Planta
Planta (Berl.) 129, 183-189 (1976)
9 by Springer-Verlag 1976
Biosynthesis and Release of Cell Wall-like Glyeoproteins during the Vegetative Cell Cycle of Chlamydomonas reinhardii* ** Winfried C. Lang*** and Maarten J. Chrispeels Department of Biology, John Muir College, University of California, San Diego, La Jolla, California 92093, USA
Summary. The culture medium of asynchronously growing Chlamydomonas reinhardii cells contains distinct proteins which are derived from the cell walls of these cells. When cultures are synchronized by a light-dark cycle cell wall proteins are synthesized throughout the cycle, but the release of these proteins into the culture medium occurs primarily in the last quarter of the cycle, after cell separation has occurred. The mutant CW-2, which does not form a normal cell wall, continuously synthesizes and secretes cell wall proteins into the culture medium. The synthesis of cell wall protein during the cell cycle appears to be modulated and peaks of synthesis occur at the end of the light period and in the second half of the dark period, shortly after cell separation. At these times the cells devote 15% of their protein-synthetic capacity to making cell wall proteins.
Introduction
The cell cycle in Chlamydomonas reinhardii is marked by the periodic expressions of discrete cytological and biochemical events. The timing of these events can be studied in light synchronized cultures. The synthesis of chloroplast DNA, chlorophyll, and other components of the photosynthetic system occur during the light period, while cell division and subsequent cell separation occur during the dark period * Abbreviations: SDS = sodium dodecyl sulfate ; TCA = trichloroacetic acid. ** Supported by a contract from E R D A (E(04-3) 34/159) to M.J.C. and a grant from the Deutsche Forschungsgemeinschaft to W.C.L. *** This work was performed while W.C.L. was on leave from the University of Kaiserslautern. Dr. Lang's permanent address is Fachbereich Biologie der Universit/it, Pfaffenbergstral3e 95, D-6750 Kaiserslautern, F. R. G. Requests for reprints may be addressed directly to W.C.L.
(for a review, see Ref. [8]). The accumulation of cellular protein is continuous during the light period, but ceases when the light is turned off. This cessation of protein accumulation results from an increased protein turnover during the dark period [7]. Although there is no protein accumulation during the dark period, individual enzyme activities have been shown to increase during this portion of the cycle [8]. There is presently no information about the timing of cell wall synthesis or about the accumulation of cell wall macromolecules during the cell cycle. The cell wall of C. reinhardii lacks a microfibrillar component, but consists of a multilayered structure composed of glycoproteins some of which form highly ordered crystalline lattices [12]. These glycoproteins, like their counterparts in higher plants, are rich in hydroxyproline, arabinose and galactose [10]. To study the timing of cell wall glycoprotein synthesis we have made use of the mutant cell line CW-2 which does not form a normal cell wall but secretes its cell wall glycoproteins into the culture medium [4].
Materials and Methods Strains
Wild type cells are derivatives of Chlamydomonas reinhardii Dangeard strain 137C mating type + . The mutant line is a derivative of strain CW-2, isolated by Davies and Plaskitt [4].
Media and Growth Conditions
Both strains were grown autotrophically on high salt medium [14] at room temperature in 3L Fernbach flasks containing 1L of culture medium. The cultures were constantly bubbled with filtered 5% CO2 in air and constantly mixed by stirring bars, driven by thermally isolated magnetic mixers. For asynchronous growth, the cells were constantly illuminated from above by fluorescent light (4,000 Lx). Synchronous growth was achieved by the method of Surzycki [15] using a 12 h light-12 h dark illumination cycle. Stock cultures,
184 maintained on yeast extract/acetate agar slants were constantly illuminated at room temperature. Cells were transferred every two months onto fresh slants. When the cells were to be labeled with 35SO2-, magnesium chloride was substituted for magnesium sulfate in the culture medium.
Cell Concentration Determinations Cell concentrations were determined by duplicate hemocytometer counting. Cells which had completed division and contained multiple daughter ceils in the mother cell wall were scored as single cells until their separation was clearly visible. Separation of Cells and Culture Medium. The cell culture was rapidly cooled to 4~ and centrifuged at 10,000xg for 10 min in a refrigerated centrifuge. The culture medium was decanted immediately after the rotor had come to a stop.
Colorimetric Determinations Protein. Protein was estimated with ninhydrin [13] after hydrolysis in 6N HC1 (18 h, ll0~ and removal of the hydrochloric acid in a rotary evaporator. Hydroxyproline. Hydroxyproline was estimated in the neutralized hydrolysates according to Kivirriko [9]. Carbohydrates. Carbohydrates were measured with the phenolsulfuric acid test using mannose as a standard [5].
