Structurefunction analysis of the cysteine string protein in Drosophila: cysteine string, linker and C terminus
1 Lehrstuhl für Genetik und Neurobiologie, Theodor-Boveri-Institut
für Biowissenschaften, Am Hubland, D-97074 Würzburg,
Germany
2 Lehrstuhl für Physiologische Chemie I, Theodor-Boveri-Institut
für Biowissenschaften, Am Hubland, D-97074 Würzburg,
Germany
Author for correspondence (e-mail:
buchner{at}biozentrum.uni-wuerzburg.de)
Accepted 26 January 2004
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Summary |
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Key words: cysteine string protein (CSP), secretory vesicle, Drosophila, in vitro mutagenesis, protein targeting, paralysis, life span
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Introduction |
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Phosphorylation of vertebrate CSP at Ser10 reduces its
interaction with syntaxin and may thus modulate exocytosis
(Evans et al., 2001). The
J-domain has been linked to a putative co-chaperone function of CSPs. CSPs
bind to and stimulate the ATPase activity of the molecular chaperone HSC70
only if the J-domain is intact (Braun et
al., 1996
; Chamberlain and
Burgoyne, 1997
). In Drosophila the Csp and
hsc70-4 genes interact (Bronk et
al., 2001
). Recently, the small glutamine-rich tetratrico-peptide
repeat protein (SGT) has been identified as a third component of this
chaperone complex (Tobaben et al.,
2001
). The function of the cysteine string has been controversial.
The extensive palmitoylation of the cysteine string suggested a role in
membrane attachment (Gundersen et al.,
1994
); however, depalmitoylation does not displace CSPs from
membranes (van de Goor and Kelly,
1996
; Chamberlain and Burgoyne,
1998
), indicating that the hydrophobic amino acids associated with
the cysteine string are sufficient to keep the protein attached to the
membrane (Mastrogiacomo et al.,
1998
). Replacement of seven of the 14 cysteines in PC12 and HeLa
cells demonstrated that the intact string is required for initial membrane
targeting in cultured cells (Chamberlain
and Burgoyne, 1998
). Information on the function of the linker
region and the C terminus is sparse so far. The inhibitory effect on regulated
exocytosis of overexpressing the mammalian CSP2 isoform in hamster insulinoma
cells is reduced by a point mutation in the linker region. The C-terminal
difference between the two known vertebrate isoforms appears to be critical
for this suppression (Zhang et al.,
1999
). Linker and/or cysteine string appear to be important for
CSP-CSP self-association (Swayne et al.,
2003
).
Deletion of the Csp gene from the Drosophila genome
causes semi-lethality during prolonged development, a severely shortened life
span of escapers, temperature-sensitive paralysis, and breakdown of evoked
synaptic transmission at elevated temperatures
(Umbach et al., 1994;
Zinsmaier et al., 1994
;
Eberle et al., 1998
). The
complexity of the null phenotype suggests that CSP function may be dissected,
i.e. that the integrity of different regions of the protein can be related to
different aspects of its function in the wild type.
In the present study we therefore mutated in vitro the regions of the Drosophila Csp cDNA that code for the cysteine string, the linker region and the C terminus, ligated the mutated cDNAs to Csp regulatory sequences, and transformed the modified gene into the germline of wild-type and null-mutant flies. The phenotype of these transgenic flies as well as the distribution and biochemical properties of the mutated proteins are investigated. We show that an intact cysteine string is required for almost all known functional aspects of the protein, whereas deletions in the linker region or the C-terminal domain cause more selective defects.
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Materials and methods |
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Antibodies were obtained from the following sources.
Anti-Drosophila CSP: DCSP1 is mouse monoclonal antibody (mAb) ab49
(Hofbauer, 1991;
Buchner et al., 1986
), DCSP2
(6D6) and DCSP3 (1G12) were kindly provided by K. Zinsmaier.
Anti-Drosophila SAP47: mouse mAb nc46
(Hofbauer, 1991
;
Reichmuth et al., 1995
).
Anti-Drosophila syntaxin: 3D8 was kindly provided by S. Benzer
(Caltech, Pasadena, CA, USA) and later obtained from Developmental Studies
Hybridoma Bank (DSHB), University of Iowa, USA.
The QuickChangeTM site-directed mutagenesis kit was obtained from Stratagene (Cambridge, UK). Restriction enzymes were purchased from Life Technologies (Karlsruhe, Germany) and Fermentas MBI (St Leon-Rot, Germany). Enhanced chemiluminescense antibody binding detection kit was obtained from Amersham Corp. (Slough, UK). Oligonucleotides were synthesized by MWG (Ebersberg, Germany).
