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Chapter 18 Phosphorylation of Chlamydomonas Flagellar Proteins
CHAPTER 18
Phosphorylation of Chlamydomonas Flagellar Proteins Robert A. Bloodgood and Nancy L. Salomonsky Department of Cell Biology University of Virginia School of Medicine Charlottesville, Virginia 22908
I. Introduction
11. In Vivo Labeling of Chlamydomonas Flagellar Proteins with
[32P]0rthopho~phoric Acid A. Solutions B. Cell Culture C. Labeling D. Flagellar Fractionation References
I. Introduction Protein phosphorylation and dephosphorylation are widely used as switches for regulating a wide variety of biological processes. Phosphorylation can be studied in uiuo using [32P]orthophosphoricacid, but steady-state labeling of phosphoproteins in uiuo depends on the coordinated actions of endogenous protein kinases and phosphatases; the extent of radioactive labeling depends on the rate at which particular phosphate groups turn over and the specific activity of the cellular ATP pool (Garrison, 1993). Protein phosphorylation also can be (Hasegawa et al., 1987) or [S-Y-~~PIATP and studied in uitro using [Y-~~PIATP [Y-~~PIATP (Segal and Luck, 1985) and endogenous or exogenous protein kinases. Cilia and flagella possess a large number of phosphoproteins, and most studies of them have focused on axonemal proteins and the role of phosphorylation in regulating axonemal motility (Chapter 63 of this volume; Satir et al., 1993). In METHODS IN CELL BIOLOGY, VOL. 47
Copyright 0 1995 by Academic Press. Inc. All rights of reproduction m any form reserved.
121
122
Robert A. Bloodgood and Nancy L. Salomonsky
Chlamydomonas, protein phosphorylation had been suggested to be involved in the regulation of axonemal motility (Hasegawa et al., 1987),the photophobic response (Segal and Luck, 1985), flagellar assembly (May and Rosenbaum, 1983; May, 1984), flagellar signaling during mating (Pasquale and Goodenough, 1987; Zhang and Snell, 1994), and flagellar glycoprotein dynamics (Bloodgood and Salomonsky, 1991; Bloodgood, 1992b). In uivo labeling with [32P]orthophosphoric acid coupled with two-dimensionalpolyacrylamide gel electrophoresis (PAGE) has revealed greater than 80 phosphorylated Chlamydomonas axonemal polypeptides, including components of dynein arms, radial spokes, the central pair complex, and beak projections within selected doublet microtubules (Adams et al., 1981; Huang et al., 1981; King and Witman, 1994; Piperno and Luck, 1976, 1981;Piperno et al., 1981; Segal et al., 1984). Extraction of purified flagella with nonionic detergents such as Nonidet P-40 and Triton X-100 solubilizes the flagellar membrane, resulting in an axonemal fraction, which can be pelleted, and a membrane-matrix fraction, which remains in the supernatant. Phosphorylated polypeptides in the latter fraction have been identified by in uiuo and in vitro labeling (May, 1984; Bloodgood, 1992a,b; Bloodgood and Salomonsky, 1994). In uiuo studies demonstrated the presence of greater than 30 phosphoproteins in the membrane-matrix compartment, including a lowabundance surface-exposed flagellar membrane phosphoglycoprotein, FMG3C, and a 60-kDa phosphoprotein associated with the 350-kDa major membrane glycoprotein (Bloodgood and Salomonsky, 1994). Important questions to be addressed before carrying out a labeling experiment include the following: (1) Do in uiuo and in uitro labeling with 32Pproduce similar patterns of flagellar protein phosphorylation? (2) Does the labeling vary according to different solution conditions? We find significant differences among the patterns of flagellar membrane-matrix polypeptides phosphorylated in uitro as a function of the concentration of free Ca2+(Bloodgood, 1992b), as well as major differences between the pattern of the membrane-matrix polypeptides phosphorylated in uiuo and the pattern of those phosphorylated in uitro by endogenous protein kinases and phosphatases at high or low free Ca2+concentrations (Fig. 1). A pattern intermediate between in uiuo and in uitro phosphorylation