©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Three Proteins in the Eye of Aplysia, Whose Synthesis Is Altered by Serotonin (5-HT)
POSSIBLE INVOLVEMENT OF THESE PROTEINS IN THE OCULAR CIRCADIAN SYSTEM (*)

Constantinos Koumenis (1)(§), Marta Nunez-Regueiro (1), Uma Raju (2), Richard Cook (3), Arnold Eskin (1)(¶)

From the (1)Department of Biochemical and Biophysical Sciences, University of Houston, Houston, Texas 77204 and the Departments of (2)Pediatrics and (3)Microbiology and Immunology, Baylor College of Medicine, Houston, Texas 77030

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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()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) .

The reversible transcription inhibitor 5,6-dichloro-1--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) .

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 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.

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.


MATERIALS AND METHODS

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.

To examine the effects of 5-HT on the synthesis of individual proteins, experimental eyes were exposed to 5-HT (5 10M 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 10M) 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.

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 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 HO, 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.).


RESULTS

Effect of 5-HT Treatments on the Synthesis of Individual Proteins

6-h treatments of 5-HT (5 10M) 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.

Groups of five or six experimental eyes were treated with 5-HT (5 10M 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.

The effects of a 5-HT treatment on incorporation of [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

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, 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.

Eyes were exposed to [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).()

The effects of a 6-h treatment with 8-bt-cAMP (2 mM) on incorporation of [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

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,()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.

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) .


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.

To confirm that the 36-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, 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

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 [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.

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 (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.


DISCUSSION

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 [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.

Four of the proteins that exhibited altered incorporation of [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.

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 [H]leucine into BiP 3 h after the end of the treatment.

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 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) .

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 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).

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 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.

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.

  
Table: Summary of the effects of 6-h treatments with 5-HT (5 10M) given at CT 6-12 on incorporation of [H]leucine into proteins

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 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

Proteins were labeled during the last 4 h of the treatment. Asterisks are as described in Table I.


  
Table: Summary of the effects of light, 5-HT, cAMP, and a reversible transcription inhibitor (DRB) on the synthesis of proteins

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).



FOOTNOTES

*
This work was supported by National Institute of Mental Health Grant MH41979 and Air Force Office Sponsored Research Grant F49620-92-J-0494. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Radiation Oncology, Stanford University School of Medicine, Stanford, CA 94305.

To whom correspondence should be addressed. Tel.: 713-743-8381; Fax: 713-743-8397; E-mail: eskin@uh.edu.

The abbreviations used are: 5-HT, serotonin; PAGE, polyacrylamide gel electrophoresis; BFSW, buffered filtered sea water; BiP, binding protein; CT, circadian time; GRP78, glucose-regulated protein 78; HSC70, heat shock cognate 70; HSP70, heat shock protein 70; VDAC, voltage-dependent anion channel; CHAPS, 3-([3-cholamidopropyl)dimethylammonio]-1-propanosulfate; DRB, 5,6-dichloro-1--D-ribobenzimidazole; 8-bt-cAMP, 8-benzylthio-cAMP.

A. Eskin, unpublished observations.

C. Koumenis, Q. Tran, and A. Eskin, submitted for publication.

C. Koumenis and A. Eskin, unpublished results.


ACKNOWLEDGEMENTS

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.