Generation of the plasmid constructs
Csp cDNA-1 [`lcz49-9'; Zinsmaier et al.
(1990); `type-I'; Zinsmaier et
al. (1994
)], was cleaved by
Bsp1407I and EcoRI and the resulting 1.9 kb fragment
containing 3' sequences of exon 3 and exons 47. This fragment was
ligated to a HindIII/Bsp1407I (long) or an
Asp718/Bsp1407I (short) genomic fragment, which contained (long or
short) regulatory sequences of the Csp gene, exon 1, intron 1, exon
2, intron 2, and the 5' sequences completing exon 3. Thus the two
constructs generate a short and a long Csp cDNA-1
rescue construct, scDna1 and lcDna1, respectively (cf.
Fig. 1C,D). The fragments were
cloned into the pW8 P-element vector
(Klemenz et al., 1987
) and
transformed into w1118 flies by standard procedures
(Ashburner, 1989
).
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For site-directed mutagenesis the type-I cDNA was used as template in the
QuickChangeTM system. The deletion of 11 cysteines was achieved in two
steps. The forward primers (reverse = complement) used were
5'-CACTGGATGCTGTTGTTGCTGCAACTTCTGCTGC-3' for the first step,
resulting in the deletion of six cysteines in the construct Scsp
(short cysteine string protein), and
5'-TGTGCCGTGATCACTGGAAACTTCTGCTGCGG-3' for the second step,
leading to the construct Cslp (cysteine string less protein). The
conversion of the two flanking pairs of cysteines to serines was again
obtained in two steps. The primers for this mutation were
5'-AAGGCGGTGGTTATCTCCTCTGCCGTGATCACTGGA-3' and its complement for
the first step and 5'-TGATCACTGGAAACTTCTCCTCCGGCAAGTTCAAGC-3' and
its complement for the second step, to obtain the construct Clp
(cysteine less protein). For the replacement of the cysteine string by a
serine string in the Ssp (serine string protein) construct the
primers were
5'-CGTGATCACTGGATCCTCTTCTTCCTCCTCCTCCTCCTCCTCTTCCAACTTCTGCTGCGG-3'
and its complement. Deletion of the eight amino acids AEQFGEEN in the linker
region of the L8 construct was achieved by the primer
5'-CGGCTTGTACATAGTCAACGCATACTTCGTGGTCACTTCACCG-3' and its
complement. In order to eliminate 27 C-terminal amino acids in the
C
27 construct the condons for the amino acids PVAA
were replaced by TAGTAGTAGCT, introducing three inframe stop codons and a
frame shift.
To confirm the fidelity of the introduced mutations the DNA of all mutant clones was sequenced prior to transformation. For transformation into the genome of Drosophila the modified cDNA was cleaved and ligated to the putative genomic promoter/enhancer region of Csp gene as described above for the rescue vector lcDna1.
Preparation and chemical treatment of cell membranes
Heads of wild-type and mutant Drosophila were either directly
homogenized in 1 µl head1 of sample buffer
(Laemmli, 1970) or collected
on ice and homogenized with 5 µl head1 buffer A (150 mmol
l1 NaCl, 10 mmol l1 Hepes, pH 7.4, 1 mmol
l1 EGTA, 0.1 mmol l1 MgCl2). A
post-nuclear supernatant was obtained by 1000 g centrifugation
for 10 min at 4°C. The supernatant was subjected to a 100 000
g centrifugation for 60 min at 4°C to separate soluble
proteins from proteins attached to membranes or cytoskeleton. For deacylation
pellets were resuspended in 1 mol l1 hydroxylamine (pH 7)
and incubated for 20 h at room temperature. After incubation, an additional 60
min centrifugation step at 100 000 g and 4°C followed in
order to separate proteins solubilized by the treatment (S3) from the membrane
fraction (P3). Pellets were finally dissolved in equivalent volumes of sample
buffer, and proteins from equal volumes of supernatants and dissolved pellets
were separated on 12.5% gels by SDS-PAGE followed by western blot analysis
using monoclonal antibodies. Separation of different membrane fractions was
achieved by glycerol gradient velocity sedimentation as described by van de
Goor et al. (1995
). All
experiments were done at least twice.
Immunochemistry
Immunohistochemistry was carried out as described earlier
(Buchner et al., 1986;
Ashburner, 1989
). The mouse
monoclonal supernatants were applied at dilutions of 1:10 to 1:100. For
visualizing antibody binding the ABC detection system (Vector Labs,
Burlingame, USA) was used. Western blots were incubated in monoclonal
supernatants at 1:100 dilution and developed using the ECL detection system as
recommended by the manufacturer.
