From the
Previous results using translation inhibitors in the ocular
circadian system of Aplysia suggest that protein synthesis may
be involved in the light and serotonin (5-HT) entrainment pathways or
perhaps in the circadian oscillator. Proteins have been previously
identified whose synthesis was altered by treatments of light capable
of perturbing the phase of the circadian rhythm in the eye of Aplysia. We extended these studies by investigating the
effects of other treatments that perturb the ocular circadian rhythm on
protein synthesis. 5-HT altered the synthesis of nine proteins.
Interestingly, five of the proteins affected by treatments with 5-HT
were previously shown to be affected by treatments with light. Four of
the proteins affected by treatments with 5-HT were also affected by
treatments with analogs of cAMP, a treatment which mimics the effects
of 5-HT on the ocular circadian rhythm. To identify the cellular
function of some of these proteins, we obtained their partial amino
acid sequences. Based on these sequences and additional
characterizations, a 78-kDa, pI 5.6 Aplysia protein appears to
be glucose-regulated protein 78/binding protein, and a 36-kDa, pI 5.7 Aplysia protein appears to be porin/voltage-dependent anion
channel. Heat shock experiments on Aplysia eyes revealed that
yet another one of the Aplysia proteins (70 kDa) affected by
5-HT appears to be a heat-inducible member (heat shock protein 70) of
the family of heat shock proteins. These findings suggest that these
three identified proteins, together or individually, may be involved in
some way in the regulation of the timing of the circadian oscillator in
the eye of Aplysia.
A major problem in circadian biology is to identify the
molecular components of the circadian oscillator. So far, very few
candidates for such components have emerged. The best candidates, the
products of the per gene and the frq gene, have come
from mutational analyses of Drosophila and Neurospora strains, respectively (for reviews, see Refs. 1-3). Such
approaches, however, may not identify all clock components, and
ultimately biochemical approaches also may need to be utilized to
identify additional molecular components. We have used a biochemical
approach to screen for proteins that may serve as components of the
circadian oscillator in the eye of Aplysia.
Our screening
strategy is based on information previously obtained concerning the
entrainment and regulation of the circadian oscillator in the eye.
Pulse treatments with the translation inhibitors anisomycin and
cycloheximide shift the phase of the eye circadian
rhythm(4, 5, 6) , and continuous treatments with
anisomycin lengthen the period of this rhythm(7) . Furthermore,
anisomycin and cycloheximide block phase advances produced by
light(8) , and anisomycin blocks phase advances produced by
serotonin and 8-bt-cAMP (which mimics the effects of 5-HT
The
reversible transcription inhibitor
5,6-dichloro-1-
A model for the circadian oscillator proposed for Drosophila and Neurospora and consistent with data
from the eye of Aplysia proposes that a (clock) gene is
transcribed into mRNA, and soon thereafter it is translated into a
protein. The oscillator feedback loop is completed when the protein
directly, or through an effect on additional protein(s), feeds back and
suppresses its own expression at the level of transcription or
translation, or both(17, 18, 19, 20) .
In Aplysia, the entraining agents light and 5-HT are proposed
to perturb the rhythm through an effect at the level of translation and
perhaps transcription as well. Based on this model, our initial
experimental strategy for the identification of putative oscillator
proteins was to search for specific proteins whose synthesis was
altered by light as well as by agents that mimic the effects of light
on the rhythm.
Using this approach, Raju et al.(8) ,
using two-dimensional polyacrylamide gel electrophoresis (PAGE),
identified a number of proteins in the eye of Aplysia whose
synthesis was altered by light at three phases of the circadian cycle.
The synthesis of a number of these proteins was also found to be
altered by an analog of cGMP and by high K
To investigate the
possible function(s) of the proteins that were affected by
phase-shifting treatments, we obtained partial amino acid sequences
from two of these proteins. These two Aplysia proteins appear
to be similar to binding protein (BiP)/glucose-regulated protein 78
(GRP78), a member of the heat shock protein 70 (HSP70) family, and
porin/voltage-dependent anion channel (VDAC), a channel protein. The
identity of a third protein was determined by heat shock experiments.
This protein appears to be similar to HSP70, the major heat-inducible
member of the HSP70 family.
To examine the effects of 5-HT on
the synthesis of individual proteins, experimental eyes were exposed to
5-HT (5
The 36-
and 78-kDa proteins were cut from 23 two-dimensional gels of eye
samples. In addition, the 36- and 78-kDa proteins were cut from two
preparative gels, which contained
6-h treatments of 5-HT (5
Groups of five or six experimental eyes were treated with 5-HT (5
The effects of a 5-HT treatment on
incorporation of [
Analogs of cAMP mimic the phase-shifting effects of 5-HT on
the circadian rhythm, and 5-HT treatments elevate levels of cAMP in
eyes (22). Also, treatments with translation inhibitors and high
K
Eyes were exposed to [
The effects of a 6-h treatment
with 8-bt-cAMP (2 mM) on incorporation of
[
When the results from experiments using 5-HT and analogs of
cAMP were compared with results previously obtained on the effects of
light (8) and DRB treatments,
Each peptide from the
78-kDa protein yielded a non-overlapping sequence of 40 amino acids (Fig. 3). A sequence search in the Genbank, PIR, and Swiss-Prot
data bases using the BLAST program (27) showed that the
sequences of the two peptides were 95 and 90% identical to sequences of
the human BiP/GRP78(28, 29) . The location of the 78-kDa
protein in our two-dimensional gels (molecular mass and pI values) was
very similar to that reported in the literature for the BiP protein
from several species(30) . In addition, Kuhl et al. (31) obtained the amino acid sequence of a protein in Aplysia sensory neurons with a pI and molecular mass very close to that of
our 78-kDa protein, which was found in eyes. The deduced complete amino
acid sequence of the protein identified by Kuhl et al.(31) was 80% identical to human BiP. The amino acid
sequences from the two peptides we obtained from the 78-kDa protein are
100% identical with corresponding regions of the deduced amino acid
sequence of the Aplysia BiP reported by Kuhl et
al.(31) .
