Immunogold Localization of Ribulose-1,5-Bisphosphate Carboxylase with Reference to Pyrenoid Morphology in Chloroplasts of Synchronized <i>Euglena gracilis</i> Cells

Osafune, Tetsuaki; Yokota, Akiho; Sumida, Shuji; Hase, Eiji

doi:10.1104/pp.92.3.802

Cited by 50 publications

(32 citation statements)

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“…More recently, the form II Rubisco in the dinoflagellate Amphidinium also has been localized to pyrenoids (Jenks and Gibbs, 2000). Because the formation of pyrenoids in both Euglena (Osafune et al, 1990), which contains form I Rubisco, and Gonyaulax (Schmitter, 1971) is clock controlled, we reasoned that the form II Rubisco in Gonyaulax might be selectively sequestered into pyrenoids on a daily basis. Thus, we first determined whether Gonyaulax pyrenoids did, in fact, contain form II Rubisco.…”

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confidence: 99%

Circadian Changes in Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Distribution Inside Individual Chloroplasts Can Account for the Rhythm in Dinoflagellate Carbon Fixation

2001

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Previous studies of photosynthetic carbon fixation in the marine alga Gonyaulax have shown that the reaction rates in vivo vary threefold between day and night but that the in vitro activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), which catalyzes the rate-limiting step in this process, remains constant. Using protein gel blotting, we confirm that Rubisco protein levels are constant over time. We present simultaneous measurements of the rhythms of CO 2 fixation and O 2 evolution and show that the two rhythms are ‫ف‬ 6 hr out of phase. We further show that the distribution of Rubisco within chloroplasts varies as a function of circadian time and that this rhythm in Rubisco distribution correlates with the CO 2 fixation rhythm. At times of high carbon fixation, Rubisco is found in pyrenoids, regions of the chloroplasts located near the cell center, and is separated from most of the light-harvesting protein PCP (for peridininchlorophyll a -protein), which is found in cortical regions of the plastids. We propose that the rhythm in Rubisco distribution is causally related to the rhythm in carbon fixation and suggest that several mechanisms involving enzyme sequestration could account for the increase in the efficiency of carbon fixation. INTRODUCTIONCircadian rhythms are daily biochemical changes that are driven by circadian clocks but are not by themselves clocks. Biological clocks are characterized by transcriptional feedback loops that maintain their own oscillation (Dunlap, 1999;Shearman et al., 2000) and that receive phasing information from changes in light intensity (Crosthwaite et al., 1997;Ceriani et al., 1999) or temperature (Liu et al., 1998) that allows them to synchronize their oscillations with natural environmental cycles. In contrast, circadian rhythms are changes in an organism's physiology or biochemistry that, although rhythmic under constant conditions, are incapable of independent oscillations and instead require the timing signals provided by circadian clocks.A useful theoretical framework for understanding many biological rhythms is the notion that a central oscillator provides periodic regulatory signals that result in the control of a rate-limiting step (RLS) in a given biochemical pathway. Thus, understanding the regulation of the RLS is vital to understanding the clock's effect on organisms. For example, previous work on the bioluminescence rhythm in the dinoflagellate Gonyaulax has shown that both dinoflagellate luciferase (the reaction catalyst) and the luciferin (substrate) binding protein LBP contribute to the RLS of the nightly bioluminescence reaction. Cellular levels of both proteins vary seven-to 10-fold between day and night in phase with the 50-to 100-fold variations in bioluminescence capacity (Johnson et al., 1984;Morse et al., 1989a). In a vertebrate example, N -acetyltransferase catalyzes the RLS in vertebrate melatonin synthesis, and N -acetyltransferase levels correlate with the circulating melatonin rhythm (Gastel et al., 1998).Rhythms are generally con...

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confidence: 99%

Circadian Changes in Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Distribution Inside Individual Chloroplasts Can Account for the Rhythm in Dinoflagellate Carbon Fixation

2001

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“…Details of the mechanism differ, however, as in Euglena the plastid-directed leader sequences contain two hydrophobic regions separated by an S/T-rich region reminiscent of a plastid transit sequence (Henze et al, 1995;Kishore et al, 1993). In vitro studies have demonstrated that the plastid directed proteins remain inserted in the ER membranes, unlike the apicomplexans where the plastid proteins are soluble inside the ER (Kishore et al, 1993;Osafune et al, 1990;Sulli et al, 1999;Sulli and Schwartzbach, 1996). The second hydrophobic region present in the Euglena leaders is absent in apicomplexan leaders (Foth et al, 2003).…”

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confidence: 99%

Plastid ultrastructure defines the protein import pathway in dinoflagellates

Nassoury

Cappadocia

Morse

2003

Journal of Cell Science

103

143

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Eukaryotic cells contain a variety of different compartments that are distinguished by their own particular function and characteristic set of proteins. Protein targeting mechanisms to organelles have an additional layer of complexity in algae, where plastids may be surrounded by three or four membranes instead of two as in higher plants. The mechanism of protein import into dinoflagellates plastids, however, has not been previously described despite the importance of plastid targeting in a group of algae responsible for roughly half the ocean's net primary production. Here, we show how nuclear-encoded proteins enter the triple membrane-bound plastids of the dinoflagellate Gonyaulax. These proteins all contain an N-terminal leader sequence with two distinct hydrophobic regions flanking a region rich in hydroxylated amino acids (S/T). We demonstrate that plastid proteins transit through the Golgi in vivo, that the first hydrophobic region in the leader acts as a typical signal peptide in vitro, and that the S/T-rich region acts as a typical plastid transit sequence in transgenic plants. We also show that the second hydrophobic region acts as a stop transfer sequence so that plastid proteins in Golgi-derived vesicles are integral membrane proteins with a predominant cytoplasmic component. The dinoflagellate mechanism is thus different from that used by the phylogenetically related apicomplexans, and instead, is similar to that of the phylogenetically distant Euglena,whose plastids are also bound by three membranes. We conclude that the protein import mechanism is dictated by plastid ultrastructure rather than by the evolutionary history of the cell.

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“…The grids were then floated on PBSwith 0.5% BSAcontaining antiserum or preimmune serum at 100-400 fold dilution for 20min at 37°C. The grids were then washed 5 times in PBS by flotation, and were then floated on PBS containing a 20-fold dilution of fifteen nm of protein Agold (E»Y Labs, USA) for 20min (6). The grids were subsequently floated on 3% (w/v) uranyl acetate for 10-20 min and after drying were viewed in a JEOL100B electron microscope at 80kV.…”

Section: Methodsmentioning

confidence: 99%