Epitope mapping
The epitopes of the three anti-CSP monoclonal antibodies used in this study
(DCSP1-3) were mapped by spotting sequential decapeptides on nitrocellulose
membranes according to a procedure described in detail elsewhere
(Munch et al., 1999), followed
by spot detection using ECL as in western blots. Amino acids common to
decapeptides binding the antibody are defining the epitope.
Behavioral analysis
Temperature sensitivity of the various genotypes was scored by transferring
groups of 1015 1-day-old flies in empty food vials to a water bath of
defined temperature and counting fully paralyzed animals after 5 min. To
prevent interference of adaptive phenomena all flies were tested only once.
Effects of variations in genetic background were minimized by using flies that
had recently been crossed into the out-crossed null mutant
CspU1oc.
Survival analysis
Normalized life span was determined by transferring groups of 10
F1 siblings from heterozygote crosses to fresh food vials at
25°C and counting dead flies daily.
Electrophysiology
For electrophysiological recordings climbing unbalanced third instar larvae
were immobilized for 20 min on ice. Preparation and recording essentially
followed the procedure described by Stewart et al.
(1994). Larvae were dissected
in an Elastosil® coated Petri dish under modified HL3 Ringer containing 1
mmol l1 Ca2+ starting with mid-dorsal incision.
`Filets' were pinned out flat and internal organs were removed carefully.
After transfer to a temperature-controlled fixed microscope stage,
intracellular recordings were made from ventral longitudinal muscle 6 of
abdominal half-segment 3 using quartz glass microelectrodes of 2030
M
resistance, prepared by a laser-based microelectrode puller (P-2000,
Sutter Instrument Co., Novato, CA, USA) and filled with 3 mol
l1 KCl. Excitatory junctional potentials (EJPs) were evoked
by stimulating the distal portion of the motor nerve taken up into a
fire-polished borosilicate suction electrode. Stimulus amplitude of 0.1 ms
pulses was adjusted to a value 2.5-fold of the initial threshold. Signals were
amplified with Neuroprobe Amplifier (Model 1600, A-M Systems, Inc., Carlsborg,
WA, USA), low-pass filtered at 50 kHz, and digitized via an A/D
interface (Data Acquisition Processor DAP 3200e/315, Microstar Laboratories,
Inc., Bellevue, WA, USA). Stimuli, signals and temperature were digitized at
10 kHz, data were analyzed with DASYLab Ver. 5.03.30 (measX, GmbH and Co. KG,
Mönchenglattbach, Germany). Temperature was measured by a temperature
sensor (Newport Electronics GmbH, Deckenpfronn, Germany), positioned in the
Ringer solution near the larval filet. For each preparation six evoked EJPs
were averaged 57 min after reaching the set temperature. If more than
six stimuli produced no response the amplitude was registered as `zero'. Each
experiment was performed with at least four larvae. Statistical analysis was
performed using GraphPad InstatTM Ver. 2.04a (GraphPad Software, San
Diego, CA, USA) and Statistica Ver. 6 (StatSoft, Inc., Tulsa, OK, USA).
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Results |
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The main aim of the present work was to determine the involvement of the cysteine string, the linker region, and the C terminus of Drosophila CSP in membrane association, synaptic vesicle targeting and function. We first tested whether a single isoform (CSP1) expressed in Csp null mutants can `rescue' the mutant phenotype. To this end we designed two different wild-type Csp cDNA `rescue' gene constructs, scDna1 and lcDna1. The various transgenic mutants generated by transforming in vitro mutagenized versions of these cDNA constructs into Csp-null background can then be compared to the `rescue' transformants in order to assess the effect of the mutations on CSP function.
cDNA-1 rescues all major phenotypes of Csp null mutants
We used cDNA-1, which contains the largest open reading frame. Since the
regulatory region of the Csp gene has not yet been characterized we
tested two genomic fragments of different lengths upstream of the gene as
depicted in Fig. 1D for their
capacity to direct wild-type expression of the cDNA. Because there was
evidence that the first intron may contain important Csp regulatory
sequences (K. E. Zinsmaier, personal communication), fragments were chosen
that extend from 2.3 kb (scDna1) or 4.5 kb (lcDna1) upstream
of the transcription start to a Bsp1407I site in the third exon and
thus include the first two introns. These genomic fragments were ligated to
the cDNA-1 fragment downstream of the Bsp1407I site (hatched in
Fig. 1D). The constructs were
transformed into the germ line of Drosophila wild type. Since no
difference has been observed in the expression or function of the two
constructs, both are referred to below as construct cDna1. In western
blots of head homogenates from cDna1 transgenic flies the monoclonal
antibody DCSP-1 detects a highly overexpressed largest CSP isoform in addition
to the three other wild-type isoforms (data not shown). Surprisingly, we do
not observe any of the phenotypes described for flies overexpressing CSP by
use of the UAS/GAL4 system (cf. Discussion). Head homogenates of flies
homozygous for the cDna1 transgene and the null allele
CspU1w produce only a single very strong signal on western
blots. This signal matches in size the largest isoform of wild-type
homogenates, and in fractionation and deacylation experiments behaves like all
four isoforms of wild-type flies (Figs
2,
3, and data not shown).