To confirm that the 36-kDa protein,
whose synthesis we had previously studied by
[
The identification of the 78-kDa protein as BiP (a member of
the HSP70 protein family) prompted us to investigate if other proteins
whose synthesis was affected by light, 5-HT, and DRB were related to
the heat shock family of proteins. The effects of heat shock on
proteins in Aplysia eyes were examined in four experiments.
Groups of six experimental and six contralateral matched control eyes
were prelabeled with [
In all four
experiments, there was a dramatic (>10-fold) increase in label
incorporation in a train of proteins around 70 kDa (Fig. 4). The
synthesis of the 78-kDa protein (BiP) was also increased but much less
than that of the 70-kDa train proteins. Two other proteins with
approximate molecular masses of 90 and 110 kDa also were increased in
synthesis in response to the heat shock treatment.
The most basic of
the 70-kDa proteins (pI 5.4) corresponded to a protein whose synthesis
was previously altered by 5-HT and by light (63 kDa)(8) . The
molecular mass and pI of the 70-kDa protein, the large increase of its
synthesis rate in response to heat shock, and its low rate of synthesis
under physiological conditions suggest that the 70-kDa Aplysia protein is an inducible member of the HSP70 family, most probably
a homolog of the mammalian HSP72. This identification is further
supported by the finding that the synthesis of the Aplysia ocular 70-kDa protein was also increased by treatments with
CdCl
We used a biochemical screen to search for proteins that may
serve as components of the ocular circadian oscillator in Aplysia. Our screen entailed correlating the effects of
treatments on the ocular circadian rhythm with effects of these
treatments on the synthesis of specific proteins. We have extended our
previous studies with light by investigating the effects of 5-HT and of
analogs of cAMP on the synthesis of specific proteins. 5-HT treatments
altered incorporation of [
Four of the proteins that
exhibited altered incorporation of [
The finding
that light, 5-HT, DRB, and analogs of cAMP altered incorporation of
label into the 34-, 70-, and 78-kDa proteins suggests that these
proteins are involved in the eye circadian system and may be components
of the circadian oscillator. Another possibility consistent with these
findings is that these proteins are components of the 5-HT/light input
pathways to the circadian oscillator, most likely at some point after
the convergence of the 5-HT and light entrainment pathways. Finally, a
third possibility is that these proteins are components of output
pathways of the circadian oscillator.
It is interesting that the
synthesis of three proteins (34, 36, and 70 kDa) was affected in
opposite ways by light and 5-HT treatments. Light increased
incorporation of label into the 70-kDa protein and decreased it into
the 34- and 36-kDa proteins, while 5-HT had the opposite effects on
these proteins. Light and 5-HT most likely phase shift by affecting the
synthesis of one or more oscillator proteins in opposite ways because
the phase-response curves for light and 5-HT are displaced by about
180° on the phase axis(13, 18) . Therefore, this
correlation adds further support for the idea that the 34-, 36-, and
70-kDa proteins play critical roles in the mechanism of the circadian
oscillator. The fourth protein (78 kDa) was affected in the same way by
light and 5-HT (increase). This interesting finding does not exclude a
role of this protein in the circadian system. It is possible, for
example, that 5-HT may initially increase the synthesis of BiP and
decrease it later through a feedback mechanism. Since the effects of
5-HT on proteins were examined up to only 3 h after the end of the
treatment, more research into longer lasting effects of 5-HT on
proteins is required to investigate this possibility. Alternatively,
the 78-kDa protein could play a role in the light and 5-HT entrainment
pathways or output pathways of the circadian oscillator.
Could the
34-, 36-, 70-, and 78-kDa proteins mediate the effects of light and
5-HT on some other system than the circadian system? For example, light
may alter the synthesis of proteins involved in phototransduction or
light adaptation, whereas 5-HT may alter proteins involved in
neuromodulation(8) . However, the finding that some proteins
were altered as a result of treatments with light, 5-HT, analogs of
cAMP, and DRB may indicate that one or more of these proteins are
involved in the circadian system.
Although additional information on
the physiological properties of these proteins may be gathered (e.g. whether a protein oscillates or not), the identification
of these proteins may ultimately yield more important information.
Previously, we characterized a 40-kDa protein because its synthesis was
affected by light treatments and it was an abundant protein. This
protein was identified as a lipocortin (annexin), and its possible role
in the eye circadian system has been discussed elsewhere(25) .
