352
Biochimica et Biophysica Acta, 390 (1975) 352--362
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98297 AMINO ACID I N C O R P O R A T I O N INTO PROTEIN BY RIBOSOMES BOUND TO C H L O R O P L A S T T H Y L A K O I D MEMBRANES: F O R M A T I O N OF DISCRETE PRODUCTS
ALLAN MICHAELS and MAURICE M. MARGULIES Radiation Biology Laboratory, Smithsonian Institution, 12441 Parklawn Drive, Rockville, Md. 20852 (U.S.A.)
(Received December 9th, 1974)
Summary A system which incorporates amino acids into proteins of chloroplast membranes of C h l a m y d o m o n a s reinhardti is described. It consists of chloroplast ribosomes b o u n d to thylakoid membranes and cell extract, m R N A is present in this thylakoid-ribosome complex, since neither initiation nor RNA synthesis seems to be required for amino acid incorporation. Incorporation requires ATP, GTP and a soluble portion of cell extract. It is inhibited b y chloramphenicol, but n o t cycloheximide. Most incorporated radioactivity remains b o u n d to t h e membranes. Although a large portion of this labeled membrane-bound protein occurs as nascent polypeptides, a portion appears as at least four products of discrete molecular weights. The major in vitro product migrates as a polypeptide of 23 000 daltons. We conclude that a portion of chloroplast membrane proteins is not only made within the chloroplast, but directly on the membranes. We had previously observed that release of membrane-bound ribosomes is partially dependent on puromycin, and concluded that some membrane-bound ribosomes were attached to the membranes through nascent protein chains. Thus, our results suggest that some chloroplast membrane proteins are inserted into the membranes as they are synthesized. This chloroplast membrane amino acid incorporation system offers a promising tool for studying biosynthesis of membrane proteins, and h o w they become inserted into chloroplast thylakoids to form functional membranes. Introduction Chloroplasts contain all the components necessary for synthesis of proteins [1--3]. Many of these components are distinct from the components of the protein synthesis system of the cytoplasm. Investigations of chloroplast protein synthesis have centered on the question as to where chloroplast asso-
353 ciated proteins are synthesized, in the cytoplasm or in the chloroplast. Both in vivo and in vitro experiments have been used to answer this question. The in vivo experiments use indirect evidence obtained from studying the effect of inhibitors of chloroplast or cytoplasm protein synthesis [2--5]. Analysis of products of chloroplast protein synthesis formed in vitro, a more direct approach, has yielded equivocal results until recently [1,2]. However, there are now a number of repSrts of chloroplast protein synthesizing systems which make discrete products in vitro [ 6,7 ]. Both stroma and membrane proteins are products of in vitro chloroplast protein synthesis. The conclusion reached from in vivo and in vitro experiments is that some proteins of the chloroplast thylakoid membranes are synthesized in the chloroplast on chloroplast ribosomes, while others are synthesized in the cytoplasm on cytoplasm ribosomes [5,7]. Chloropiast ribosomes may comprise 50 percent of the total cellular ribosome population [8], but in Chlamydomonas reinhardti they comprise a somewhat smaller proportion [9]. Some of the chloroplast ribosomes are bound to chloroplast thylakoids [10--13]. A large portion of chloroplast ribosomes in Chlamydomonas are isolated bound to chloroplast membranes, only if cells are first treated with the inhibitor of chloroplast protein synthesis, chloramphenicol [11,12]. From this observation, and the dependence on puromycin for release of ribosomes, it was suggested that the membrane bound ribosomes possibly are synthesizing membrane protein [ 11,12 ]. In this report, we present evidence that ribosomes bound to chloroplast membranes are active in protein synthesis; that protein synthesis is carried out on message already bound to the ribosome thylakoid complex; and that the products may indeed include membrane proteins. Our results also suggest that those membrane peptides synthesized by the membrane-ribosome complex are inserted into the membrane as they are synthesized. Methods