Immunohistochemistry employing mAb DCSP1 on head and body sections of these
flies shows an expression pattern of CSP1 both in nervous and non-nervous
tissues that is indistinguishable from wild-type stainings
(Eberle et al., 1998), with
high concentrations of CSP1 in synaptic terminals on muscles and in the
neuropil of brain and thoracic ganglia
(Fig. 4 and data not shown).
All tested cDna1 transformants rescue the premature death
(Fig. 5A) and the
temperature-sensitive paralytic phenotype
(Fig. 6) of the null mutant
CspU1, demonstrating that the 2.3·kb upstream
genomic region and the first two introns contain all essential regulatory
regions of the Csp gene and that the CSP1 isoform is sufficient to
maintain all known functions of the four natural isoforms. Thus cDna1
constructs can be used as targets for introducing site-directed mutations into
the Csp gene in vitro in order to study the effects of such
mutations in vivo after transformation of the modified gene into
CspU1 flies. cDna1 transformants and
CspU1 null mutants serve as positive and negative
controls, respectively.
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Generation of transgenic flies with deletions in the linker, cysteine string and C-terminal regions of CSP
Mutation of conserved amino acids in a protein is likely to modify its
function. We therefore designed Csp transgene constructs coding for
the following modified proteins: a variant with a deletion of eight amino
acids (underlined in Fig. 1A)
in the linker region (L8), four different variants with mutated
cysteine string (Fig. 1E), and
a variant with a deletion of the 27 C-terminal amino acids (underlined in
Fig. 1A; C
27). In the
`short cysteine string protein' (SCSP)
variant, six amino acids of the cysteine string are deleted while the entire
string of 11 cysteines is removed in the `cysteine
string-less protein' (CSLP) variant. In the variant
`serine string protein' (SSP), these 11 cysteines
are replaced by 11 serines. Serine is a small, uncharged amino acid with
structural similarity to cysteine. Replacement of the cysteine string by a
serine string possibly produces smaller overall structural changes in the
protein than its deletion. In the variant `cysteine-less
protein' (CLP) the 11 string cysteines have been deleted and the two
flanking pairs of cysteines mutated to serines
(Fig. 1E) such that the encoded
protein contains no cysteines. Transgenic flies with these constructs were
generated as described above for the lcDna1 construct and were
crossed into CspU1w and CspU1oc mutant
background (cf. Materials and methods for difference between these two mutant
strains).
Analysis of the mutated proteins
Head homogenates of wild-type and transgenic flies in
CspU1w null mutant or wild-type background were analyzed
on immunoblots stained with DCSP1 and DCSP2 antibody (Figs
2,
3). No qualitative differences
were observed between blots stained with these two antibodies except for the
C27 isoform, which is not recognized by DCSP1. Apparently, the epitope
at the very C-terminal end of the truncated protein cannot bind the antibody
(Fig. 1A). The calculated
molecular masses of the serine string protein (SSP), the short cysteine string
protein (SCSP), the cysteine string-less protein (CSLP) and the cysteine-less
protein (CLP) differ from the largest wild-type isoform CSP1 only by 0.176,
0.726, 1.331 and 1.395 kDa, respectively. The apparent molecular mass as seen
in the SDS gel, however, is approximately 6, 4, 7 and 7 kDa smaller,
respectively, compared to the 36 kDa CSP1 isoform. This discrepancy is
presumably due to the fact that most cysteines of wild-type CSP are
palmitoylated. Indeed, the untreated SSP migrates at about the same position
as the deacylated 36 kDa CSP1 isoform or the largest deacylated isoform of
wild-type head homogenate (Fig.