In light of the 5-HT study presented here, the synthesis of this
protein does not appear to be affected to a large extent by 5-HT
treatments administered during CT 6-12. It is possible, however,
that the synthesis of the 40-kDa protein may be affected even later
than we have assayed here. Alternatively, this protein may be part of
the light entrainment pathway or may be unrelated to the circadian
system.
The 78-kDa Aplysia protein identified through our
experiments appears to be BiP. The name BiP is derived from the term
``binding protein'' because this protein was originally
identified as the protein that binds to the heavy immunoglobulin chain
in the endoplasmic reticulum(38) . BiP, which resides in the
lumen of the endoplasmic reticulum, has extensive sequence homology
with the HSP70 family members HSP70 and HSC70 (39) (see also
below), which are mainly cytosolic. Like the other members of the HSP70
family, BiP functions as a molecular chaperone(40, 41) .
Its main function is to assist in the correct folding of unfolded
proteins as well as in the correct assembly of oligomeric proteins into
multimeric complexes that pass through the endoplasmic
reticulum(42, 43) . At the same time, BiP also binds to
and prevents further processing of incorrectly folded or assembled
proteins (44, 45). BiP is expressed constitutively under physiological
conditions, but its synthesis is induced under a number of stress
conditions (for a review see Refs. 30, 46, and 47). Recently, a number
of treatments with agents that are not usually classified as
``stressors'' has been reported to induce the synthesis of
BiP. Kuhl et al.(31) demonstrated that treatments with
5-HT, which mimic the effects of long-term sensitization in Aplysia, increased the levels of BiP protein in sensory
neurons 3 h after the end of 5-HT treatments, and long-term behavioral
training also increased levels of BiP mRNA. Their finding that BiP
protein synthesis is increased 3 h after the end of 5-HT treatments is
similar to our finding that phase-shifting treatments with 5-HT altered
incorporation of [
The 70-kDa Aplysia protein, whose
synthesis was decreased by 5-HT treatments in our experiments and
increased by light in others, appears to be a member of the HSP70
family. The 70-kDa protein affected by 5-HT is the most basic protein
in a train of five to six 70-kDa proteins. The synthesis of all
proteins that are members of this train was dramatically increased by
heat shock and CdCl
The 36-kDa Aplysia protein, whose synthesis was increased
by 5-HT treatments in our experiments and decreased by light in others,
appears to be porin. Porin is a channel protein first isolated and
characterized from the outer membrane of mitochondria (for reviews see
Refs. 54 and 55). The eucaryotic porin has an unusually high
conductance (around 0.4 ns), and its conductance is voltage regulated
(hence the name VDAC for voltage-dependent anion channel) (56, 57). Its
voltage dependence is regulated by a soluble mitochondrial protein in Neurospora crassa(58, 59) and by NADH in human
mitochondria(60) . Recently, extramitochondrial porin has been
found in a variety of cell types and organs, including the plasma
membrane of human B lymphocytes(61) , the plasma membrane of
human astrocytes(62) , and regions of rat brain(63) .
Furthermore, porin has been copurified with the peripheral
benzodiazepine receptor complex (63) and the GABA
What could the role
of BiP, HSP70, and porin be in the eye circadian system? One role for
BiP is that of a component of the oscillator. BiP may be a limiting
factor in the processing of protein in the endoplasmic reticulum. If
the amount of BiP limits the production of a component of the
oscillator during some phase, then a treatment with light/5-HT that
alters its synthesis would consequently affect the levels of other
oscillator components and result in phase shifting. This mechanism is
also consistent with BiP playing a role in the input pathway to the
oscillator. Finally, it is also possible that BiP could be an output
pathway component. The effects of 5-HT, light, and DRB treatments on
the synthesis of BiP could be due to effects on the oscillator, which
are then transduced to BiP. Elucidating the precise role of BiP in the
circadian system will require techniques to inhibit the expression of
BiP.
Since BiP and HSP70 share very similar cellular functions
(albeit in different cellular compartments), the possible roles in the
circadian system discussed for BiP may also apply to the HSP70 protein.
A difference in their putative roles is that the levels of HSP70 would
influence the levels or functions of cytosolic or nuclear proteins,
whereas levels of BiP would affect only proteins that are processed in
the endoplasmic reticulum. The nuclear localization of HSP70 and its
association with DNA binding proteins like p53 (64) raise the
intriguing possibility that HSP70 could influence the expression of
other proteins that are important for clock function. An interesting
possibility is that HSP70 levels may influence the levels of porin,
since some mitochondrial membrane proteins require cytosolic chaperones
to be maintained in a translocation-competent conformation(65) .
Similarly, the recent findings that porins are present in cell
membranes (see below) raise the possibility that porin may be processed
in the endoplasmic reticulum, and therefore its levels could be
affected by BiP.
In the case of HSP70, a number of results indicate
that a possible link may exist between the expression of this protein
and circadian rhythms. First, Rensing et al.(66) have
shown that heat shock treatments that induce expression of HSP69 in Neurospora phase shift the conidiation rhythm in a
phase-dependent manner. The sensitivity of the rhythm to heat shock
treatments correlated well with the inducibility of HSP69 by heat shock
treatments. Second, in the unicellular alga Chlamydomonas
reinhardtii, Gromoff et al.(67) showed that light
treatments induced the expression of three heat shock genes (HSP68,
HSP70, and HSP80). Although no studies on the phase dependence of these
effects or on a possible correlation of HSP expression to the circadian
clock were performed, these results demonstrate that light can affect
the expression of HSP70 genes in other systems, and they are in
agreement with our results that light increases the expression of HSP70
proteins.