REFERENCES
  1. Rosbash, M., and Hall, J. C.(1989) Neuron3, 387-398 [Medline] [Order article via Infotrieve]
  2. Dunlap, J. C.(1993) Annu. Rev. Physiol.55, 683-728 [CrossRef][Medline] [Order article via Infotrieve]
  3. Page, T. L.(1994) Science263, 1570-1572 [Medline] [Order article via Infotrieve]
  4. Rothman, B. S., and Strumwasser, F.(1976) J. Gen. Physiol.68, 359-384 [Abstract]
  5. Jacklet, J. W.(1977) Science198, 69-71 [Medline] [Order article via Infotrieve]
  6. Yeung, S. J., and Eskin, A.(1988) J. Biol. Rhythms3, 225-236
  7. Jacklet, J. W.(1980) J. Exp. Biol.84, 1-15 [Abstract]
  8. Raju, U., Yeung, S. J., and Eskin, A.(1990) Am. J. Physiol.258, R256-R262
  9. Eskin, A., Yeung, S. J., and Klass, M. R.(1984) Proc. Natl. Acad. Sci. U. S. A.81, 7637-7641 [Abstract]
  10. Takahashi, J. S., Murakami, N., Nicaido, S. S., Pratt, B. L., and Robertson, L. M.(1989) Recent Prog. Horm. Res.45, 279-352 [Medline] [Order article via Infotrieve]
  11. Feldman, J. F.(1989) The Science of Photobiology (Smith, K. C., ed) pp. 193-213, Plenum Press, New York
  12. Khalsa, S. B. S., Whitmore, D., and Block, G. D.(1992) Proc. Natl. Acad. Sci. U. S. A.89, 10862-10866 [Abstract]
  13. Rensing, L., and Hardeland, R.(1990) Chronobiol. Int.7, 353-370 [Medline] [Order article via Infotrieve]
  14. Raju, U., Koumenis, C., Nunez-Regueiro, M., and Eskin, A.(1991) Science253, 673-675 [Medline] [Order article via Infotrieve]
  15. Khalsa, S. B. S., and Block, G. D.(1990) Soc. Neurosci. Abstr.16, 640
  16. Ohi, K., and Takahashi, J. S.(1991) Soc. Neurosci. Abstr.17, 675
  17. Hardin, P. E., Hall, J. C., and Rosbash, M.(1990) Nature343, 536-540 [CrossRef][Medline] [Order article via Infotrieve]
  18. Koumenis, C., and Eskin, A.(1992) Chronobiol. Int.3, 201-221
  19. Hardin, P. E., Hall, J. C., and Rosbash, M.(1993) Molecular Genetics of Biological Rhythms (Young, M. W., ed) pp. 155-169, Dekker, New York
  20. Aronson, B. D., Johnson, K. A., Loros, J. J., and Dunlap, J. C.(1994) Science263, 1578-1584 [Medline] [Order article via Infotrieve]
  21. Corrent, G., Eskin, A., and Kay, I.(1982) Am J. Physiol.242, R326-R332
  22. Eskin, A., Corrent, G., Lin, C-Y., and McAdoo, D. J.(1982) Proc. Natl. Acad. Sci. U. S. A.79, 660-664 [Abstract]
  23. Yeung, S. J., and Eskin, A.(1987) Proc. Natl. Acad. Sci. U. S. A.84, 279-283 [Abstract]
  24. Kennedy, T. E., Gawinowicz, M. A., Barzilai, A., Kandel, E. R., and Sweatt, J. D.(1988) Proc. Natl. Acad. Sci. U. S. A.85, 7008-7012 [Abstract]
  25. Raju, U., Nunez-Regueiro, M., Cook, R., Kaetzel, M. A., Yeung, S.-C. J., and Eskin, A.(1994) J. Neurochem.61 1236-1245 [Medline] [Order article via Infotrieve]
  26. Eskin, A.(1982) J. Neurobiol.13, 241-249 [Medline] [Order article via Infotrieve]
  27. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol.215, 403-410 [CrossRef][Medline] [Order article via Infotrieve]
  28. Ting, J., and Lee, A. S(1988) DNA7, 275-286 [Medline] [Order article via Infotrieve]
  29. Chao, C. C. K., and Lin-Chao, S.(1992) Nucleic Acids Res.20, 6481-6485 [Abstract]
  30. Nover, L.(1991) Heat Shock Response, pp. 5-40, CRC Press, Boca Raton, FL
  31. Kuhl, D., Kennedy, T. E., Barzilai, A., and Kandel, E. R.(1992) J. Cell Biol.119, 1069-1076 [Abstract]
  32. Welch, W. J., Garrels, J. I., Thomas, G. P., Lin, J. J. C., and Feramisco, J. R.(1983) J. Biol. Chem.258, 7102-7111 [Abstract/Free Full Text]
  33. Lee, A. S., Bell, J., and Ting, J.(1984) J. Biol. Chem.259, 4616-4621 [Abstract/Free Full Text]
  34. Bureau, M. H., Khrestchatisky, M., Heeren, M. A., Zambrowicz, E. B., Kim, H., Grisar, T. M., Colombini, M., Tobin, A. J., and Olsen, R. W. (1992) J. Biol. Chem.267, 8679-8684 [Abstract/Free Full Text]
  35. Blachly-Dyson, E., Zambrowicz, E. B., Yu, W. H., Adams, V., McCabe, E. R., Adelman, J., Colombini, M., and Forte, M.(1993) J. Biol. Chem.268, 1835-1841 [Abstract/Free Full Text]
  36. Courgeon, A. M., Maisonhaute, C., and Best-Belpomme, M.(1984) Exp. Cell. Res.153, 515-521 [Medline] [Order article via Infotrieve]
  37. Cervera, J.(1985) Cell Biol. Int. Rep.9, 131-142 [Medline] [Order article via Infotrieve]
  38. Haas, I. G., and Wabl, M.(1983) Nature306, 387-389 [Medline] [Order article via Infotrieve]
  39. Munro, S., and Pelham, H. R. B.(1986) Cell46, 291-300 [Medline] [Order article via Infotrieve]