patterns was obtained when [Y-~~PIATP was added to purified whole flagella (Bloodgood, unpublished data); presumably, [Y-~~PIATP permeated the flagella because of partially disrupted membranes. In other studies, axonemal and membrane-matrix tubulin is phosphorylated in uitro but not in uiuo (Segal and Luck, 1985; Bloodgood, 1992b). Axonemal and membrane-matrix proteins are labeled primarily on serine residues both in uiuo and in uitro (Segal and Luck, 1985; Bloodgood and Salornonsky, 1994). If one does label flagella or axonemes in uitro, it is important to include sodium vanadate to inhibit the high ATPase activity produced by axonemal dynein and thereby maintain the amount of [-Y-~~P]ATP available for phosphorylation (Wang et al., 1994). Based on the presence of polypeptides phosphorylated in uitro that do not appear to be labeled in uiuo and vice versa, it is preferable to perform in uiuo
18. Flagellar Protein Phosphorylation
123
Fig. 1 Autoradiograms of two-dimensionalpolyacrylamide gels comparing Chlamydornonas reinhardtii flagellar membrane-matrix proteins phosphorylated (left) in uitro (in the presence of micromolar CaZ+and [3zP]ATP)and (right) in uiuo (using [3ZP]orthophorphoricacid). There is a significant difference in the patterns of polypeptides phosphorylated in uiuo and in uitro. The large spot of label at the bottom of the in uiuo autoradiogram represents labeled phospholipids. Reprinted with permission from Bloodgood (1992b).
phosphorylation (with [32P]orthophosphoricacid). Caution still must be taken, however, as for most in uiuo labeling studies, cells are grown in medium containing 10% or less of the normal levels of phosphate used in normal culture conditions. Total phosphate starvation of Chlamydomonas reinhardtii results in the appearance of new phosphatases, some of which are secreted into the culture medium (Matagne e f al., 1976; Dumont et al., 1990), so one must be vigilant to prevent dephosphorylation of flagellar proteins during deflagellation and subsequent purification and fractionation of the flagella.
11. In Vivo Labeling of Chlamydomonas Flagellar Proteins
with [32P]Orthopho~phoric Acid
A. Solutions Medium I of Sager and Granick containing 10% of the normal potassium phosphate plus 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid (Hepes), pH 7.2-7.4 (Table I) Medium I containing 20 mM Hepes, pH 7.2, and none of the potassium phosphate
124
Robert A. Bloodgood and Nancy L. Salomonsky
Table I Composition of Low Phosphate and Phosphate-Free Media 10% Phosphate medium Component
Stock concn (w/v)
ml/8 liters
Final concn (mM)
1. Trace metals 2. Na citrate . 2H20 3. FeC13 . 6H20 4. CaC12 . 2H20 5. MgS04 * 7HzO 6. NH4NO3 7. KHzP04 8. K2HPOI 9. Hepes, pH 7.4
(see below) 10% 17% 5.3% 10% 10% 10% 10% 1M
8 40 8 8 24 24 0.8 0.8 80
(see below) 1.7 0.37 0.36 1.2 3.7 0.074 0.057 10
Phosphate-free medium ml/8 liters
8 40
8 8 24 24 0 0 160
Final concn (mM) (see below) 1.7 0.37 0.36 1.2 3.7 0 0 20
Trace metal stock solution Component
mg/liter
H3BO3 ZnSO, . 7H20 MnSO, * H 2 0 CoC12 6H20 NazMoO4. 2H20 cuso,
lo00 lo00 303 200 200 40
5mCi [32P]orthophosphoricacid (New England Nuclear NEX-O53,8500-9100 Ci/mmole in water) HMDS: 10 mM Hepes, pH 7.4, 5 mM MgSO,, 1 mM dithiothreitol, 4% sucrose (Witman, 1986) STOP solution: 20 mM Hepes, pH 7.2, 20 mM ethylene glycol bis(p-aminoethyl ether)-iV,N’-tetraaceticacid (EGTA), 200 mM sodium fluoride, 4% sucrose, 200 U/ml Trasylol (aprotinin, Sigma), 1 p M Microcystin-LR (Calbiochem), 0. l p M okadaic acid (Gibco-BL, No. 31665A) 0.05% Nonidet P-40, 20 mM Hepes, pH 7.2 (for electrophoresis) 100 mM NaC1, 5 mM EGTA, 1 mM MgC12, 100 U/ml aprotinin, 20 mM Tris-HC1, pH 8.3 (for immunoprecipitation)
B. Cell Culture 1. Grow C. reinhardtii for 3 days at 21°C (light/dark cycle of 14 : 10 hours) in 200-ml cultures in medium I containing only 10% of the normal level of phosphate (Table I). Inoculate 8-liter bottles containing the same medium from the small culture and grow for an additional 3 days on the same light/dark cycle.