Both light and 5-HT have been shown to phase shift the eye
circadian rhythm through opposite effects on membrane potential. Our
finding that light and 5-HT affect the synthesis of porin in opposite
ways raises the possibility that effects on the expression of porin
could be mediating such effects. In Bulla, Khalsa et al. (68) demonstrated that treatments that inhibit Cl
The results
discussed above raise the following question. Could the expression of
the HSP70 genes (HSP and BiP) and porin be part of the clock and have
additional roles as well? Such an idea is possible, especially in light
of accumulating evidence that elevation of the levels of HSPs in cells
can protect them from environmental insults such as further heat shock
ischemia, anoxia (reviewed in Ref. 30), and light damage in the
retina(69) . A link, therefore, between the circadian clock and
the expression of HSPs could offer cells and organisms an evolutionary
advantage in that extreme temperature fluctuations (as well as other
damaging effects of light like UV irradiation) could be anticipated by
the elevation of HSP levels, which then would protect the cell from the
ensuing stresses.
The idea that HSPs may comprise part of the
oscillator might offer an additional (and perhaps complementary)
perspective to the current theories on the evolution of the eye
circadian system. Pittendrigh (70), in his ``escape from
light'' theory for the evolution of circadian organization, very
elegantly states that `` . . . the daily cycles of temperature and
light in the outside world must have imposed significant and
predictable periodicity on the chemical milieu of early cells. Such
order, not yet organization, would derive from inevitable variation in
the temperature coefficients of the cell's constituent reactions,
and the bottle-neck created by the cold at night.'' Pittendrigh
then proposes that the ``flood'' of UV and visible radiation
provided the evolutionary ``pressure'' for natural selection
of cells that restricted their genome reading and propagating processes
during the subjective night period.
Using the same rationale, one
can also envision a ``flight from heat'' theory, in which the
anticipatory elevation of ``stress proteins'' by the
oscillator would offer a selective advantage to cells that lived in
extreme environmental conditions and was therefore gradually
incorporated into the oscillator's causal loop. In this respect,
it is noteworthy that heat shock proteins are among the most highly
conserved proteins in nature, from Archaebacteria to humans, and that
prokaryotic and eucaryotic porins share common structural and
functional properties. The evolutionary conservation of these proteins
is therefore a property that is consistent with their possible role in
the evolution of the circadian oscillator.
Proteins were labeled during the last 4 h (CT 8-12)
of the 5-HT treatment, for 2 h (CT 13-15) 1 h after the 5-HT
treatment, or for 2 h (CT 15-17) 3 h after the end of the 5-HT
treatment. The values are the normalized geometric mean of the % change
between the experimental and the control proteins [(E - C)/C
Proteins were labeled during the last 4 h of the treatment.
Asterisks are as described in Table I.
Proteins are designated by their molecular mass and pI.
Data for light were taken from Ref. 8, and data for DRB were taken from
Koumenis, Tran, and Eskin (submitted for publication). The * protein
was previously identified as a 63-kDa protein (8). The** protein was
previously identified as a 95-kDa protein (8).
We thank Zhong Chen for providing help with
densitometric and statistical analysis of the autoradiograms and Dr.
Tim Liu and Dr. Kathleen Quigley for reviewing this manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)on the rhythm)(9) . Taken together, these
findings suggest that translation is part of the oscillator mechanism
in the Aplysia eye. This conclusion appears to be a general
one, for translation appears to play a role in many other circadian
systems(10, 11, 12, 13) .
-D-ribobenzimidazole (DRB) also phase
shifts and lengthens the period of the ocular nerve impulse rhythm,
indicating that transcription may also play a role in the mechanism of
the Aplysia circadian oscillator(14) . Similar results
using transcription inhibitors have been obtained in other systems
including the Bulla eye (15) and the chick pineal
gland(16) .
solutions,
which mimic the effects of light on the rhythm. To extend these
previous studies, we investigated the effects of 5-HT on the synthesis
of individual proteins at a phase when 5-HT advances the phase of the
rhythm(21) . We looked for proteins whose synthesis was altered
during the 5-HT treatment as well as 1 and 3 h after the end of the
treatment. Since analogs of cAMP mimic the effects of 5-HT on the phase
of the circadian rhythm(22) , we also searched for proteins
whose synthesis was altered by analogs of cAMP.
Animals
Aplysia californica were obtained from Alacrity Marine Biological Services (Redondo
Beach, CA), Marinus Inc. (Long Beach, CA), and Marine Specimens
Unlimited (Pacific Palisades, CA). Animals were maintained at 15 °C
in artificial sea water and entrained to 12:12-h light-dark cycles for
at least 72 h before they were sacrificed for experiments. Circadian
time (CT) is defined such that CT 0 to CT 12 corresponded to the
projected time in constant darkness that the light portion of the
previous light-dark cycle would have occurred. All dissections were
performed during the last 3 h of the light portion of the light-dark
cycle.