  40. Gething, M.-J., and Sambrook, J. F.(1992) Nature355, 33-45 [CrossRef][Medline] [Order article via Infotrieve]
  41. Craig, E. A., Gambill, B. D., and Nelson, R. J.(1993) Microbiol. Rev.57, 402-414 [Abstract]
  42. Kim, P. S., Bole, D., and Arvan, P.(1992) J. Cell Biol.118, 541-549 [Abstract]
  43. Gething, M.-J., Blond-Elguindi, S., Mori, K., and Sambrook, J. F. (1994) in The Biology of Heat Shock Protein and Molecular Chaperones (Morimoto, R. I., Tissieres, A., and Georgopoulos, C., eds) pp. 111-135, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  44. Hurtley, S. M., Bole, D. G., Hoover-Litty, H., Helenius, A., and Copeland, C. S.(1989) J. Cell Biol.108, 2117-2126 [Abstract]
  45. Navarro, D., Qadri, I., and Pereira, L.(1991) Virology184, 253-284 [Medline] [Order article via Infotrieve]
  46. Lee, A. S.(1987) Trends Biol. Sci.12, 20-23
  47. Lee, A. S.(1992) Curr. Opin. Cell Biol.4, 267-273 [Medline] [Order article via Infotrieve]
  48. Loomis, W. F., Wheeler, S., and Schmidt, J. A.(1982) Mol. Cell. Biol.3, 1540-1543
  49. Nover, L., and Scharf, K.-D.(1984) Eur. J. Biochem.139, 302-313
  50. Greenberg, S. G., and Lasek, R. J.(1985) J Neurosci.5, 1239-1245 [Abstract]
  51. Welch, W. J., and Feramisco, J. R.(1984) J. Biol. Chem.259, 4501-4513 [Abstract/Free Full Text]
  52. Welch, W. J., and Feramisco, J. R.(1985) Mol. Cell. Biol.5, 1229-1237 [Medline] [Order article via Infotrieve]
  53. Hendrick, J. P., and Hartl, F. U.(1993) Annu. Rev. Biochem.62, 349-384 [CrossRef][Medline] [Order article via Infotrieve]
  54. Colombini, M.(1979) Nature279, 643-645 [Medline] [Order article via Infotrieve]
  55. Benz, R.(1985) CRC Crit. Rev. Biochem.19, 145-190 [Medline] [Order article via Infotrieve]
  56. Benz, R.(1990) Experientia46, 131-137 [Medline] [Order article via Infotrieve]
  57. Colombini, M.(1989) J. Membr. Biol.111, 103-111 [Medline] [Order article via Infotrieve]
  58. Holden, M. J., and Colombini, M.(1988) FEBS Lett.241, 105-109 [CrossRef][Medline] [Order article via Infotrieve]
  59. Liu, M. Y., and Colombini, M.(1992) J Bioenerg. Biomembr.24, 41-46 [Medline] [Order article via Infotrieve]
  60. Zizi, M., Forte, M., Blachly-Dyson, E., and Colombini, M(1994) J. Biol. Chem.269, 1614-1616 [Abstract/Free Full Text]
  61. Thinnes, F. P., Götz, H., Kayser, H., Benz, R., Schmidt, W. E., Kratzin, H. D., and Hilschmann, N.(1989) Biol. Chem. Hoppe-Seyler370, 1253-1264 [Medline] [Order article via Infotrieve]
  62. Dermietzel, R., Hwang, T.-K. Buettner, R., Hofer, A., Dotzler, E., Kremer, M., Deutzmann, R., Thinnes, F. P., Fishman, G. I., Spray, D. C., and Siemen, D.(1994) Proc. Natl. Acad. Sci. U. S. A.91, 499-503 [Abstract]
  63. McEnery, M. W., Snowman, A. M., Trifilleti, R. R., and Snyder, S. (1992) Proc. Natl. Acad. Sci. U. S. A.89, 3170-3174 [Abstract]
  64. Hupp, T. R., Meek, D. W., Midgley, C. A., and Lane, D. P.(1992) Cell71, 875-886 [Medline] [Order article via Infotrieve]
  65. Langer, T., and Neupert, W.(1994) in The Biology of Heat Shock Protein and Molecular Chaperones (Morimoto, R. I., Tissieres, A., and Georgopoulos, C., eds) pp. 53-83, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  66. Rensing, L., Bos, A., Kroeger, J., and Cornelius, G.(1987) Chronobiol. Int.4, 543-549 [Medline] [Order article via Infotrieve]
  67. Gromoff, E. D., Treier, U., and Beck, C. F.(1989) Mol. Cell. Biol.9, 3911-3918 [Medline] [Order article via Infotrieve]
  68. Khalsa, S. B. S., Ralph, R. R., and Block, G. D.(1990) Brain Res.520, 166-169 [Medline] [Order article via Infotrieve]
  69. Barbe, F. M., Tytel, M., Gower, D. J., and Welch, W. J.(1988) Science241, 1817-1820 [Medline] [Order article via Infotrieve]
  70. Pittendrigh, C. S.(1993) Annu. Rev. Physiol.55, 17-54 [CrossRef]

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







This Article
Abstract
Full Text (PDF)
Purchase Article
View Shopping Cart
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Copyright Permissions
Google Scholar
Articles by Koumenis, C.
Articles by Eskin, A.
Articles citing this Article
PubMed
PubMed Citation
Articles by Koumenis, C.
Articles by Eskin, A.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   Biochemistry and Molecular Biology Education 
Copyright © 1995 by the American Society for Biochemistry and Molecular Biology.