Culture o f cells. The arginine auxotroph of Chl. reinhardti (arg-1), was cultured on arginine medium, as previously described [14]. The cell wail mutant, CW-15 [15], was obtained from Professor D.R. Davies, and was grown on minimal medium [14]. Membrane preparation. Chloroplast thylakoids were isolated according to the procedure of Hoober [5] as modified by Margulies and Michaels [12]. Membranes were obtained from cells t h a t h a d been treated for 60 rain with 100 #g/ml of chloramphenicol (plus chloramphenicol membranes), or from cells that were not treated with chloramphenicol (minus chloramphenicol membranes). In some cases, to prepare the large quantities of plus chloramphenicol membranes we required, membranes were floated using a Beckman Ti-15 zonal rotor instead of carrying out membrane flotation in swinging bucket rotors [5,12]. The Ti-15 rotor contained: 640 ml buffer solution (25 mM KC1, 25 mM MgCl~, 25 mM Tris • HC1, 1 mM dithiothreitol, pH 7.5) containing 1.25 M sucrose; crude membranes (about 60 mg chlorophyll), in buffer solution containing 1.5 M sucrose, and 130 ml of buffer solution containing 2.0 M sucrose. The rotor was centrifuged at 32 000 rev./min for 15 h. It was then unloaded
354 and divided into 20-ml fractions. The green material, which appeared at the interface of the 1.25 M and 1.5 M sucrose, was collected b y centrifugation, and the pellet resuspended in buffer without sucrose. Plus chloramphenicol membranes were also prepared from homogenates of CW-15 cells as described [12]. However, the procedure used to break cells was modified to minimize damage to mRNA. The principal changes included breaking cells in a TenBrock homogenizer and adding rat liver ribonuclease inhibitor (0.05 ml/ml) [ 1 6 , 1 7 ] , and heparin {0.08 mg/ml) [16] to the homogenization medium. The preservation of m R N A was improved by this procedure as judged by the increased yields of cytoplasmic polyribosomes (Michaels, unpublished). Unless stated otherwise, " m e m b r a n e s " refers to membranes that were obtained from arg-1 cells that were treated with chloramphenicol. Preparation of cell extract. A cell extract fraction which contained soluble factors necessary for in vitro amino acid incorporation, was prepared from late log phase cells (approx. 106 cells/ml) of arg-1. Cells were broken in a French pressure cell. The homogenate was centrifuged for 15 rain at 30 000 × g, and the supernatant decanted and centrifuged for 120 min at 1 5 0 0 0 0 × g. The supernatant from the second centrifugation was fractionated on a column (1 cm X 24 cm) of Sephadex G-25, and the peak of ultraviolet absorbing material which was eluted with the void volume was combined, frozen in solid CO2 and acetone, and stored a t - - 8 0 ° C in 1 ml aliquots until needed. Amino acid incorporation. The amino acid incorporation reaction mixture contained the following in pmol/ml: ATP, 0.4; CTP, UTP, GTP, 0.02 each; phosphoenol pyruvate, 4.4; 2-mercaptoethanol, 5; sucrose, 50; KC1 25; MgC12 20; and Tris • HC1 (pH 7.6) 40. In addition, each ml of reaction mixture contained 20 pg pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40), 0.8 A260 units of cell extract, 4 pCi of a tritiated amino acid mixture (Table I), and membranes containing 0.3 mg chlorophyll. The reaction was routinely carried out in a final volume of 0.51 ml or multiples of it. Reaction mixtures were incubated with shaking in a water bath maintained at 25 ° C. The reaction was started by warming the reaction mixture from 0°C to 25°C. Duplicate 0.05 ml samples were pipetted onto filter paper discs at at least five time points from 0 to 60 min, were extracted and counted according to Mans and Novelli [18]. To obtain enough labeled material for analysis of radioactive products, the membrane concentration was increased to 1 mg chlorophyll/ml, the amount of tritiated amino acid was increased to 40--80/~Ci/ml, and the incubation period extended from 60 to 120 min (Table II, Figs 1--3). Incorporation was terminated by cooling rapidly to 0 ° C. The reaction mixture was processed immediately, or it was frozen in solid C O 2 / a c e t o n e and stored a t - - 8 0 ° C until processed further. The reaction mixture was centrifuged for 30 min at 2°C at 150 000 X g. The green pellet was resuspended in buffer solution and recentrifuged. The supernatants from the t w o centrifugations were combined and freeze-dried. Both pellet and the freeze
Biochimica et Biophysica Acta, 390 (1975) 352--362
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98297 AMINO ACID I N C O R P O R A T I O N INTO PROTEIN BY RIBOSOMES BOUND TO C H L O R O P L A S T T H Y L A K O I D MEMBRANES: F O R M A T I O N OF DISCRETE PRODUCTS
ALLAN MICHAELS and MAURICE M. MARGULIES Radiation Biology Laboratory, Smithsonian Institution, 12441 Parklawn Drive, Rockville, Md. 20852 (U.S.A.)