18. Flagellar Protein Phosphorylation
125
2. Collect cells by centrifugation and wash into medium I containing 20 mM Hepes, pH 7.2, and potassium phosphate. Bubble cells with air in front of bright fluorescent lights for - 1 hour. Adjust cell concentration to 4-4.5 x lo7 cells/ml.
C. Labeling
1. Add 5 mCi of [32P]~rthophosphoric acid to 200 ml cells, for a final concentration of 25 pCi/ml as follows: (a) Divide cells into two 100-ml aliquots (control and experimental). (b) Dilute 1 ml containing 5 mCi of [32P]orthophosphoric acid with 9 ml of culture medium. (c) Add dropwise to cells while they are being mixed on a platform shaker ( 5 ml for each aliquot). 2. Incubate cells for up to 60 minutes at room temperature in 250-ml roundbottomed polycarbonate centrifuge bottles on a platform shaker illuminated with fluorescent cool-white lights. Adequate labeling for resolution on one- or two-dimension polyacrylamide gels is obtained with a 10-minute labeling period. Labeling for up to 70 minutes produces no significant difference in the pattern of labeled polypeptides. If cells are to be treated with drugs, antibodies, or a biotinylation reagent, label first with the [32P]orthophosphoricacid. 3. At the end of the labeling period, dilute cells with an equal volume of icecold phosphate-free medium. 4. Centrifuge at 1500g for 7 minutes 4”C, aspirate the supernatant, and suspend the pellet in 10 ml of HMDS.
D. Flagellar Fractionation 1. Deflagellate cells by adding 2 ml of ice-cold 25 mM dibucaine in water to the cell suspension in 10 ml of HMDS. Pull up and down in a 10-ml plastic disposable pipet 10 times to deflagellate the cells. 2. Check deflagellation using a phase microscope. As soon as cells are deflagellated, add an equal volume of ice-cold STOP solution. Keep flagella at 4°C and add 1 puM Microcystin-LR for all remaining steps. 3. For membrane-matrix fractions, extract flagella on ice for 15 minutes in either 0.05% Nonidet P-40, 20 mM Hepes, pH 7.2 (for electrophoresis), or in 100 mM NaCl, 5 mM EGTA, 1 mM MgC12, 100 U/ml aprotinin, 20 mMTris-C1, pH 8.3 (for immunoprecipitation). Pellet axonemes by centrifugation at 89,OOOg for 20 minutes. The supernatant is defined as the “membrane-matrix fraction.” Overnight incubation of the fraction in the immunoprecipitation buffer at 4°C resulted in minimal loss of protein-associated 32P. A typical experiment involving a 60-minute labeling period and 4.5 x lo9cells yields approximately 300 pl of flagellar membrane-matrix extract containing approximately 50,000 cpmlpl extract. With typical protein concentrations of
126
Robert A. Bloodgood and Nancy L. Salomonsky
0.5-1.0 mg/ml, the specific activity of this extract is 25,000-50,000 cpm/pg of protein. We typically load 100,000-500,000 cpm of 32Pon each isoelectric focusing tube gel, which requires a typical exposure time for the second-dimension slab gel of 12-48 hours using Kodak XAR film and a DuPont Cronex enhancing screen. Spots on the second-dimension gels have sufficient label to allow phosphoamino acid analysis with exposure times of 1-2 weeks (Hemmings et al.,
-
1984).