Treatments and Radiolabeling of Isolated
Eyes
Eyes were removed from animals and secured in small
(1.5 ml volume) polypropylene tubes half-filled with sylgard elastomer
(DowCorning) containing 500 µl of buffered filtered sea water
(BFSW) (artificial seawater containing 30 mM HEPES,
streptomycin sulfate (100 mg/ml), and penicillin G (100 units/ml)
(BioWhitaker), pH 7.65, at 25 °C). Groups of 5-6 control and
5-6 contralateral experimental eyes were used for each
experiment. All experiments began with isolated eyes being placed into
constant darkness in a 15 °C incubator at the end of the light
portion of the light-dark cycle.
10
M in BFSW) from CT
6-12. Control eyes were rinsed with BFSW. In the experiments in
which labeling was performed during the 5-HT treatments, a solution of
40 µCi/ml [
H]leucine (>150 mCi/mmol, ICN)
in 5-HT (5
10
M) replaced the 5-HT
solution in experimental eyes, while a solution of
[
H]leucine in BFSW replaced the BFSW solution in
control eyes. In experiments in which labeling began 1 and 3 h after
the end of the 5-HT treatment, both experimental and control eyes were
rinsed four times with BFSW after the end of the 5-HT treatment (CT
12), and [
H]leucine in BFSW was added to the eyes
of each group. At the end of the labeling periods, eyes from both
groups were rinsed five times with ice-cold BFSW and homogenized for 2
min in 50 µl of grinding buffer (50 mM Tris, 1 mM EGTA, 5 mM EDTA, 0.125 mg/ml bacitracin, 0.125 mg/ml
soybean trypsin inhibitor, 1.5 mg/ml benzamidine, 0.8 units/ml
aprotinin, and 0.175 mg/ml phenylmethylsulfonyl fluoride; pH 7.4).
Then, the eyes were ground in 50 µl of lysis buffer (9.1 M ultra-pure urea, 2% Pharmalyte ampholytes (3 parts pH 3-10
and 2 parts pH 4-6.5), 5% 2-mercaptoethanol, 3% CHAPS) for
another 2 min. Urea was then added to each sample to a final
concentration of 9 M. The samples were frozen in liquid
N
and thawed three times and then stored at -80
°C.
Two-dimensional PAGE of Eye
Proteins
Two-dimensional PAGE of all the samples was
performed as described(8) . In the first dimension, proteins
were separated in a pH gradient established with a mixture of
ampholytes (3 parts pH 3-10 and 2 parts pH 4-6.5)
(Pharmacia Biotech Inc.). In the second dimension, proteins were
separated by a 10% polyacrylamide, 1% SDS slab gel (minislab gels, Idea
Scientific, Corvallis, OR). Equal counts of
[H]leucine (as determined by trichloroacetic acid
precipitation) were loaded in each experimental and control gel.
Trichloroacetic acid precipitation was as described(23) . The
gels were dried and then exposed to films for various durations of
time.
Analysis of Autoradiograms
The
autoradiograms of two-dimensional gels were scanned with an automated
densitometer (Technology Resources, Inc., Nashville, TN), and an
integrated optical density (volume) was automatically computed for each
protein. The OD values of the proteins of interest from each gel were
normalized to the aggregate OD value of 4 or more non-changing proteins
to correct for errors in sample loading, differing film development,
etc. Non-changing proteins were proteins that were not significantly
affected by treatments and demonstrated 10% change in OD ((E
- C)/C
100) between the experimental and control gels
across all experiments. The values reported for proteins represent the
geometric mean of the % change between the normalized ODs of the
experimental and the control proteins ((E - C)/C
100)
± S.E. The p values were calculated using a two-tailed
Student's t test. The reported N numbers
represent the number of times each experiment was independently
repeated. Different N numbers were due to the fact that some
proteins were not visible or could not be reliably analyzed in all
experiments.
Amino Acid Sequencing of Peptides
To
obtain amino acid sequences of the 36- and 78-kDa proteins, preparative
two-dimensional PAGE of Aplysia eye homogenates was performed
to purify and collect individual proteins. Details for this procedure
as well as for the subsequent V8 digestion, electroblotting of
peptides, and sequencing procedures have been previously described (24,
25). For the preparative two-dimensional gel procedure, eyes were
processed in grinding and lysis buffers as described above for
analytical two-dimensional PAGE, except that the grinding buffer
contained 50 µg/ml RNase A and 0.1 mg/ml DNase 1. About 500 µg
of protein (the amount of protein extracted from approximately 25 eyes)
was loaded on each tube gel of the first dimension. After the second
dimension of separation by SDS slab gels, the gels were stained with
Coomassie Brilliant Blue R-250, and the proteins of interest were cut
out of the gels and stored in 50% MeOH at -20 °C.
C-labeled eye proteins.