2C). Hydroxylamine treatment of S2 supernatants of SSP and CLP did
not alter apparent molecular masses demonstrating that these proteins are not
significantly acylated (data not shown). The C-terminal deletion of 27 amino
acids leads to a shift in western blots of about 5 kDa, somewhat larger than
the calculated loss of 3.868 kDa. In contrast, the protein with a deletion in
the linker region (L
8) shows the expected approximate molecular mass
difference of 1.048 to the 36 kDa wild-type isoform
(Fig. 2A). Note that the
strength of the western signals of all mutated proteins except C
27 is
strongly reduced (Fig. 2A; cf.
Discussion).
In order to test if the mutated proteins associate with membranes,
homogenates of heads from transformants and wild type were separated into
cytosolic and membrane fractions and analyzed by immunoblotting
(Fig. 2B). In contrast to the
wild-type CSP isoforms from wild-type flies or cDna1 transformants,
the CLP and SSP mutant forms can be detected only in the soluble fraction.
This is confirmed by glycerol gradient velocity sedimentation experiments
using cDna1, Ssp and Clp flies in CspU1w
and wild-type background (Fig.
3). Endogenous or transgenic wild-type CSPs migrate predominantly
in the synaptic vesicle fractions (van de
Goor et al., 1995) whereas SSP and CLP comigrate with the loading
control protein SAP47, which is soluble
(Reichmuth et al., 1995
).
Transgenic cysteine string-less protein CSLP, on the other hand, is found in
similar concentrations in soluble and membrane fractions
(Fig. 2B). The protein with the
short cysteine string (SCSP) is found in the pellet, similar to the wild-type
isoforms. The fractionation of wild-type CSPs is apparently not significantly
influenced by the overexpression in the same cells of the CSP1 isoform in a
wild-type background. The proteins with intact cysteine string but deletions
in the linker or C-terminal domains, L
8 and C
27, are found in
the synaptic vesicle fractions and, compared to wild-type CSPs, appear to be
somewhat enriched in the plasma membrane fractions (Figs
2,
3). Fractions containing plasma
membranes are identified in the gradient by use of an antibody against
syntaxin (mAb 8C3, SYX in Fig.
3).
Tissue distribution of mutated proteins
In wild-type flies, CSPs are highly concentrated in synaptic neuropil,
synaptic terminals on muscles, and various non-nervous structures
(Eberle et al., 1998). No
differences in the immunohistochemical staining patterns have been observed
between wild-type flies and cDna1;CspU1w transformants
(Fig. 4A,B, and data not
shown). In these experiments head sections of wild-type and transgenic flies
were stained with mAb DCSP1 on the same microscope slide to ensure identical
immunohistochemical treatment. Comparison of
Fig. 4C with D and F reveals
the selective disruption of synaptic terminal targeting of the cysteine string
mutant proteins SSP and CLP in the respective transformants in null mutant
background. In Ssp;CspU1w or
Clp;CspU1w the modified proteins distribute rather
homogeneously throughout all parts of the brain, including cellular rind,
axonal bundles (e.g. in the optic chiasms) and neuropil
(Fig. 4D,F). In the
Cslp;CspU1w transformant a certain fraction of the
modified protein is apparently targeted to the synaptic terminals while the
remaining fraction distributes to other parts of the neurons
(Fig. 4E). The deletion of six
cysteines of the string (SCSP) (Fig.
4G) or of eight amino acids in the linker region (L
8) (not
shown) strongly reduces staining intensity but does not alter the distribution
of the protein. Deletion of the C terminus (C
27) (not shown) does not
affect immunohistochemical staining patterns. For comparison we show stainings
of two mutants. The CspU2 mutation
(Eberle et al., 1998
) leaves
the coding region intact but carries a B104 transposon insertion in intron 4
of the Csp gene. This mutant shows selective CSP accumulation in
mushroom body neuropil (not shown) and in synaptic terminals of photoreceptors
and visual interneurons (Fig.
4H), but CSP expression in the rest of the brain is drastically
reduced. The CspU1 null mutant serves as a negative
control (Fig. 4I),
demonstrating that fat body staining is unspecific.
Phenotypes of cysteine-string mutant transformants
Longevity
A severely reduced life span of adult flies has been observed previously as
a major phenotype of Csp null mutants. We noted that quantitative
aspects of this and other phenotypic traits of CspU1
flies, such as the degree of developmental delay and semi-lethality, the
critical temperature for the failure of larval synaptic transmission or adult
paralysis, all significantly depend on genetic background. The originally
semilethal CspU1 and CspU1w stocks
(Eberle et al., 1998) that had
been kept at 18°C in the homozygous condition could after several years be
maintained at 25°C, larvae no longer showed reversible synaptic failure in
nerve muscle preparations at 32°C, and adults lived significantly longer
than observed previously. We therefore out-crossed for 12 generations the
CspU1 stock against white
(w1118;Csp+ in Canton-S background) until we
noted that homozygous flies again showed the strong phenotype described
earlier (Umbach et al., 1994
;
Zinsmaier et al., 1994
). To
further reduce the influence of genetic background we performed the
experiments analyzing phenotypes of the transformants on siblings whenever
possible. With P(tg) transgene insertions on the X
chromosome (tg=cDna1, Ssp, L
8 or
C
27), crosses of TM3 balanced flies heterozygous for
the out-crossed CspU1oc mutation and the
P insertion of the transformation (all in w
background) were used:
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The survival functions at 25°C for the balanced flies (one copy of the
Csp+ gene), the homozygous CspU1oc
flies and the four P transgenics in homozygous CspU1oc
background are shown in Fig.