References Adams, G. M. W., Huang, B., Piperno, G., and Luck, D. J. L. (1981). Central pair microtubular complex of Chlamydomonas flagella: polypeptide composition as revealed by analysis of mutants. J . Cell Biol. 91, 69-76. Bloodgood, R. A. (1992a). Calcium-regulatedphosphorylation of proteins in the membrane-matrix compartment of the Chlamydomonas flagellum. Exp. Cell Res. 198, 228-236. Bloodgood, R. A. (1992b). Directed movements of ciliary and flagellar membrane components. Biol. Cell 76, 291-301. Bloodgood, R. A., and Salomonsky, N. L. (1991). Regulation of flagellar glycoprotein movements by protein phosphorylation. Eur. J . Cell Biol. 54, 85-89. Bloodgood, R. A., and Salomonsky, N. L. (1994). The transmembrane signaling pathway involved in directed movements of Chlamydomonas flagellarmembrane glycoproteins involves the dephosphorylation of a 60 kDa phosphoprotein that binds to the major flagellar membrane glycoprotein. J . Cell B i d . 127, 803-81 1. Dumont, F., Loppes, R., and Kremers, P. (1990). New polypeptides and in-uitro-translatable mRNAs are produced by phosphate-starved cells of the unicellular alga Chlamydomonas reinhardtii. PIanta 182, 610-616. Garrison, J. C. (1993).Study of protein phosphorylation in intact cells. In “Protein Phosphorylation. A Practical Approach” (D. G. Hardie, ed.), pp. 1-29. IRL Press, Oxford. Hasegawa, E., Hayashi, H., Asakura, S., and Kamiya, R. (1987). Stimulation of in uitro motility of Chlamydomonas axonemes by inhibition of CAMP-dependent phosphorylation. Cell Motil. Cytoskel. 8, 302-311. Hemmings, H. C., Nairn, A. C., and Greengard, P. (1984). DARPP-32, a dopamine- and adenosine 3’ :5‘-monophosphate-regulated neuronal phosphoprotein. J . Biol. Chem. 259, 14491-14497. Huang, B., Piperno, G., Ramanis, Z., and Luck, D. J. L. (1981). Radial spokes of Chlamydomonas flagella: genetic analysis of assembly and function. J . Cell Biol. 88, 80-88. King, S . M., and Witman, G. B. (1994). Multiple sites of phosphorylation within the y heavy chain of Chlamydomonas outer arm dynein. J. Biol. Chem. 269, 5452-5457. Matagne, R. F., Loppes, R., and Deltour, R. (1976).Phosphatases of Chlamydomonas: Biochemical and cytochemical approach with specific mutants. J . Bacteriol. 125, 937-950. May, G. S . (1984). Flagellar protein kinases and protein phosphorylation during flagellar regeneration and resorption in Chlamydomonas reinhardti Ph.D. Dissertation. Yale University. University Microfilms No. 8509723. May, G. S., and Rosenbaum, J. L. (1983). Flagellar protein phosphorylation during flagellar regeneration and resorption in Chlamydomonas reinhardtii. J . Cell Biol. 97, 195a. Pasquale, S. M., and Goodenough, U. W. (1987). Cyclic AMP functions as a primary sexual signal in gametes of Chlamydomonas reinhardtii. J . Cell Biol. 105, 2279-2292. Piperno, G., and Luck, D. J. (1976). Phosphorylation of axonemal proteins in Chlamydomonas reinhardtii. J . Biol. Chem. 251, 2161-2167. Piperno, G . , and Luck, D. J. (1981). Inner arm dyneins from flagella of Chlamydomonas reinhardtii. Cell 27, 331-340.
18. Flagellar Protein Phosphorylation
127
Piperno, G., Huang, B., Ramanis, Z., and Luck, D. J. L. (1981). Radial spokes of Chlamydomonas flagella: polypeptide composition and phosphorylation of stalk components. J . Cell Biol. 88, 73-79. Sager, R., and Granick, S. (1953). Nutritional studies with Chlnrnydomonas reinhardtii. Ann. N . Y . Acad. Sci. 56,831-838. Satir, P., Barkalow, K., and Hamasaki, T. (1993). The control of ciliary beat frequency. Trends Cell Biol. 3, 409-412. Segal, R. A., and Luck, D. J. (1985). Phosphorylation in isolated Chlamydomonas axonemes: a phosphoprotein may mediate the Ca2+-dependent photophobic response. J . Cell Biol. 101, 1701-1712. Segal, R. A., Huang, B., Ramanis, Z., and Luck, D. J. L. (1984). Mutant strains of Chlamydomonas reinhardtii that move backwards only. J . Cell Biol. 98, 2026-2034. Tash, J. S. (1989). Protein phosphorylation: the second messenger signal transducer of flagellar motility. Cell Motil. Cytoskel. 14, 332-339. Wang, W., Himes, R. H., and Dentler, W. L. (1994). The binding of a ciliary microtubule plusend binding protein complex to microtubules is regulated by ciliary protein kinase and phosphatase activities. J . Biol. Chem. 269, 21,460-21,466. Witman, G. 8. (1986). Isolation of Ch/amydomonas flagella and flagellar axonemes. Methods Enzymol. 134,280-290. Zhang, Y. H . , and Snell, W. J. (1994). Flagellar adhesion dependent regulation of Chlamydomonas adenylyl cyclase in uitro: A possible role for protein kinases in sexual signaling. J. Cell Biol. 125, 617-624.