The
C-labeled and unlabeled gel pieces of each protein
were mixed, put in Eppendorf tubes, and a reaction buffer (20%
glycerol, 0.05 M Tris (pH 6.8), 0.2% bromphenol blue, 0.1%
SDS, and 3.5 mg/ml (2.5 units/ml) V8 protease (sequencing grade,
Sigma)) was added. The gel pieces were ground, and the slurry was
loaded onto a one-dimensional SDS gel. Molecular weight markers
(Rainbow, low MW, Amersham Corp.) were loaded in an adjacent well. The
material was run at 75 V through the stacking gel. When the dye front
reached the interface between the stacking and separating gels, the run
was stopped, the slurry was removed from the wells, and the run was
then continued at 250 V. The peptides from the V8 digestion were
electroblotted at 0.5 A for 50 min onto a Problott membrane (Applied
Biosystems Inc.) using a Transblot system apparatus (Bio-Rad). After
the transfer, the membrane was rinsed with double distilled
H
O, 100% MeOH and finally stained with Coomassie Brilliant
Blue R-250 for 30 s. The membrane was destained in 50% MeOH, dried, and
stored at -20 °C. The peptide bands were cut out and
sequenced with a 477-A pulsed liquid protein sequencer (Applied
Biosystems Inc.).
Effect of 5-HT Treatments on the Synthesis of
Individual Proteins
10
M) shift the phase of the eye circadian rhythm in a
phase-dependent manner. 5-HT produces its largest effect when given at
CT 6-12, and it advances the phase of the rhythm by about 2.5 h
at this time(21) . Therefore, we looked for proteins whose
synthesis was affected by 5-HT treatments given during CT 6-12.
10
M in BFSW) during CT 6-12.
Five or six contralateral matched control eyes were treated only with
BFSW. Because the inhibition of translation produced by anisomycin
treatments does not reverse completely until 9 h after the end of the
treatment(6) , it is possible that, in the experiments where
anisomycin blocked the phase-shifting effects of 5-HT
treatments(9) , protein synthesis was inhibited not only during
the 5-HT treatment but also for a number of hours after the end of the
6-h 5-HT treatment. This means that proteins whose synthesis is
important for 5-HT to produce phase shifts may have been affected by
5-HT during the treatment or for sometime after the end of the 5-HT
treatment. Therefore, we looked for proteins that were affected by 5-HT
during the treatment by administering [
H]leucine
during the last 4 h of the 5-HT treatment. Also, we looked for proteins
that were affected by 5-HT in a delayed manner by administering
[
H]leucine for 2 h, beginning at 1 or 3 h after
the end of the 5-HT treatment.
H]leucine into individual
proteins are shown in Fig. 1. summarizes the effects
of 5-HT treatments on incorporation of [
H]leucine
when proteins were labeled at the three different times. 5-HT altered
[
H]leucine incorporation into four proteins when
labeling was performed during the last 4 h of the 5-HT treatment (CT
8-12). The effects of 5-HT ranged from an increase in
incorporation of 602% (34-kDa protein) to a decrease in incorporation
of 26% (55-kDa protein).
Figure 1:
Effect of treatment with 5-HT given at
CT 6-12 on incorporation of [H]leucine into
proteins. Shown are autoradiograms of two-dimensional gels of ocular
proteins. Six experimental and six control eyes were used in this
experiment. Proteins were labeled during the last 4 h of the 5-HT
treatment. The arrows indicate proteins (molecular mass) whose
synthesis was significantly affected by 5-HT treatments when eyes were
labeled during, 1 h, or 3 h after the 5-HT treatment (see text and
Table I). The film was slightly overexposed so that both lightly
labeled and heavily labeled proteins could be
visualized.
When eyes were exposed to label between CT
13-15 (that is from 1-3 h after the 5-HT treatment),
incorporation of [H]leucine into four proteins
was affected. One of these four proteins (34 kDa) was also affected
during the 5-HT treatment, while three of the proteins affected were
``new'' proteins. When eyes were exposed to
[
H]leucine between CT 15-17 (that is
3-5 h after the 5-HT treatment), incorporation of
[
H]leucine into two ``new'' proteins
(which were not affected during or 1 h after the 5-HT treatment) was
affected. No ``old'' proteins (i.e. those affected
during or 1 h after the 5-HT treatment) were significantly affected 3 h
after the 5-HT treatment. The effects of 5-HT when proteins were
labeled 1 or 3 h after the 5-HT treatment ranged from an increase in
incorporation of 470% (78-kDa protein) to a decrease in incorporation
of 32% (70-kDa protein).
Effect of 4-h Treatments with cAMP Analogs on the
Synthesis of Individual Proteins
, which block the effects of 5-HT on the rhythm,
block the effects of analogs of cAMP on the
rhythm(9, 26) . Thus, it is very likely that elevation
of cAMP mediates the effect of 5-HT on the circadian rhythm. Therefore,
we predicted that treatments with analogs of cAMP would affect the
synthesis of some proteins in the same manner as that produced by 5-HT.
The effects of analogs of cAMP on the proteins affected by 5-HT were
examined.
H]leucine
during the last 4 h of the treatments with analogs of cAMP, which were
given from CT 6-12. 8-bt-cAMP was used in the first three
experiments, and 8-bromo-cAMP was used in the last two experiments.
When the effects of each analog on proteins were examined separately,
the results were very similar. In addition, the two analogs of cAMP
produced statistically indistinguishable phase shifts of the circadian
rhythm (22).
(
)
H]leucine into proteins are shown in Fig. 2. summarizes the effects of treatments with
analogs of cAMP on five of the proteins affected by 5-HT. Four of the
proteins in which incorporation of label was affected by 5-HT (the 34-,
49-, 70-, and 78-kDa proteins) were also affected by treatments with
cAMP analogs. A fifth protein (36-kDa protein) was not significantly
affected by treatments with analogs of cAMP. It is possible that a
larger number of experiments may reveal a significant effect of analogs
of cAMP on the 36-kDa protein. Analysis of the gels indicated that
analogs of cAMP increased incorporation of label into the 36-kDa
protein in four out of five experiments.
