Absence of Heat Shock Protein Synthesis in Isolated Mitochondria and ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 262, No. 25, Issue of September 5, pp. 12288-12292,1987 Printed in U.S.A.

Absence of HeatShock Protein Synthesis in Isolated Mitochondria and Plastids from Maize* (Received for publication, February 27, 1987)

Jorge Nieto-SoteloSand Tuan-Hua DavidHo From the Plant Biology Program, Department of Biology, Washington Uniuersity, St. Louis, Missouri 63130

Examination of the proteins synthesizedby isolated tion in viuo of this protein has notgiven us consistentresults. mitochondria, chloroplasts, or proplastids from maize Wewere therefore prompted to reinvestigate its induction tissues showed that a heat treatment at 40 "C does not using both in vivo and in vitro systems. While the synthesis induce or enhance the synthesis of any protein when of mitochondrial (asubunit of the F,-ATPase) or chloroplascompared to preparations treated at the control temtic (large subunit of RBUp,Case) markers was shown to be perature of 28 "C. These observations are consistent dependent on external energy addition, our results showed with the results obtained by labeling proteins in vivo that in either organelle there is no external energy-dependent under sterile conditions. In vivo labding in the presence of cycloheximide during heat shock showed no synthesis of anyhsp under heat shock conditions. These heat shock proteinsynthesis. Labeling in the presence results correlate with studies on protein synthesis in viuo. Protein synthesis in the presence of cycloheximide and heat of chloramphenicol during heat shock showed a similar shock failed to show any hsp synthesis in maize roots that heatshockproteinpattern as intheabsenceofthe inhibitor. It is concluded that maize organellesdo not weregrown and handled under sterile conditions. On the synthesize heat shock proteins that, and if present, they contrary, tissues that were not handled with aseptic techniques showed the induction of a 62-kDa protein. We conclude may be due tobacterial contamination. that thepresence of hsp of a molecular mass of approximately 62 kDa in maize is the product of bacterial contamination. Living organisms respond to supraoptimal temperaturesby inducing or enhancing the expression of a setof genes encoding heat shock proteins (hsp)' (1).In view of the number of correlations found between the induction of hsp andthe establishment of thermotolerance in the organism it is generally believed that hsp are important in overcoming heat stress (2). Although nothing is known about the function of the hsp in higher plants, some of these proteins have been identified in other organisms: lysyl tRNA synthetase in Escherichia coli (3), ATP-dependent protease in E. coli (4), ubiquitin in chicken embryo fibroblasts (5),an isoprotein of enolase in yeast (6), and the uncoating ATPase that releases triskelia from coated vesicles in mammals (7). Other properties of hsp include the binding of these proteins to poly(A) mRNA in HeLa cells (8), to fatty acids in rat (9), and to collagen in chicken embryo fibroblasts (10). In maize there areat least 10 new polypeptides synthesized at heat shock temperatures with molecular masses in the range of70-110 kDa and 18-33 kDa (11, 12). However, in some instances hsp of molecular mass near 62 kDa have been observed in maize (13-15). The synthesis of this protein has been shown to be induced by heat shock in isolated mitochondria in vitro, indicating that its gene may be encoded in the mitochondrial genome (14, 15). In our laboratory the induc* This work wassupported inpart by National Science Foundation Grant DCB-8316319 (to T.-H. D. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ''oduertisement" in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. 4 Supported by a fellowship from the Division of Biology and Biomedical Sciences a t Washington University and a program training grant from the Monsanto Co., St. Louis, MO. 'The abbreviations used are: hsp, heat shock protein($; RBUp,Case, ribulose bisphosphate carboxylase; EGTA, [ethylenebis (oxyethylenenitrilo)]tetraacetic acid; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DCMU, 3-(3,4-dichlorophenyl)-1,l-dimethylurea.