(Received December 9th, 1974)
Summary A system which incorporates amino acids into proteins of chloroplast membranes of C h l a m y d o m o n a s reinhardti is described. It consists of chloroplast ribosomes b o u n d to thylakoid membranes and cell extract, m R N A is present in this thylakoid-ribosome complex, since neither initiation nor RNA synthesis seems to be required for amino acid incorporation. Incorporation requires ATP, GTP and a soluble portion of cell extract. It is inhibited b y chloramphenicol, but n o t cycloheximide. Most incorporated radioactivity remains b o u n d to t h e membranes. Although a large portion of this labeled membrane-bound protein occurs as nascent polypeptides, a portion appears as at least four products of discrete molecular weights. The major in vitro product migrates as a polypeptide of 23 000 daltons. We conclude that a portion of chloroplast membrane proteins is not only made within the chloroplast, but directly on the membranes. We had previously observed that release of membrane-bound ribosomes is partially dependent on puromycin, and concluded that some membrane-bound ribosomes were attached to the membranes through nascent protein chains. Thus, our results suggest that some chloroplast membrane proteins are inserted into the membranes as they are synthesized. This chloroplast membrane amino acid incorporation system offers a promising tool for studying biosynthesis of membrane proteins, and h o w they become inserted into chloroplast thylakoids to form functional membranes. Introduction Chloroplasts contain all the components necessary for synthesis of proteins [1--3]. Many of these components are distinct from the components of the protein synthesis system of the cytoplasm. Investigations of chloroplast protein synthesis have centered on the question as to where chloroplast asso-
353 ciated proteins are synthesized, in the cytoplasm or in the chloroplast. Both in vivo and in vitro experiments have been used to answer this question. The in vivo experiments use indirect evidence obtained from studying the effect of inhibitors of chloroplast or cytoplasm protein synthesis [2--5]. Analysis of products of chloroplast protein synthesis formed in vitro, a more direct approach, has yielded equivocal results until recently [1,2]. However, there are now a number of repSrts of chloroplast protein synthesizing systems which make discrete products in vitro [ 6,7 ]. Both stroma and membrane proteins are products of in vitro chloroplast protein synthesis. The conclusion reached from in vivo and in vitro experiments is that some proteins of the chloroplast thylakoid membranes are synthesized in the chloroplast on chloroplast ribosomes, while others are synthesized in the cytoplasm on cytoplasm ribosomes [5,7]. Chloropiast ribosomes may comprise 50 percent of the total cellular ribosome population [8], but in Chlamydomonas reinhardti they comprise a somewhat smaller proportion [9]. Some of the chloroplast ribosomes are bound to chloroplast thylakoids [10--13]. A large portion of chloroplast ribosomes in Chlamydomonas are isolated bound to chloroplast membranes, only if cells are first treated with the inhibitor of chloroplast protein synthesis, chloramphenicol [11,12]. From this observation, and the dependence on puromycin for release of ribosomes, it was suggested that the membrane bound ribosomes possibly are synthesizing membrane protein [ 11,12 ]. In this report, we present evidence that ribosomes bound to chloroplast membranes are active in protein synthesis; that protein synthesis is carried out on message already bound to the ribosome thylakoid complex; and that the products may indeed include membrane proteins. Our results also suggest that those membrane peptides synthesized by the membrane-ribosome complex are inserted into the membrane as they are synthesized. Methods