Phosphorylation of Chlamydomonas Flagellar Proteins Robert A. Bloodgood and Nancy L. Salomonsky Department of Cell Biology University of Virginia School of Medicine Charlottesville, Virginia 22908
I. Introduction
11. In Vivo Labeling of Chlamydomonas Flagellar Proteins with
[32P]0rthopho~phoric Acid A. Solutions B. Cell Culture C. Labeling D. Flagellar Fractionation References
I. Introduction Protein phosphorylation and dephosphorylation are widely used as switches for regulating a wide variety of biological processes. Phosphorylation can be studied in uiuo using [32P]orthophosphoricacid, but steady-state labeling of phosphoproteins in uiuo depends on the coordinated actions of endogenous protein kinases and phosphatases; the extent of radioactive labeling depends on the rate at which particular phosphate groups turn over and the specific activity of the cellular ATP pool (Garrison, 1993). Protein phosphorylation also can be (Hasegawa et al., 1987) or [S-Y-~~PIATP and studied in uitro using [Y-~~PIATP [Y-~~PIATP (Segal and Luck, 1985) and endogenous or exogenous protein kinases. Cilia and flagella possess a large number of phosphoproteins, and most studies of them have focused on axonemal proteins and the role of phosphorylation in regulating axonemal motility (Chapter 63 of this volume; Satir et al., 1993). In METHODS IN CELL BIOLOGY, VOL. 47
Copyright 0 1995 by Academic Press. Inc. All rights of reproduction m any form reserved.
121
122
Robert A. Bloodgood and Nancy L. Salomonsky
Chlamydomonas, protein phosphorylation had been suggested to be involved in the regulation of axonemal motility (Hasegawa et al., 1987),the photophobic response (Segal and Luck, 1985), flagellar assembly (May and Rosenbaum, 1983; May, 1984), flagellar signaling during mating (Pasquale and Goodenough, 1987; Zhang and Snell, 1994), and flagellar glycoprotein dynamics (Bloodgood and Salomonsky, 1991; Bloodgood, 1992b). In uivo labeling with [32P]orthophosphoric acid coupled with two-dimensionalpolyacrylamide gel electrophoresis (PAGE) has revealed greater than 80 phosphorylated Chlamydomonas axonemal polypeptides, including components of dynein arms, radial spokes, the central pair complex, and beak projections within selected doublet microtubules (Adams et al., 1981; Huang et al., 1981; King and Witman, 1994; Piperno and Luck, 1976, 1981;Piperno et al., 1981; Segal et al., 1984). Extraction of purified flagella with nonionic detergents such as Nonidet P-40 and Triton X-100 solubilizes the flagellar membrane, resulting in an axonemal fraction, which can be pelleted, and a membrane-matrix fraction, which remains in the supernatant. Phosphorylated polypeptides in the latter fraction have been identified by in uiuo and in vitro labeling (May, 1984; Bloodgood, 1992a,b; Bloodgood and Salomonsky, 1994). In uiuo studies demonstrated the presence of greater than 30 phosphoproteins in the membrane-matrix compartment, including a lowabundance surface-exposed flagellar membrane phosphoglycoprotein, FMG3C, and a 60-kDa phosphoprotein associated with the 350-kDa major membrane glycoprotein (Bloodgood and Salomonsky, 1994). Important questions to be addressed before carrying out a labeling experiment include the following: (1) Do in uiuo and in uitro labeling with 32Pproduce similar patterns of flagellar protein phosphorylation? (2) Does the labeling vary according to different solution conditions? We find significant differences among the patterns of flagellar membrane-matrix polypeptides phosphorylated in uitro as a function of the concentration of free Ca2+(Bloodgood, 1992b), as well as major differences between the pattern of the membrane-matrix polypeptides phosphorylated in uiuo and the pattern of those phosphorylated in uitro by endogenous protein kinases and phosphatases at high or low free Ca2+concentrations (Fig. 1). A pattern intermediate between in uiuo and in uitro phosphorylation patterns was obtained when [Y-~~PIATP was added to purified