cells, respectively(1) . Although calcium is the trigger for regulated secretion, the underlying mechanisms of exocytosis are controlled at the level of protein-protein interactions. The identification and characterization of proteins involved in secretion has recently been the focus of intensive research(2, 3) , and adrenal chromaffin cells have been an important model for the study of neuroendocrine secretion (2) .
Cysteine-string proteins (Csps) ()were
originally discovered in Drosophila, where they were
apparently found localized exclusively at synaptic
terminals(4) . Csps are unique in that they contain a
cysteine-rich motif, which in Drosophila consists of 11
cysteine residues flanked on either side by another pair of cysteines.
The proteins also incorporate a ``J'' domain, homologous to
DnaJ proteins which interact with the bacterial homologue of the
chaperone protein Hsp70(5) . Drosophila Csps exist in
at least two forms generated by alternative RNA splicing(4) ,
and a single related Csp was found in Torpedo(6) .
An independent study found that Torpedo csp antisense cRNA virtually abolished expression of an N-type calcium channel in Xenopus oocytes injected with Torpedo mRNA (6) . Torpedo Csp was, therefore, proposed to be an essential subunit or modulator of presynaptic calcium channels. However, Csps were subsequently found to copurify with synaptic vesicles and not with presynaptic membranes(7) , prompting the proposal that Csps on the synaptic vesicle membrane may interact with presynaptic calcium channels converting them from an inactive to an active state. Torpedo Csp has been shown to be post- translationally modified by the palmitoylation of 11 or 12 of the 13 cysteine residues, and this fatty acylation is thought to tether Csps to the synaptic vesicle membrane(8) .
Genetic analysis found that deletion of the entire Drosophila csp locus or the promoter sequence and first exon conveyed a temperature-sensitive lethal phenotype which was subsequently characterized as causing a defect in presynaptic neurotransmission(9, 10) . The fact that the mutant phenotype was more pronounced at 30 °C than at 22 °C suggests that Csps may stabilize components of the neurotransmitter release machinery. Interestingly, deletion of dnaJ in Escherichia coli causes a temperature-sensitive phenotype suggestive of chaperone activity (11) .
A Csp has recently been cloned from rat brain, suggesting that these proteins may also play an important role in mammalian presynaptic neurotransmission(12) . It has been suggested that Csps function in membrane fusion(13) , but their exact role in the nerve terminal is still not known. In this paper we report the cloning of two csp coding sequences from bovine adrenal medullary chromaffin cells. The first encodes a protein identical in size with the rat brain form, whereas the other, which appears to be a splice variant of the first, encodes a truncated protein approximately 3.3 kDa smaller in size. Subsequent PCR, Northern, and immunoblotting analysis revealed that Csps are not brain-specific as previously reported(4) , but are in fact found in a range of non-neuronal tissues, suggesting a more general function for these proteins.
Since Csps are highly conserved, a strategy was adopted to
allow the PCR amplification of the coding sequence of bovine chromaffin
Csp using oligonucleotide primers based on the rat brain csp sequence but incorporating restriction sites to allow direct
cloning into vectors for expression of His-tagged proteins.
In initial experiments, mRNA was isolated from purified bovine adrenal
medullary chromaffin cells, the rat clonal PC12 pheochromocytoma cell
line, and rat brain. Reverse transcription of the mRNA was used to
generate complementary cDNA which was subsequently amplified by PCR
using oligonucleotide primers designed to the 5` and 3` regions of the
rat csp coding sequence. The result of the PCR amplifications
is shown as Fig. 1. Only one size of DNA fragment was amplified
from rat brain, and this is in agreement with a previous
study(12) , suggesting the presence of only a single csp species in rat brain. However, a slightly larger DNA molecule was
amplified from chromaffin and PC12 cell cDNA in addition to a fragment
apparently identical in size with that from rat brain.
Figure 1: Amplification of rat brain, chromaffin cell, and PC12 cell csp DNA. The DNA species were amplified by PCR from cDNA using primers designed to the 5` and 3` ends of the rat brain csp coding sequence with restriction sites incorporated. Amplification from chromaffin cell and PC12 cell cDNA generated two DNA species (indicated by arrows), the smaller of these being identical in size with that generated from rat brain cDNA.