Analysis of Amino Acids and Sugars Analysis of the amino acids was performed on neutralized acid hydrolysates with an automated amino acid analyzer according to the method of Moore and Stein [11], Identification and quantitative determination of neutral sugars was performed by gas-liquid chromatography of acetylated sugar alcohols according to Albershelm [I].
W.C. Lang and M.J. Chrispeels: Synthesis of Cell Wall Proteins nonradioactive precursor in 5% TCA and radioactivity was determined as described. The radioactivity in aqueous samples was determined by mixing a 1 ml aliquot with 7 ml of Aquasol (New England Nuclear Corp.)
Results and Discussion The Accumulation of Cell Wall Proteins in the Culture Medium During the Cell Cycle The accumulation of protein in the cells and in the culture medium of wild type and CW-2 cells is shown in Fig. 1. The results show that cellular protein accumulated continuously during the light period, and that protein accumulation stopped during the dark in both the wild type and the mutant cells. Accumulation of protein in the culture medium was also continuous during the cell cycle of both mutant and wild type cells. However, a comparison of the two cell lines shows that in the wild type most rapid accumulation of medium proteins occurred in the middle of the dark period, around the time of cell separation, while in the mutant accumulation was most rapid during the light period. To find out whether the medium contained proteins similar to the cell wall glycoproteins characterized by Roberts [12] we analyzed the amino acid and sugar composition of a dialyzed preparation of medium macromolecules (Table 1). The proteins contained about 9~ hydroxyproline, which is somewhat less than the value of 13.4% reported by Roberts [12] for purified cell walls. This suggests that some non-cell wall proteins way
SDS Gel Electrophoresis The cell free culture medium was dialyzed against water (4~ 24 h), then made 0.01% SDS and fteeze-dried. The dry material was redissolved in 3 to 5 ml of water, again dialyzed, freeze-dried and finally redissolved in 0.6 ml water. After the addition of 0.1 ml 10% /~-mercaptoethanol, 0.1 ml glycerol and 0.1 ml bromphenolblue (as tracking dye), the sample was boiled for 2 rain. For preparation of the gels and separation of the polypeptides the method of Weber and Osborn [161 was used with the gel buffer at 1/3 of the recommended concentration. The gels were run at 10 mA/gel for approximately 8 hrs, stained with 0.25% Coomassie Blue and destained.
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Planta (Berl.) 129, 183-189 (1976)
9 by Springer-Verlag 1976
Biosynthesis and Release of Cell Wall-like Glyeoproteins during the Vegetative Cell Cycle of Chlamydomonas reinhardii* ** Winfried C. Lang*** and Maarten J. Chrispeels Department of Biology, John Muir College, University of California, San Diego, La Jolla, California 92093, USA
Summary. The culture medium of asynchronously growing Chlamydomonas reinhardii cells contains distinct proteins which are derived from the cell walls of these cells. When cultures are synchronized by a light-dark cycle cell wall proteins are synthesized throughout the cycle, but the release of these proteins into the culture medium occurs primarily in the last quarter of the cycle, after cell separation has occurred. The mutant CW-2, which does not form a normal cell wall, continuously synthesizes and secretes cell wall proteins into the culture medium. The synthesis of cell wall protein during the cell cycle appears to be modulated and peaks of synthesis occur at the end of the light period and in the second half of the dark period, shortly after cell separation. At these times the cells devote 15% of their protein-synthetic capacity to making cell wall proteins.
Introduction
The cell cycle in Chlamydomonas reinhardii is marked by the periodic expressions of discrete cytological and biochemical events. The timing of these events can be studied in light synchronized cultures. The synthesis of chloroplast DNA, chlorophyll, and other components of the photosynthetic system occur during the light period, while cell division and subsequent cell separation occur during the dark period * Abbreviations: SDS = sodium dodecyl sulfate ; TCA = trichloroacetic acid. ** Supported by a contract from E R D A (E(04-3) 34/159) to M.J.C. and a grant from the Deutsche Forschungsgemeinschaft to W.C.L. *** This work was performed while W.C.L. was on leave from the University of Kaiserslautern. Dr. Lang's permanent address is Fachbereich Biologie der Universit/it, Pfaffenbergstral3e 95, D-6750 Kaiserslautern, F. R. G. Requests for reprints may be addressed directly to W.C.L.