Culture o f cells. The arginine auxotroph of Chl. reinhardti (arg-1), was cultured on arginine medium, as previously described [14]. The cell wail mutant, CW-15 [15], was obtained from Professor D.R. Davies, and was grown on minimal medium [14]. Membrane preparation. Chloroplast thylakoids were isolated according to the procedure of Hoober [5] as modified by Margulies and Michaels [12]. Membranes were obtained from cells t h a t h a d been treated for 60 rain with 100 #g/ml of chloramphenicol (plus chloramphenicol membranes), or from cells that were not treated with chloramphenicol (minus chloramphenicol membranes). In some cases, to prepare the large quantities of plus chloramphenicol membranes we required, membranes were floated using a Beckman Ti-15 zonal rotor instead of carrying out membrane flotation in swinging bucket rotors [5,12]. The Ti-15 rotor contained: 640 ml buffer solution (25 mM KC1, 25 mM MgCl~, 25 mM Tris • HC1, 1 mM dithiothreitol, pH 7.5) containing 1.25 M sucrose; crude membranes (about 60 mg chlorophyll), in buffer solution containing 1.5 M sucrose, and 130 ml of buffer solution containing 2.0 M sucrose. The rotor was centrifuged at 32 000 rev./min for 15 h. It was then unloaded
354 and divided into 20-ml fractions. The green material, which appeared at the interface of the 1.25 M and 1.5 M sucrose, was collected b y centrifugation, and the pellet resuspended in buffer without sucrose. Plus chloramphenicol membranes were also prepared from homogenates of CW-15 cells as described [12]. However, the procedure used to break cells was modified to minimize damage to mRNA. The principal changes included breaking cells in a TenBrock homogenizer and adding rat liver ribonuclease inhibitor (0.05 ml/ml) [ 1 6 , 1 7 ] , and heparin {0.08 mg/ml) [16] to the homogenization medium. The preservation of m R N A was improved by this procedure as judged by the increased yields of cytoplasmic polyribosomes (Michaels, unpublished). Unless stated otherwise, " m e m b r a n e s " refers to membranes that were obtained from arg-1 cells that were treated with chloramphenicol. Preparation of cell extract. A cell extract fraction which contained soluble factors necessary for in vitro amino acid incorporation, was prepared from late log phase cells (approx. 106 cells/ml) of arg-1. Cells were broken in a French pressure cell. The homogenate was centrifuged for 15 rain at 30 000 × g, and the supernatant decanted and centrifuged for 120 min at 1 5 0 0 0 0 × g. The supernatant from the second centrifugation was fractionated on a column (1 cm X 24 cm) of Sephadex G-25, and the peak of ultraviolet absorbing material which was eluted with the void volume was combined, frozen in solid CO2 and acetone, and stored a t - - 8 0 ° C in 1 ml aliquots until needed. Amino acid incorporation. The amino acid incorporation reaction mixture contained the following in pmol/ml: ATP, 0.4; CTP, UTP, GTP, 0.02 each; phosphoenol pyruvate, 4.4; 2-mercaptoethanol, 5; sucrose, 50; KC1 25; MgC12 20; and Tris • HC1 (pH 7.6) 40. In addition, each ml of reaction mixture contained 20 pg pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40), 0.8 A260 units of cell extract, 4 pCi of a tritiated amino acid mixture (Table I), and membranes containing 0.3 mg chlorophyll. The reaction was routinely carried out in a final volume of 0.51 ml or multiples of it. Reaction mixtures were incubated with shaking in a water bath maintained at 25 ° C. The reaction was started by warming the reaction mixture from 0°C to 25°C. Duplicate 0.05 ml samples were pipetted onto filter paper discs at at least five time points from 0 to 60 min, were extracted and counted according to Mans and Novelli [18]. To obtain enough labeled material for analysis of radioactive products, the membrane concentration was increased to 1 mg chlorophyll/ml, the amount of tritiated amino acid was increased to 40--80/~Ci/ml, and the incubation period extended from 60 to 120 min (Table II, Figs 1--3). Incorporation was terminated by cooling rapidly to 0 ° C. The reaction mixture was processed immediately, or it was frozen in solid C O 2 / a c e t o n e and stored a t - - 8 0 ° C until processed further. The reaction mixture was centrifuged for 30 min at 2°C at 150 000 X g. The green pellet was resuspended in buffer solution and recentrifuged. The supernatants from the t w o centrifugations were combined and freeze-dried. Both pellet and the freeze