The DNA fragments obtained from PCR amplification of chromaffin cell cDNA were purified, ligated to BamHI/HindIII-digested pQE-30, and transformed into E. coli M15[pREP4] cells. Of 9 clones that were isolated, 3 contained a smaller PCR product. From sequencing, 2 encoded the likely bovine homologue of the rat brain csp (Fig. 2). This species which we term bovine Csp1 showed 88% nucleotide and 95% amino acid sequence identity with the rat brain Csp. Six clones encoded a likely splice variant, which we term Csp2, since it was identical with the first coding sequence apart from a 72-base insert (GGAGGGCACTGACCTGTGCGGGAGTGTTTGTGGTGGCAGCGGGACGGTTGAGGTGTGAACGTGGACGCTGGA after nucleotide 492). One clone containing a smaller PCR product possessed this insert but also had an 88-base deletion at position 234 which would result in a reading frameshift after amino acid 78. This last clone was not characterized further. All sequences shown (Fig. 2) are consensuses of multiple sequence runs of all clones to eliminate problems due to PCR amplification errors. In addition, all nucleotide differences between bovine Csp1 and rat Csp1 were also present in Csp2. The additional insert present in csp2 introduces two stop codons into the amino acid sequence at positions 168 and 181, and thus the Csp1 and Csp2 proteins have expected molecular masses of 22,221 Da (Csp1) and 18,904 (Csp2).
Figure 2: Comparison of the amino acid sequences of bovine, rat, Torpedo, and Drosophila Csps. Amino acid sequences are indicated in the single-letter code. The identical amino acids among the six proteins are highlighted with asterisks. Gaps introduced to generate this alignment are represented by dashes.
In
order to confirm that the cloned sequences encoded
His-tagged proteins of the predicted molecular mass,
expression of the cloned Csps was induced by addition of
isopropyl-1-thio-
-D-galactopyranoside to transformed
bacterial cultures. The expression of a new protein after induction was
clearly visible, the induced proteins were subsequently partially
purified using Ni
-NTA agarose resin, and the bound
proteins were analyzed (data not shown). The induced proteins binding
to the Ni
-NTA agarose have apparent molecular masses
of 27 and 22 kDa, respectively. Although the apparent molecular sizes
of the proteins are higher than the expected values, this has also been
found for other identified Csps(12) . The data on the expressed
proteins confirm that the cloned cDNAs encode proteins of the expected
size and that plasmids containing the coding sequence with the 72-base
insert encode a truncated protein as predicted.
Csps have previously
been suggested to be specifically localized to synapses(4) .
The data presented so far already show that they are also expressed by
the non-neuronal chromaffin and PC12 cells, and this was investigated
further by isolating mRNA from several different rat tissues and using
the primers designed to the rat csp coding sequence to amplify
the cDNA by PCR. The result of this is shown in Fig. 3a. All tissues examined resulted in the
amplification of two cDNA bands identical in size with DNA-encoding
Csp1 and Csp2, but with differing ratios of the two products from the
various tissues. The presence of mRNA-encoding Csps was confirmed by
Northern blotting. RNA blots loaded with
poly(A)-enriched RNA from various rat tissues were
hybridized at high stringency with an RNA probe complementary to the
Csp1 coding region, and mRNAs were detected in heart, spleen, lung,
liver, muscle, kidney, and testis in addition to brain (Fig. 3b). In each tissue, an mRNA species of around 5
kilobases was detected which was more abundant in brain and brain also
contained a larger mRNA.
Figure 3:
PCR and Northern blot analysis of csp1 and csp2 distribution in rat tissues. a,
amplification of csp DNA from the various named tissues was
achieved using PCR with primers designed to the 5` and 3` ends of the
coding sequence of rat brain csp. csp DNA was amplified from
all tissues examined but with apparent differential expression of the
two csps in the different tissues. b, RNA blots
containing poly(A)-enriched RNA were hybridized with a
probe encompassing the coding region of Csp1 and the hybridization
signal was detected using a PhosphorImager.
To directly confirm expression of Csp in non-neuronal tissues, an antiserum was raised using purified expressed Csp1 as the antigen. The antiserum recognized both Csp1 and Csp2 recombinant proteins, and, in immunoblotting on rat brain homogenates, recognized a polypeptide of around 36 kDa as expected for the post-translationally modified Csp1 (Fig. 4). The antiserum also specifically recognized a larger polypeptide of around 70 kDa, the amount of which varied between experiments. It is well established that Csps in Torpedo and rat brain form dimers detectable by immunoblotting(12, 16) , and it is likely that the antiserum recognizes only Csp1 and its dimer in brain homogenates. The antiserum also recognized an abundant 36-kDa polypeptide and the dimer in chromaffin cells. Since chromaffin cells are derived from neuronal precursor cells, we examined the possible non-neuronal expression of Csp in kidney and spleen, tissues that would not be expected to contain any neuronal-like cells. Immunoblotting showed less abundant but detectable expression of Csp1 in both kidney and spleen. Polypeptides corresponding to Csp dimers were also seen in these tissues. Immunoreactivity was also detected in liver and pancreas, but this was at the limit of detectability. An immunoreactive polypeptide, corresponding in size to that expected for post-translationally modified Csp2, was detected in spleen (Fig. 4).