5A. Obviously, the dramatic life-shortening phenotype of the
CspU1oc mutation is rescued by the wild-type
cDna1 construct and by the constructs with a deletion in the linker
region and in the C terminus, L8 and
C
27. Flies with only the Ssp construct, on
the other hand, live merely 1 day longer than the null mutants. Life
expectancy (50% survival time) of white flies at 25°C is about 35
days under these conditions. For characterization of the Clp
transformant, which mapped to the third chromosome and was recombined with the
original CspU1w mutation, siblings from the following
crosses were analyzed:
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In the F1 generation we can compare longevity of Csp+ (balanced) flies and Csp-null flies of mixed genetic background (CspU1w/CspU1oc), with or without the Clp transgene. The survival curves for these four genotypes are shown in Fig. 5B. Again, the construct without cysteines (Clp) cannot rescue the short life phenotype and increases life expectancy of the null mutant only by 1020%. Transformants with Cslp and Scsp constructs in CspU1 background could not be tested for longevity because several independent transformant lines were difficult to maintain, indicating that these constructs may produce a dominant negative effect (cf. Discussion). Life expectancy of the hypomorphic CspU2 mutant at 18°C is about 44 days, compared to 12 days for CspU1 and 88 days for wild type under identical conditions. This demonstrates that small amounts of intact CSP can sustain much of its vital function whereas relatively normal expression of CLP or SSP is almost ineffective for sustaining the life of adults.
Temperature-sensitive paralysis of adult flies
Paralysis at elevated temperatures represents a second prominent phenotypic
aspect of Csp null mutants. Temperature sensitivity was quantified as
shown in Fig. 6 for wild type
(w1118;;Csp+), two null mutant lines
(CspU1w and CspU1oc), and five transgenic lines
in CspU1oc background (cDna1, L8,
C
27, Clp, Ssp). For the transgenic lines, each curve
represents the average of two independent insertion lines that did not differ
significantly. The difference between the two null mutants reflects the
influence of genetic background mentioned above (cf. Discussion). This
phenomenon makes it rather difficult to interpret the small difference between
the transgenic lines Clp and Ssp or
C
27 and L
8. Yet it is clear
that the C
27 and L
8
constructs essentially rescue temperature-sensitive paralysis whereas
constructs with mutations in the cysteine string do not. A weak dominant
negative effect is apparent in the two independent insertions of the
Clp construct in the Clp;CspU1oc lines.
Qualitative tests of the few surviving adult Cslp;CspU1w
and Scsp;CspU1w transformants reveal temperature-sensitive
paralysis similar to the null mutant. Again the hypomorphic
CspU2 mutant displays a much weaker phenotype than the
string mutants as it paralyzes at 37°C only after 610 min.
Electrophysiology
The influence of the various mutations in the Csp transgene on
neuromuscular transmission has been studied by intracellular recordings from
body wall muscle 6 of third instar larvae. The motor neurons innervating this
muscle were cut and stimulated by a suction electrode. All transgenes were
analyzed in homozygous CspU1oc genetic background. The
results in Fig. 7 demonstrate
that replacement of the cysteine string in Ssp;CspU1oc
transgenic mutants causes significantly reduced excitatory junction potential
(EJP) amplitude at 18°C and break-down of synaptic transmission at
32°C, similar to the null phenotype
(Umbach et al., 1994).
(Control experiments with wild type and null mutant had shown that responses
were similar at 0.2 and 1.0 Hz). Contrary to expectations on the basis of the
biochemical data and adult phenotypes, larval neuromuscular transmission in
Clp;CspU1oc larvae persisted in four out of five
preparations while the fifth preparation showed the expected null mutant
phenotype. Behavioral observation verified the paralysis of intact
Ssp;CspU1oc transgenic larvae at 32°C while
Clp;CspU1oc larvae appeared almost unaffected by this
temperature. Again unexpectedly, synaptic transmission failed at 32°C in
three out of seven transgenic C
27;CspU1oc
larvae. Equivocal results were obtained with two independent insertion lines
of the L
8 construct.