EXPERIMENTAL PROCEDURES

Maize (&a mays L.) seeds (hybrid 222, 1983 crop) were purchased from Crow's Hybrid Corn Co. (Milford, IL). Seeds were surface sterilized in 7% Chlorox for 10 min and grown in the dark at 28 "C under sterile conditions on paper towels moistened with 0.1 mM CaC1, in covered glass trays. All manipulations of plant material from growth to labeling in vivo were carried out in a sterile laminar flow hood using sterile solutions and glassware. To label proteins in uiuo, five1.5-cm root tips from 40-h-old seedlings were excised with a razor blade. They were transferred to flasks containing 20 mM sodium succinate, pH 5.0, and incubated a t 28 "C for 3 h prior to pulse labeling with 30-50 pCi/ml of [%SI methionine for 2 h either at 28 or 40 "C. Incubation medium was removed, tissue washed twice with 1 mM methionine, and homogenized in SDS sample buffer as described by Laemmli (16) containing 1mM phenylmethylsulfonyl fluoride and 1mM p-hydroxymercuribenzoate sodium salt. Mitochondria were prepared from 3- to 4-day-old seedlings using a protocol similar to theone described by Leaver et al. (17) by means of discontinuous Percoll or sucrose gradients. The final mitochondrial pellets were used immediately for in organello protein synthesis. Proteins were labeled a t 28 or 40 "C for 90 min in a reaction mixture containing [35S]methionineto a final concentration of 80-100 pCi/ ml and sodium succinate/ADP/GTP (17). At the end of the incubation period mitochondria were centrifuged and thepellet resuspended in SDS sample buffer (16). Chloroplasts were isolated from 8- to 12-day-old plants grown in the greenhouse using the procedure of Fish and Jagendorf (18) except that EDTA, EGTA, and Mn2+ were omitted from the extraction medium. Chloroplasts were further purified in discontinuous Percoll gradients as described by Reinero and Beachy (19). Proteins were labeled by incubating chloroplasts at 28 or 40 "C in the incubation medium described by Ramirez et al. (20) with the addition of [35S] methionine a t a final concentration of 80 pCi/ml. At the end of the incubation period chloroplasts were pelleted by centrifugation and proteins extracted with SDS sample buffer (16). Proplastids were isolated from roots of 3-day-old etiolated seedlings following the procedure of Emes and England (21). Proplastid protein synthesis in in vitro reactions were performed in the incubation medium for mitochondrial protein synthesis (17). After labeling for 60 min, pellets were resuspended in SDS sample buffer (16) or in

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Triton X-100 extraction buffer (4% Triton X-100, 100 mM Tris, pH 6.8,30% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 1 mM p hydroxymercuribenzoate sodium salt). Triton X-100 extracts were incubated for 20 min a t 4 "C and centrifuged. The resulting superX-100 extract. The resulting pellet natant is referred to as the Triton is referred to as Triton X-100 non-extractablefraction and was resuspended with SDS sample buffer. Proteins were separated by SDS-PAGE in a 11.5% gel according to Laemmli (16). Molecular mass markers were run alongside the extracted proteins. To visualize labeled proteins, gels were fixed and infiltrated for fluorography by the procedure of Jen and Thach(22). Gels were dried and exposed to Kodak XAR-5 x-ray film a t -80 "C.