Figure 4: Expression of Csp proteins detected by immunoblotting with anti-Csp1 antiserum. Homogenates of rat brain (a), adrenal chromaffin cells (b), kidney (c), and spleen (d) were analyzed by SDS-PAGE and immunoblotting with anti-Csp1 at 1:1000 dilution. Polypeptides corresponding to the expected size of Csp1 were detected in all tissues as well as dimer forms of Csp. An additional smaller polypeptide was also detected in spleen, corresponding in size to Csp2.
We have cloned and characterized two Csp variants from bovine adrenal chromaffin cells. PCR amplification of cDNA from the clonal PC12 cell line also generated two DNA species identical in size with csp1 and csp2 confirming that one cell type can express two distinct Csps. DNA sequencing demonstrated that the two plasmid inserts encode variant Csps, which are highly homologous at the nucleotide and amino acid level to the previously identified Csps. Protein expression confirmed that proteins of the correct molecular mass were synthesized and most notably that csp2 encodes a truncated protein, as predicted by DNA sequence analysis. The truncated Csp2 has intact ``J'' and cysteine-rich domains and is thus a Csp, but lacks the extreme C terminus possessed by the other members of the Csp family. Future studies of the functions of Csp1 and Csp2 should indicate the role of the C-terminal domain.
Previous studies have only detected one Csp protein isoform in both rat brain and Torpedo electric lobe(12, 15) . However, in both cases, detection was by Western blotting using antibodies specific for the C-terminal region of Torpedo Csp. These antibodies would clearly not bind to the truncated Csp2 which lacks the C terminus. The present study failed to identify by PCR a larger DNA species (similar to that encoding Csp2) in rat brain, and it may well be that brain expresses only the full-length Csp1.
Using PCR we have detected
expression of the two csp mRNAs in rat kidney, liver, spleen,
pancreas, lung, and adrenal gland (all tissues examined), and Northern
blotting detected mRNAs in brain, heart, spleen, lung, liver, muscle,
kidney, and testis, implying that Csps are widespread. This finding is
in contrast to previous work which has claimed that Drosophila Csps are synapse-specific(4) , and that csp mRNA
is not present in electric organ, liver, or muscle of Torpedo(15, 16) . Analysis of protein
expression by immunoblotting with antiserum raised against recombinant
Csp1 confirmed expression in chromaffin cells and other non-neuronal
tissues. Csp2 was readily detected only in spleen suggesting that this
variant protein is expressed at low levels. In immunoblotting, the
polypeptide corresponding to Csp2 was detected in spleen but not in
kidney despite similar amounts of the two PCR products being detected
in both these and other tissues. It should be noted, however, that the
PCR method used was not quantitative and the relative amounts of the
two PCR products would not reflect the relative amounts of the mRNAs
for the two Csps in these tissues. The ratio of the Csp dimer to
monomer detected by immunoblotting varied considerably between
different tissues. The significance of these differences is not clear
since the ratio of dimer to monomer varied widely between experiments
even with the recombinant Csp1. We were unable to find conditions that
prevented dimer formation. While this paper was under review,
expression of Csp1 was demonstrated in adrenal medulla (17) consistent with our findings. Interestingly, a number of
proteins involved in exocytosis previously thought to be brain-specific
have since been found in non-neuronal cell types, most recently the
Ca-sensing protein synaptotagmin(18) ,
setting a precedent for the present finding.
The implications of the
apparently ubiquitous tissue distribution of csp are manyfold.
It has been suggested that Csps may regulate voltage-dependent
Ca channels (6) and mediate membrane
fusion(13) . Our finding that Csps are not restricted to
synapses makes their prime function unlikely to be the regulation of
Ca
channels as many of the tissues examined do not
have such channels. Alternatively, Csps may be universal membrane
traffic proteins such as NSF and SNAPS, which function in both
constitutive and regulated secretion. It is possible that Csps could
mediate membrane fusion as suggested previously(13) , but our
findings do not restrict the proteins to the synapse. If Csps do
function in membrane fusion at the synapse during
neurotransmission(13) , then it seems possible that they also
play a fundamental and more general role in membrane traffic within the
cell.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X92666 [GenBank]and X92667[GenBank].
Note Added in Proof-The bovine Csp1 sequence shows greater identity, with only four amino acid differences, to a more recently reported rat brain Csp sequence, and non-neuronal expression of Csp was shown in pancreatic zymogen granules(19) .