L
81 responses were similar to those of the
null mutant, L
82 showed wild-type-like
EJPs. It was noted that expression of the transgene was strongly reduced in
L
81 larvae compared to
L
82 larvae but similar in adults of the
two lines. It is therefore assumed that synaptic failure in
L
81 larvae is due to low expression levels
rather than the deletion in the linker region.
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Discussion |
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Linker region and C terminus are not involved in membrane binding
CSPs from the electric organ of Torpedo were shown to be membrane
associated, and the demonstration that at least 11 residues of the cysteine
string were thioester-linked with palmitoic acids suggested that this highly
hydrophobic region was responsible for the observed properties of CSPs as
integral membrane proteins (Gundersen et
al., 1994; Mastrogiacomo et
al., 1994
). Injection of unmodified CSPs into frog oocytes
supported this notion, but deacylation of membrane proteins in experiments
using PC12 cells, HeLa cells or Drosophila heads failed to release
CSPs into the supernatant (van de Goor and
Kelly 1996
; Chamberlain and
Burgoyne 1998
). These findings suggested that the palmitoylated
cysteine string is not required for stable membrane attachment of CSPs and it
was proposed that a different domain might be responsible for membrane
association (Chamberlain and Burgoyne
1998
; van de Goor and Kelly
1996
). None of the mutations introduced in the present work
outside the cysteine string resulted in a release of membrane-bound CSP
variants into the supernatant during a deacylation experiment. Thus neither
the linker nor the C-terminal domain are relevant for membrane attachment of
deacylated CSP.
A cDNA transgene construct coding for the largest CSP isoform rescues the null mutant
In the present study two wild-type Csp cDNA gene constructs were
designed and transformed into Csp null-mutant flies. Both constructs
express the 36 kDa CSP1 isoform. In the wild type this isoform represents the
sole CSP in non-neural tissues, whereas in the brain three additional smaller
isoforms of 34, 33 and 32 kDa are expressed more abundantly
(Eberle et al., 1998). On
sections of cDna1-rescued null mutants the distribution of CSP as
detected by mAb DCSP1 in neuronal (and non-neuronal, data not shown) tissues
matches the stainings described for wild-type flies. We conclude that the
essential sequences for regulation of CSP expression are contained in a region
from 2.3 kb upstream of the transcription start to the end of the second
intron of the Csp gene. Both cDna1 constructs rescue the
salient features of the null-mutant phenotype, i.e. temperature-sensitive
failure of synaptic transmission and early death. Thus expression of the 36
kDa isoform is sufficient to rescue the major cellular functions of the
Csp gene. The cell-specific expression of alternatively spliced CSP
isoforms in the brain (Zinsmaier et al.,
1994
; Eberle et al.,
1998
) may fine-tune synaptic transmission to achieve yet unknown
functional specializations. The strong overexpression of the 36 kDa isoform in
head homogenates of cDna1 transformants is likely to result
predominantly from the absence of alternatively spliced introns such that all
transcripts translate into the same protein isoform. This overexpression of
the 36 kDa isoform has been analyzed in 12 independent strains with different
insertion sites and does not cause any obvious new phenotypes, in either
null-mutant background or when co-expressed with the three smaller isoforms in
the wild type (Fig. 2B and data
not shown). Nie et al. (1999
)
demonstrate rescue of the attenuated excitatory junction potential (EJP)
amplitude of CspU1 larval nerve muscle preparations by
expressing any of the three known cDNAs under control of the elav
promotor (mediated by the GAL4-UAS system), but find dramatic effects on eye
and wing development and on survival rate by overexpressing these cDNAs. Since
CSP overexpression from the Csp promotor in genomic rescue
transformants (Zinsmaier et al.,
1994
; Nie et al.,
1999
) or our cDna1 transformants does not produce these
effects, we propose that they may be caused by higher levels of overexpression
or temporal and/or spatial misexpression due to the elav GAL4
driver.
Phenotypic analysis of mutants or transgenic animals has to take into consideration the influence of genetic background effects. The loss of both semi-lethality and larval synaptic failure at 32°C in the CspU1 null mutant cultured in the homozygous condition, indicates that genetic modifiers suppressing Csp-related phenotypes have been selected for. Extensive out-crossing against wild type was required to reestablish a line that showed phenotypic characteristics similar to the original null mutant. This suggests that important genetic modifiers may be present on the third chromosome in the vicinity of the Csp gene. All electrophysiological experiments and most behavioral tests were therefore performed on flies in the out-crossed CspU1oc null mutant background.