oxidizable substrate (e.g sodium succinate) (lanes 11 and 12). In addition, the patternof protein synthesis in the preparation does not correspond to the patternsreported in the literature for mitochondrial protein synthesis (17, 23). The a-subunit of the F,-ATPase (58 kDa) is considered to be the largest labeled polypeptidesynthesized in uitro by mitochondria (24). Experiments using mitochondrial preparations obtained by centrifugation in discontinuous sucrose gradients showed results differing from those previously mentioned. Fig. 2 shows the dependence of such preparations on the addition of a mitochondrial oxidizable substrate (sodium succinate) for protein synthesis (lanes 5 and 11). Inhibition of protein RESULTS synthesis by 100 p~ chloramphenicol (lunes 8 and 13) and Fig. 1shows a fluorogram of the proteins synthesized by a insensitivity to 100 p~ cycloheximide, 100pM streptomycin, mitochondrial preparation isolated from the roots of 3-day100 p~ erythromycin (lanes 7,9, and 10, respectively) are and old etiolated maize seedlings.The mitochondria were purified also properties of mitochondrial protein synthesis in uitro by centrifugation in discontinuous Percoll gradients. Follow(23). Heat shock at 40 "C showed no major changes in the ing a heat shock of 40 "C the pattern of protein synthesis of the preparation changed, showingthe enhancement of a major pattern of protein synthesis by mitochondria isolated in suband of 62 kDa as reported previously (see lane 4 ) (14, 15). crose gradients (compare lanes 6 and 12). The 10,000 X g In addition, other minor bands appeared to be enhanced (88, pellet obtained prior to centrifugation in the sucrose gradient 74, 38, 26, 18, and 17 kDa). Protein synthesis in this prepa- was still capable of synthesizing a hsp of 62 kDa (lanes 1 and ration was not inhibited by 100 p~ cycloheximide (lanes 5 2). However, the synthesis of this hsp did not meet the criteria and 6) but by100 p~ chloramphenicol (lanes 7 and 8 ) as previously mentioned for a mitochondrial protein-synthesizexpectedfor a mitochondrial protein-synthesizing system. ing system (data notshown). To test thepossibility that the62-kDa hsp was encoded by However, in this experiment as in others using mitochondrial preparations obtained by centrifugation in Percoll gradients, plastid DNA we isolated chloroplasts from green leavesof 13the criteria of energy dependence for protein synthesis was day-old maize seedlings and performed protein synthesis in not met: the preparation did not require a mitochondrial uitro. Fig. 3 shows that chloroplasts are not able to synthesize hsp in such an in uitro system. Protein synthesis in this

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FIG. 1. Proteins synthesized in vitro by mitochondria isolated in Percoll gradients. Mitochondria were incubated a t 28 or 40 "C in the presence or absence of energy-generating substrates (sodium succinate/ADP/GTP) and several protein synthesis inhibitors. Protein extracts were electrophoresed in an 11.5% polyacrylamide SDS gel. The gel was prepared for fluorography and exposed to x-ray film. Lanes I and 2, proteins synthesized in vivo by excised maize root tips. Lanes 3-12, proteins synthesized in vitro by mitochondria after centrifugation in a discontinuous Percoll gradient. Reactions were supplemented with (lanes 3-10) or without (lanes 11 and 12) energy-generating substrates. Numbers on the top indicate temperature of incubation in "C.Numbers on the right are molecular mass standards in kDa. Arrow indicates position of the 62-kDa protein. E l , energy-generating system 1 (sodium succinate/ADP/ GTP); E2, energy-generating system 2 (sodium acetate/GTP); CX, 100 p M cycloheximide; CAM, 100 p M chloramphenicol.

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FIG. 2. Proteins synthesized in vitro by mitochondria isolated in sucrose gradients. Mitochondria were incubated a t 28 or 40 "C in the absence or presence of energy-generating substrates (sodium succinate/ADP/GTP) and several protein synthesis inhibitors. Protein extracts were electrophoresed in an 11.5% polyacrylamide SDS gel. The gel was prepared for fluorography and exposed to x-ray film. Lanes I and 2, proteins synthesized invitro by a mitochondria-enriched preparation prior to sucrose gradient centrifugation. Lanes 3 and 4, proteins synthesized in vivo by excised maize root tips. Lanes 5-13, proteins synthesized in vitro by mitochondria after centrifugation in a sucrose gradient. Numbers on the top indicate temperature of incubation in "C. Numbers on theright are molecular mass standards in kDa. Arrow indicates position of the 62-kDa protein. Asterisk indicates a-subunit of the F1-ATPase. E, energygenerating substrates (sodium succinate/ADP/GTP); CX, 100 p~ cycloheximide; CAM, 100 p~ chloramphenicol; ST,100 p~ streptomycin; ER, 100 pM erythromycin.