Figure 2:
Effect of a treatment with 8-bt-cAMP given
at CT 6-12 on incorporation of [H]leucine
into proteins. The arrows indicate proteins (molecular mass)
whose synthesis was significantly affected by 8-bt-cAMP and
8-bromo-cAMP treatments when eyes were labeled during the treatment
(see text).
Incorporation of
[H]leucine into the 34-, 49-, and 78-kDa proteins
was increased (increases ranged from 150 to 1101% compared with control
values), while it was decreased into the 70-kDa protein (49% relative
to control values) as a result of treatments with the analogs of cAMP.
Interestingly, the 34-, 49-, 70-, and 78-kDa proteins were affected in
the same way by 5-HT treatments. However, with the exception of the
78-kDa protein, treatments with analogs of cAMP resulted in much larger
changes than those produced by 5-HT.
Microsequencing of Proteins
(
)it was found that
the 27-, 34-, 36-, 70-, and 78-kDa proteins were altered by both light
and 5-HT (the 70-kDa protein corresponds to a protein previously named
as 63k, and the 78-kDa protein corresponds to a protein previously
named 95k(8) ) (see I). The 34-, 70-, and 78-kDa
proteins were also affected by analogs of cAMP, while 34-, 36-, 70-,
and 78-kDa proteins were affected by treatments with DRB. To
investigate the possible functions of the proteins affected by these
phase-shifting treatments, we obtained partial amino acid sequences of
some of them. The 36- and 78-kDa proteins were selected first for
microsequencing because they were altered by both light and 5-HT and
because they are relatively abundant proteins.
Identification of the 78-kDa Protein
78-kDa
proteins were excised from 23 preparative two-dimensional gels and
digested with Staphylococcus aureus V8 protease as described
under ``Materials and Methods.'' The peptides resulting from
the digestion were separated by one-dimensional gel electrophoresis and
electroblotted onto a polyvinylidene difluoride membrane. Two peptide
bands (26 and 11 kDa) (10 and 12 pmol) were excised from the
membrane and placed into a microsequencer.
Figure 3:
Comparison of the amino acid sequences of
peptides derived from Aplysia proteins with amino acid
sequences of known proteins. Top, the amino acid sequences of
two peptides derived from the 36-kDa protein are aligned with porin
sequences from human and rat. The 20-kDa peptide is 72% identical to
rat porin, and the 10-kDa peptide is 72% identical to human porin (34,
35). Bottom, the sequences of two peptides derived from the
78-kDa protein are aligned with human BiP/GRP78. The 26-kDa peptide is
95% identical, and the 11-kDa peptide is 90% identical to human
BiP/GRP78 (28, 29). Identical amino acids are indicated by solidlines, and similar amino acids are indicated by a colon. The percent identities and P values (the
probabilities that matches as good or better than those found would
occur by chance) were obtained using the BLAST program at NCBI
(27).
How do we know that the 78-kDa protein, whose
synthesis we had previously studied by [H]leucine
labeling in experiments with entraining agents and DRB, and the
sequenced protein are the same proteins? Three pieces of evidence
suggest that this indeed is the case. First, in heat shock experiments
in which eyes were exposed to elevated temperatures for 30 min (see
below), the incorporation of [
H]leucine into the
78-kDa protein, which had been previously studied in light, 5-HT and
DRB experiments, was found to be greatly increased (Fig. 4).
Since the synthesis of BiP has been shown to increase in response to
heat shock in a number of systems(32, 33) , this result
is consistent with the sequencing information we obtained for this
protein. Second, the patterns of peptide maps resulting from digestion
of Coomassie-stained and [
H]leucine-labeled
78-kDa protein spots are very similar (results not shown). The darkest
Coomassie-stained peptides corresponded to the most intensely
[
H]leucine-labeled peptides. This also strongly
suggests that the protein we sequenced (BiP) is the same protein we had
previously studied in [
H]leucine labeling
experiments. Third, we recently found that 5-HT treatments at CT
6-12 increase the levels of BiP mRNA in Aplysia eyes.
(
)This finding is consistent with the
fact that 5-HT treatments from CT 6-12 affected the synthesis of
the 78-kDa protein, which appears to be BiP.
Figure 4:
Effect
of a 30-min heat shock on the synthesis of proteins in the eye of Aplysia. Eyes in the experimental group were prelabeled with
[H]leucine at 15 °C for 30 min, and then the
eyes were heat shocked by exposing them to 37 °C for 30 min. At the
end of this period, the eyes were returned to 15 °C for another
hour. Eyes in the control group were labeled as those in the
experimental group but remained at 15 °C throughout the treatment
period. The arrows in the figure indicate two proteins (70 kDa
and 78 kDa) whose synthesis was affected by heat shock as well as by
light and 5-HT. The 36-kDa protein, which was not affected by heat
shock, is also shown. The smallarrows in the figure
indicate additional proteins whose synthesis showed a dramatic increase
following heat shock. Four independent experiments were
performed.