Mutated CSPs cannot fully restore normal function
To elucidate the role of the linker region, the cysteine string (CS), and
the C-terminal domain in CSP function, we analyzed expression and membrane
association of six mutant isoforms in transgenic flies as well as the
associated phenotypes. For each construct several independent transformants
were obtained, and at least two were tested in each experiment in order to
detect possible effects of the site of transgene insertion. Deletion of the
entire cysteine string (11 out of 15 cysteines of the CS region) results in a
protein (CSLP) that is found both in the soluble and membrane fractions.
Replacement of the 11 cysteines by serines in SSP, however, eliminates
membrane targeting. These results suggest that the relative positions of the
remaining two pairs of cysteines may be critical for the residual targeting of
CSLP. The SSP and the CLP are detected only in the soluble fraction and are
not enriched in synaptic terminals. An earlier study used a mammalian mutant
CSP construct in which 7 of the 14 cysteines of the string region were
replaced by serines. When this mutant protein was expressed in PC12 or HeLa
cells, it was not palmitoylated and was not associated with membranes
(Chamberlain and Burgoyne,
1998). Our data make clear that the cysteine string is required
for correct targeting of Drosophila CSPs to synaptic vesicles in an
intact organism. In Drosophila the short cysteine string mutant
protein (SCSP), in which 6 out of 15 cysteines in the string region have been
deleted, appears to be efficiently targeted to membranes. A small increase in
electrophoretic mobility is observed after depalmitoylation of SCSP. In spite
of partial (CSLP) or normal (SCSP) targeting to membranes, transgenic flies
expressing these proteins in null mutant background display paralysis similar
to the null mutant, strongly indicating that an intact cysteine string region
is necessary for cellular functions of CSPs beyond protein targeting. Such
functions may, for example, depend on the tightness of membrane attachment by
the palmitoylated cysteine residues. The fact that several transgenic lines
containing the Cslp or Scsp constructs were difficult or
impossible to maintain in homozygous CspU1w genetic
background indicates that mutant CSPs with intact or partially intact
targeting but modified or deleted cysteine string disturb cellular processes
that can be maintained in the absence of CSPs. SSP and CLP, on the other hand,
are not targeted but slightly improve viability.
The present experiments demonstrate that for the most visible consequences
of CSP function in adult Drosophila an intact linker region and the C
terminus are not essential. Both regions contain amino acids that are highly
conserved in evolution, demonstrating fitness-related function. The
temperature sensitivity of neuromuscular transmission in some
C27;CspU1oc larvae could relate to such a
function and indicates that in larval synapses CSPs may function differently
compared to adult synapses. The unexpected `rescue' of synaptic transmission
and prevention of paralysis at 32°C by CLP in most larvae but not in
adults also points in this direction. The intra-strain variability of some of
the electrophysiological data might be due to polymorphic genetic background
effects, which can never be entirely eliminated. The only defect noticed in
the L
82;CspU1oc line concerns a
moderate temperature sensitivity of adults. The synaptic failure in
L
81;CspU1oc larvae is assumed
to be caused by the low larval expression of the transgene in this strain due
to its specific insertion site.
Western blot signals from head homogenates of all mutated proteins except
C27 are significantly weaker. Since this effect is independent of
transgene insertion sites it is assumed that it reflects reduced stability of
the mutated proteins. Instability of mutated or truncated proteins is a
general phenomenon. The normal stability of the C
27 protein is not
unexpected because a natural mammalian CSP isoform lacks the conserved
C-terminal domain (31 amino acids; Chamberlain and Burgoyne, 1996;
Coppola and Gundersen, 1996
).
We asked whether the reduced abundance of the mutated proteins might simply be
responsible for the phenotype of the transgenic flies in Csp-null
background. However, the hypomorphic mutation CspU2, which
reduces CSP abundance about tenfold but leaves the coding region intact,
displays strongly attenuated phenotypes compared to null mutants, such as
viability at 25°C, longevity reduced at this temperature only to about
50%, delayed temperature-sensitive paralysis and fast recovery
(Eberle, 1995
). These
observations support the above proposal, that the strong phenotype shown by
the cysteine string mutants is not mainly due to incomplete or incorrect
targeting of the mutated protein but that the cysteine string is important for
CSP function beyond its demonstrated role in targeting of the protein to the
synaptic vesicle membrane. This hypothesis can possibly be tested by replacing
the cysteine string with other synaptic vesicle membrane-targeting
signals.
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Acknowledgments |
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Footnotes |
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References |
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