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FIG. 3. Proteins synthesized in vitro by isolatedchloroplasts. Chloroplasts isolated in Percoll gradients were incubated a t 28 or 40 "C in the light or dark in the presence or absence of an external ATP-generating system (ATP/creatinine phosphokinase). Proteins were separated on an 11.5% polyacrylamide SDS gel. Labeled products were visualized by fluorography. Lanes 1 and 2, proteins labeled in vivo in excised root tips. Lanes 3-10, proteins labeled by isolated chloroplasts in vitro. Numbers on the top indicate temperature of incubation in "C. Numbers on the left are molecular mass markers in kDa. Arrow indicates position of the 62-kDa protein. 0, dark; L, light (800 p E m-' s-'); ATP, ATP-generating system (ATP/ creatine phosphate/creatine phosphokinase).

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FIG. 4. Proteins synthesized in vitro by partially purified chloroplasts. Intact chloroplast fraction (interface a t 60-80% Percoll) was mixed with the fraction at theinterface of 45-6075 Percoll. The preparation was incubated a t 28 or 40 "C in the light or dark in the presence or absence of an external ATP-generatingsystem (ATP/ creatine phosphate/creatine phosphokinase). Some metabolic inhibitors were included in some samples. Proteins were separated on an 11.5% polyacrylamide SDS gel and visualized by fluorography. Lanes 1 and 2, proteins labeled in vivo by excised root tips. Lanes 3-16, proteins labeled by partially purified chloroplasts in vitro. Numbers on the top indicate temperature of incubation in "C. Numbers on the right are molecular mass standards in kDa. Arrow indicates position of the 62-kDa protein. ATP, ATP-generating system (ATP/creatine phosphate/creatinephosphokinase);L, light (100p E m-' s-'); DCMU, 100 p~ DCMU; CX, 100 p~ cycloheximide; CAM, 100 p~ chloramphenicol; ER, 100 pM erythromycin.

preparations. Incorporation of [35S]methionineinto protein in proplastids was extremely low compared to that obtained in chloroplast or mitochondrial preparations. Fluorograms required prolonged exposures. Dueto the lack of appropriate preparation was totally light energy driven (lanes 5 and 9) as metabolic controls (i.e dependence of protein synthesis on incubation in the dark failed to stimulate protein synthesis some ATP-generating system specific for proplastids) it was (lanes 3 and 7). Addition of an ATP-generating system (ATP/ uncertain to consider the proplastids as the site of synthesis creatine phosphate/creatine phosphokinase) in the dark re- of the proteins observed in the fluorograms. Since mitochonestablished protein synthesis (lanes 4 and 8). dria and plastids lack cell walls it is possible to disrupt the Fig. 4 shows that partially purified chloroplast preparations organelles with mild detergents (i.e. Triton X-100). When can show hsp synthesis. However, as in the case of mitochon- proplastids were resuspended in buffer containing 4% Triton drial protein synthesis under heat shock, the synthesis of X-100, almost all of the proteins in SDS gels which were these hspfailed to meet all the criteria for a chloroplast stained with Coomassie BlueR were Triton X-100 extractable protein-synthesizing system. Although in this preparation (supernatant) from the proplastid preparations. However, all protein synthesis was insensitive to 100 PM cycloheximide the 35S-labeledprotein remained in thepellets as seen on the (lanes 11 and 12) and sensitive to 100 PM chloramphenicol fluorograms (data not shown). Bacterial cultures labeled unand 100 PM erythromycin (lanes 13-16). The synthesis of the der similar conditions showed no extractability of protein by hsp decreased when incubated in the light (compare lanes 4 4% Triton X-100 either at the level of Coomassie Blue R and S ) , was not stimulated by ATP in the dark (lane 4 versus stained protein or at the level of 35S-labeledprotein as ex6),nor inhibited by the electron transport inhibitor DCMU pected for cell walled organisms (data not shown). Protein in the light ( l a n e 8 versus 10). In contrast, the synthesis of synthesis patterns of bacterial cultures incubated at 28 or similar to theones obtained using proplastid the large subunit of RBUp,Case (around 54 kDa) and other 40 "C looked very chloroplast marker proteins (i.e. 32-kDa protein) was stimu- preparations. To further test thepossibility that hsp 62 synthesis is due lated by light (compare lane 3 with lane 7)) by ATP in the dark (lane 3 versus 5 ) , and inhibited by DCMU in the light to bacterial contamination, proteins were labeled in vivo using (lune 7 versus 9).'Therefore, these observations indicate that two different batches of seedlings: one in which sterile conthe synthesis of these hsp is not occurring in the chloroplast. ditions were maintained throughout planting of seeds to laIn other experiments proplastids were isolated from 2-day- beling, and anotherin which seedlingswere exposed to normal old maizeroots and proteins were labeledin vitro using these laboratory air conditions during growth and labeling. Fig. 5