whole flagella (Bloodgood, unpublished data); presumably, [Y-~~PIATP permeated the flagella because of partially disrupted membranes. In other studies, axonemal and membrane-matrix tubulin is phosphorylated in uitro but not in uiuo (Segal and Luck, 1985; Bloodgood, 1992b). Axonemal and membrane-matrix proteins are labeled primarily on serine residues both in uiuo and in uitro (Segal and Luck, 1985; Bloodgood and Salornonsky, 1994). If one does label flagella or axonemes in uitro, it is important to include sodium vanadate to inhibit the high ATPase activity produced by axonemal dynein and thereby maintain the amount of [-Y-~~P]ATP available for phosphorylation (Wang et al., 1994). Based on the presence of polypeptides phosphorylated in uitro that do not appear to be labeled in uiuo and vice versa, it is preferable to perform in uiuo
18. Flagellar Protein Phosphorylation
123
Fig. 1 Autoradiograms of two-dimensionalpolyacrylamide gels comparing Chlamydornonas reinhardtii flagellar membrane-matrix proteins phosphorylated (left) in uitro (in the presence of micromolar CaZ+and [3zP]ATP)and (right) in uiuo (using [3ZP]orthophorphoricacid). There is a significant difference in the patterns of polypeptides phosphorylated in uiuo and in uitro. The large spot of label at the bottom of the in uiuo autoradiogram represents labeled phospholipids. Reprinted with permission from Bloodgood (1992b).
phosphorylation (with [32P]orthophosphoricacid). Caution still must be taken, however, as for most in uiuo labeling studies, cells are grown in medium containing 10% or less of the normal levels of phosphate used in normal culture conditions. Total phosphate starvation of Chlamydomonas reinhardtii results in the appearance of new phosphatases, some of which are secreted into the culture medium (Matagne e f al., 1976; Dumont et al., 1990), so one must be vigilant to prevent dephosphorylation of flagellar proteins during deflagellation and subsequent purification and fractionation of the flagella.
11. In Vivo Labeling of Chlamydomonas Flagellar Proteins
with [32P]Orthopho~phoric Acid
A. Solutions Medium I of Sager and Granick containing 10% of the normal potassium phosphate plus 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid (Hepes), pH 7.2-7.4 (Table I) Medium I containing 20 mM Hepes, pH 7.2, and none of the potassium phosphate
124
Robert A. Bloodgood and Nancy L. Salomonsky
Table I Composition of Low Phosphate and Phosphate-Free Media 10% Phosphate medium Component
Stock concn (w/v)
ml/8 liters
Final concn (mM)
1. Trace metals 2. Na citrate . 2H20 3. FeC13 . 6H20 4. CaC12 . 2H20 5. MgS04 * 7HzO 6. NH4NO3 7. KHzP04 8. K2HPOI 9. Hepes, pH 7.4
(see below) 10% 17% 5.3% 10% 10% 10% 10% 1M
8 40 8 8 24 24 0.8 0.8 80
(see below) 1.7 0.37 0.36 1.2 3.7 0.074 0.057 10
Phosphate-free medium ml/8 liters
8 40
8 8 24 24 0 0 160
Final concn (mM) (see below) 1.7 0.37 0.36 1.2 3.7 0 0 20
Trace metal stock solution Component
mg/liter
H3BO3 ZnSO, . 7H20 MnSO, * H 2 0 CoC12 6H20 NazMoO4. 2H20 cuso,
lo00 lo00 303 200 200 40
5mCi [32P]orthophosphoricacid (New England Nuclear NEX-O53,8500-9100 Ci/mmole in water) HMDS: 10 mM Hepes, pH 7.4, 5 mM MgSO,, 1 mM dithiothreitol, 4% sucrose (Witman, 1986) STOP solution: 20 mM Hepes, pH 7.2, 20 mM ethylene glycol bis(p-aminoethyl ether)-iV,N’-tetraaceticacid (EGTA), 200 mM sodium fluoride, 4% sucrose, 200 U/ml Trasylol (aprotinin, Sigma), 1 p M Microcystin-LR (Calbiochem), 0. l p M okadaic acid (Gibco-BL, No. 31665A) 0.05% Nonidet P-40, 20 mM Hepes, pH 7.2 (for electrophoresis) 100 mM NaC1, 5 mM EGTA, 1 mM MgC12, 100 U/ml aprotinin, 20 mM Tris-HC1, pH 8.3 (for immunoprecipitation)
B. Cell Culture 1. Grow C. reinhardtii for 3 days at 21°C (light/dark cycle of 14 : 10 hours) in 200-ml cultures in medium I containing only 10% of the normal level of phosphate (Table I). Inoculate 8-liter bottles containing the same medium from the small culture and grow for an additional 3 days on the same light/dark cycle.