Identification of the 36-kDa Protein
The
36-kDa protein was excised from 23 preparative two-dimensional gels and
digested with S. aureus V8 protease. Two peptides (20 kDa and
10 kDa) derived from this protein (5 and 30 pmol, respectively), were
sequenced. The 20-kDa peptide yielded a sequence of 30 amino acids,
which did not overlap with a sequence of 25 amino acids obtained from
the 10-kDa peptide (Fig. 3). A search of the PIR and Swiss-Prot
data bases using the BLAST program (27) showed that these
sequences were each 72% identical (over a 25- and 22-amino acid overlap
region, respectively) to rat and human
porin/VDAC(34, 35) . Porins are channel proteins found
in several eucaryotic membranes and are analogous to porins found in
the plasma membrane of bacteria.
H]leucine labeling in experiments with
entraining agents and DRB, and the sequenced protein are the same
proteins, peptide maps of [
H]leucine-labeled and
Coomassie-stained proteins corresponding to the 36-kDa protein were
compared. The peptide maps of the 36-kDa protein exhibited very similar
patterns, with a good correspondence in the degrees of labeling and
staining (results not shown).
Effect of 30-min Heat Shock on the Synthesis of
Individual Proteins and the Identification of the 70-kDa Protein
H]leucine for 30 min. Then,
at CT 6 the experimental group was ``heat shocked'' at 37
°C for 30 min, while the control group remained at 15 °C. After
the heat treatment, the experimental group was returned to 15 °C,
and labeling was continued for another hour.
(50 µM) (data not shown). CdCl
treatments have been reported to increase the synthesis of HSP70
proteins(36, 37) . Because of the very low amount of the Aplysia ocular 70-kDa protein, no protein staining with
Coomassie Brilliant Blue could be identified that corresponded to the
[
H]leucine protein we studied. Therefore,
microsequencing of this protein is not feasible at this time.
H]leucine into four
proteins when labeling was performed during the last 4 h of the
treatment, while incorporation into six proteins (including one protein
from the group of four mentioned above) was affected in a delayed
manner, that is after the end of the 5-HT pulse. Alteration of
incorporation of [
H]leucine into a protein by a
5-HT treatment does not necessarily mean an increase or decrease in the
synthesis of that protein. Other mechanisms, such as decreased or
increased rate of protein degradation, may be responsible for such an
effect. More research is required to elucidate the precise mechanisms
by which the effects of 5-HT occur.
H]leucine as
a result of 5-HT treatments, the 34-, 36-, 70-, and 78-kDa proteins,
had been previously identified as proteins that were affected by light
treatments(8) . All of these proteins except the 36-kDa protein
were also affected by analogs of cAMP. It is possible that additional
experiments would yield a significant effect of analogs of cAMP on the
36-kDa protein since analogs of cAMP appeared to produce substantial
increases in incorporation of [
H]leucine in four
of five experiments. In addition, incorporation of label into the 34-,
36-, and 70-kDa proteins was also affected by DRB treatments that can
shift the phase of the circadian rhythm.
H]leucine into BiP 3 h after
the end of the treatment.
treatments. It is very likely that the
proteins in this train are phosphorylated forms of the 70-kDa
protein(48, 49) . These results are in agreement with
those of Greenberg and Lasek (50) on the expression of proteins
in Aplysia abdominal and pleural ganglia cells in response to
heat shock. HSP70 is a member of the HSP70 family of heat shock
proteins, which includes BiP. It is mainly cytosolic, expressed at very
low levels under physiological conditions, and its synthesis is greatly
induced by a variety of stressors (reviewed in Ref. 28). Upon heat
shock, HSP70 is also present in the nucleus(51, 52) .
Like BiP, HSP70 also functions as a molecular chaperone(53) .
receptor
complex(34) . Because of the large unit conductance of porin and
its anionic selectivity, a change in its expression levels could have
an impact on the membrane potential (see below).
conductance shortened the free running period, suggesting that a
Cl
conductance is involved in the circadian system.
The anionic selectivity and large conductance of porin and the recent
reports of localization of porin in the plasma membrane of nerve cells
are consistent with a putative role of porin as a circadian oscillator
component. More research on the localization of porin at both the
subcellular and cellular levels in the Aplysia and Bulla eye as well as on the precise mechanism of the effects of light
and 5-HT on its expression is required to explore the possibility of an
involvement of porin in these circadian systems.
Table: Summary of the effects of 6-h treatments with
5-HT (5 10
M) given at CT
6-12 on incorporation of [
H]leucine into
proteins
100] for N number of experiments and are reported
along with S.E. and p values. The p values were
calculated using a two-tailed Student's t-test *, p < 0.05;**, p < 0.01. The different N numbers for some proteins are due to the fact that not all
proteins were visible or could be reliably analyzed in all experiments.
Table: Summary of the
effects of 6-h treatments with analogs of cAMP (CT6-12) on
incorporation of [H]leucine into proteins
Table: Summary of the effects of light, 5-HT, cAMP,
and a reversible transcription inhibitor (DRB) on the synthesis of
proteins
-D-ribobenzimidazole; 8-bt-cAMP,
8-benzylthio-cAMP.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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Copyright © 1995 by the American Society for Biochemistry and Molecular Biology.
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Molecular and Cellular Proteomics
Journal of Lipid Research
Biochemistry and Molecular Biology Education