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.

Highlights

  • How do we know that the 78-kDa protein, whose synthesis we had previously studied by [3Hneucine labeling in experiments with entraining agents and DRB, and the sequenced protein are the same proteins? Three pieces of evidence suggest that this is the case

  • To confirm that the 36-kDa protein, whose synthesis we had previously studied by [3Hneucine labeling in experiments with entraining agents and DRB, and the sequenced protein are the same proteins, peptide maps of [3Hneucine-Iabeled and Coomassie-stained proteins corresponding to the 36-kDa protein were compared

  • We used a biochemical screen to search for proteins that may serve as components of the ocular circadian oscillator in Aplysia

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Summary

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-cholamidopropyIJdimethylammonioJ-l-propanosulfate; DRB, 5,6-dichloro-I-I3-D-ribobenzimidazole; 8-bt-cAMP, 8-benzylthio-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)/. This protein appears to be similar to HSP70, the major heat-inducible member of the HSP70 family

MATERIALSAND METHODS
RESULTS
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DISCUSSION
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