The Role of Chloroplast‐Membrane‐Protein Synthesis in the Circadian Clock

Abstract

A polypeptide (polypeptide P39), which is presumed to be involved in the photosynthetic circadian rhythm in the green alga Acetabularia, was purified from the EDTA-insoluble chloroplast membrane fraction by means of preparative dodecylsulfate gel electrophoresis and then partially characterized.The purity of the isolated polypeptide P39 was confirmed by a further electrophoresis on an analytical dodecylsulfate gel and further elucidated by amino-terminal analysis which shows that glycine is the only amino-terminal amino acid of the purified polypeptide material. The molecular weight of the polypeptide P39 was found to be about 39000 on analytical gel electrophoresis and the value was further supported by those obtained from amino acid composition and peptide mapping.The amino acid composition of polypeptide P39 showed that the proportion of intermediate amino acid groups is high while the proportion of hydrophilic amino acid groups is well balanced by that of hydrophobic amino acid groups, a property characteristic of membrane proteins.Cycloheximide is known to cause phase shifts in the photosynthetic circadian rhythm of Acetabulavia in a phase and temperature-dependent manner [l]. Previous reports [2-41 indicate that at least one protein (polypeptide P39, designated as polypeptide X in [3,4]) is synthesized in the presence of cycloheximide and is retained within the chloroplast membrane fraction. The time and amount of the synthesis of polypeptide P39 in the presence of cycloheximide coincide with the time and duration, respectively, of sensitivity towards cycloheximide for phase-shifting the photosynthetic circadian rhythm. This polypeptide (polypeptide P39) was suggested to play an essential role in the circadian clock [4,5]. It was, therefore, tempting to purify this polypeptide and to study its characteristics and biochemical properties in order to further understand the regulating mechanism underlying the photosynthetic circadian rhythm.Although much is known about the structure, function and bioenergetics of the chloroplast [6 -141, only a few protein components have been isolated from the chloroplast membrane and characterized [15 -221. Most of these are chlorophyll-protein functional complexes related to the photosystems I and 11.

On the role of gene expression in the circadian oscillator mechanism

“…In the course of such investigations, the interest was directed to a protein of molecular weight 39,000, which is localized in the chloroplast membrane of Acetabularia (Leong et al, 1977;Leong, 1978: Leong and Schweiger, 1978, 1979. A quite unusual response to cycloheximide was ascribed to this protein, which was therefore suggested to play an important role within the oscillator.…”

Chronobiology at the cellular and molecular levels: Models and mechanisms for circadian timekeeping

“…Indeed, Leong and Schweiger (1979) have reported a periodically appearing membrane protein (polypeptide P39, or polypeptide X) which was synthesized in the presence of CHX and was retained in the chloroplast membrane fraction. Moreover, the timing and amount of synthesis of this polypeptide coincided with the timing and the duration, respectively, of the phase of the circadian rhythm of PA sensitive to phase shifting by pulses of CHX and depended on temperature in a similar manner.…”

“…Moreover, the timing and amount of synthesis of this polypeptide coincided with the timing and the duration, respectively, of the phase of the circadian rhythm of PA sensitive to phase shifting by pulses of CHX and depended on temperature in a similar manner. This protein has been purified and found to have a molecular weight of about 39,000 on the basis of analytical gel electrophoresis, amino acid composition, and peptide mapping (Leong and Schweiger, 1979). Furthermore, the proportion of intermediate amino acid groups was high, while the proportion of hydrophilic amino acid groups was well balanced by that of hydrophobic amino acid groups, a property characteristic of membrane proteins.…”

“…Further, it incorporated the recent reemphasis on the necessary role of protein synthesis in the maintenance (if not generation) of persisting circadian rhythms in Acetabularia, Gonyaulax, and Neurospora. Of course, the search for "essential" proteins may itself be very time-consuming (Leong and Schweiger, 1979), with no guarantee of immediate success (although the advent of the powerful techniques of recombinant DNA research and gene "cloning" may offer some solace).…”