18. Flagellar Protein Phosphorylation
125
2. Collect cells by centrifugation and wash into medium I containing 20 mM Hepes, pH 7.2, and potassium phosphate. Bubble cells with air in front of bright fluorescent lights for - 1 hour. Adjust cell concentration to 4-4.5 x lo7 cells/ml.
C. Labeling
1. Add 5 mCi of [32P]~rthophosphoric acid to 200 ml cells, for a final concentration of 25 pCi/ml as follows: (a) Divide cells into two 100-ml aliquots (control and experimental). (b) Dilute 1 ml containing 5 mCi of [32P]orthophosphoric acid with 9 ml of culture medium. (c) Add dropwise to cells while they are being mixed on a platform shaker ( 5 ml for each aliquot). 2. Incubate cells for up to 60 minutes at room temperature in 250-ml roundbottomed polycarbonate centrifuge bottles on a platform shaker illuminated with fluorescent cool-white lights. Adequate labeling for resolution on one- or two-dimension polyacrylamide gels is obtained with a 10-minute labeling period. Labeling for up to 70 minutes produces no significant difference in the pattern of labeled polypeptides. If cells are to be treated with drugs, antibodies, or a biotinylation reagent, label first with the [32P]orthophosphoricacid. 3. At the end of the labeling period, dilute cells with an equal volume of icecold phosphate-free medium. 4. Centrifuge at 1500g for 7 minutes 4”C, aspirate the supernatant, and suspend the pellet in 10 ml of HMDS.
D. Flagellar Fractionation 1. Deflagellate cells by adding 2 ml of ice-cold 25 mM dibucaine in water to the cell suspension in 10 ml of HMDS. Pull up and down in a 10-ml plastic disposable pipet 10 times to deflagellate the cells. 2. Check deflagellation using a phase microscope. As soon as cells are deflagellated, add an equal volume of ice-cold STOP solution. Keep flagella at 4°C and add 1 puM Microcystin-LR for all remaining steps. 3. For membrane-matrix fractions, extract flagella on ice for 15 minutes in either 0.05% Nonidet P-40, 20 mM Hepes, pH 7.2 (for electrophoresis), or in 100 mM NaCl, 5 mM EGTA, 1 mM MgC12, 100 U/ml aprotinin, 20 mMTris-C1, pH 8.3 (for immunoprecipitation). Pellet axonemes by centrifugation at 89,OOOg for 20 minutes. The supernatant is defined as the “membrane-matrix fraction.” Overnight incubation of the fraction in the immunoprecipitation buffer at 4°C resulted in minimal loss of protein-associated 32P. A typical experiment involving a 60-minute labeling period and 4.5 x lo9cells yields approximately 300 pl of flagellar membrane-matrix extract containing approximately 50,000 cpmlpl extract. With typical protein concentrations of
126
Robert A. Bloodgood and Nancy L. Salomonsky
0.5-1.0 mg/ml, the specific activity of this extract is 25,000-50,000 cpm/pg of protein. We typically load 100,000-500,000 cpm of 32Pon each isoelectric focusing tube gel, which requires a typical exposure time for the second-dimension slab gel of 12-48 hours using Kodak XAR film and a DuPont Cronex enhancing screen. Spots on the second-dimension gels have sufficient label to allow phosphoamino acid analysis with exposure times of 1-2 weeks (Hemmings et al.,
-
1984).
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