(for a review, see Ref. [8]). The accumulation of cellular protein is continuous during the light period, but ceases when the light is turned off. This cessation of protein accumulation results from an increased protein turnover during the dark period [7]. Although there is no protein accumulation during the dark period, individual enzyme activities have been shown to increase during this portion of the cycle [8]. There is presently no information about the timing of cell wall synthesis or about the accumulation of cell wall macromolecules during the cell cycle. The cell wall of C. reinhardii lacks a microfibrillar component, but consists of a multilayered structure composed of glycoproteins some of which form highly ordered crystalline lattices [12]. These glycoproteins, like their counterparts in higher plants, are rich in hydroxyproline, arabinose and galactose [10]. To study the timing of cell wall glycoprotein synthesis we have made use of the mutant cell line CW-2 which does not form a normal cell wall but secretes its cell wall glycoproteins into the culture medium [4].
Materials and Methods Strains
Wild type cells are derivatives of Chlamydomonas reinhardii Dangeard strain 137C mating type + . The mutant line is a derivative of strain CW-2, isolated by Davies and Plaskitt [4].
Media and Growth Conditions
Both strains were grown autotrophically on high salt medium [14] at room temperature in 3L Fernbach flasks containing 1L of culture medium. The cultures were constantly bubbled with filtered 5% CO2 in air and constantly mixed by stirring bars, driven by thermally isolated magnetic mixers. For asynchronous growth, the cells were constantly illuminated from above by fluorescent light (4,000 Lx). Synchronous growth was achieved by the method of Surzycki [15] using a 12 h light-12 h dark illumination cycle. Stock cultures,
184 maintained on yeast extract/acetate agar slants were constantly illuminated at room temperature. Cells were transferred every two months onto fresh slants. When the cells were to be labeled with 35SO2-, magnesium chloride was substituted for magnesium sulfate in the culture medium.
Cell Concentration Determinations Cell concentrations were determined by duplicate hemocytometer counting. Cells which had completed division and contained multiple daughter ceils in the mother cell wall were scored as single cells until their separation was clearly visible. Separation of Cells and Culture Medium. The cell culture was rapidly cooled to 4~ and centrifuged at 10,000xg for 10 min in a refrigerated centrifuge. The culture medium was decanted immediately after the rotor had come to a stop.
Colorimetric Determinations Protein. Protein was estimated with ninhydrin [13] after hydrolysis in 6N HC1 (18 h, ll0~ and removal of the hydrochloric acid in a rotary evaporator. Hydroxyproline. Hydroxyproline was estimated in the neutralized hydrolysates according to Kivirriko [9]. Carbohydrates. Carbohydrates were measured with the phenolsulfuric acid test using mannose as a standard [5].
Analysis of Amino Acids and Sugars Analysis of the amino acids was performed on neutralized acid hydrolysates with an automated amino acid analyzer according to the method of Moore and Stein [11], Identification and quantitative determination of neutral sugars was performed by gas-liquid chromatography of acetylated sugar alcohols according to Albershelm [I].
W.C. Lang and M.J. Chrispeels: Synthesis of Cell Wall Proteins nonradioactive precursor in 5% TCA and radioactivity was determined as described. The radioactivity in aqueous samples was determined by mixing a 1 ml aliquot with 7 ml of Aquasol (New England Nuclear Corp.)
Results and Discussion The Accumulation of Cell Wall Proteins in the Culture Medium During the Cell Cycle The accumulation of protein in the cells and in the culture medium of wild type and CW-2 cells is shown in Fig. 1. The results show that cellular protein accumulated continuously during the light period, and that protein accumulation stopped during the dark in both the wild type and the mutant cells. Accumulation of protein in the culture medium was also continuous during the cell cycle of both mutant and wild type cells. However, a comparison of the two cell lines shows that in the wild type most rapid accumulation of medium proteins occurred in the middle of the dark period, around the time of cell separation, while in the mutant accumulation was most rapid during the light period. To find out whether the medium contained proteins similar to the cell wall glycoproteins characterized by Roberts [12] we analyzed the amino acid and sugar composition of a dialyzed preparation of medium macromolecules (Table 1). The proteins contained about 9~ hydroxyproline, which is somewhat less than the value of 13.4% reported by Roberts [12] for purified cell walls. This suggests that some non-cell wall proteins way
SDS Gel Electrophoresis The cell free culture medium was dialyzed against water (4~ 24 h), then made 0.01% SDS and fteeze-dried. The dry material was redissolved in 3 to 5 ml of water, again dialyzed, freeze-dried and finally redissolved in 0.6 ml water. After the addition of 0.1 ml 10% /~-mercaptoethanol, 0.1 ml glycerol and 0.1 ml bromphenolblue (as tracking dye), the sample was boiled for 2 rain. For preparation of the gels and separation of the polypeptides the method of Weber and Osborn [161 was used with the gel buffer at 1/3 of the recommended concentration. The gels were run at 10 mA/gel for approximately 8 hrs, stained with 0.25% Coomassie Blue and destained.
/
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Determination of Radioactivity m
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