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14.41 2 3 4 5 6 7 8 FIG. 5. Proteins synthesized by excised root tips in vivo under heat shock. Plant material was handled under sterile conditions. Maize root tips were labeled with [35S]methioninefor 2 h a t either 28 or 40 "C in the presence or absence of protein synthesis inhibitors. Proteins were extracted,separated by SDS-PAGE a t 11.5% polyacrylamide and visualized by fluorography. Lanes 1 and 2, proteins synthesized in vivo by a bacterial isolate from maize roots. Lanes 3-8, proteins synthesized maize root tips in vivo. Numbers on the top indicate temperature of incubation in "C. Numbers on the left are molecular mass markers in kDa. Arrow indicates position of the 62-kDa protein. CX, 100 pM cycloheximide; CAM, 100 p M chloramphenicol.

14.41 2 3 4 5 6 7 8 FIG. 6. Proteins synthesized by excised root tips in vivo under heat shock. Plant material was manipulated under nonsterile conditions. Treatments are the same as described in the legend to Fig. 5.

organelles or by bacteria (17, 25). The concern of purity in the preparation is obvious in virtue of the universality of the heat shock response. Practically all organisms studied so far, from bacteria to higher eukaryotes, are able to synthesize hsp (La.

A hsp of 62.5 kDa is known to be induced in E. coli by heat shock. This protein is the most abundant heat shock protein shows the lack of induction by heat shock of hsp 62 on the in E. coli (26).This may explain why a hspof similar molecular batch of sterile seedlings (lane 4 ) . Addition of 100 PM cyclo- mass appeared to be induced by heat shock in contaminated heximide during heat shock completely blocked protein syn- organellar preparations or during labeling of proteins in vivo thesis (lane 6 ) and addition of 100 p~ chloramphenicol did when using nonsterile tissues. not change the pattern of protein synthesis (lane 8 ) . Fig. 6 This work raises the question of how cellular organelles can shows that seedlings grown and manipulated under non- cope with the effects of heat shock since they are not able to sterile conditions synthesized a 62-kDa protein under heat synthesize thier own hsp. Sensitivity of chloroplast functions shock (lane 4 ) . Cycloheximide (100 PM) did not inhibit the to high temperature is well documented (27,28). It is considinduction by heat shock of the 62-kDa protein as well as ered that the Photosystem 11-water-splitting complex, the others of 88, 84, 76, 75, and 66 kilodaltons (lane 6). Finally, photophosphorylation reactions, and the light activation of addition of 100 p~ chloramphenicol inhibited the synthesis the stromal RBUpz Case are among the most heat-sensitive of the hsp 62 (lane 8). photosynthetic processes. The effects of heat shock on mitochondrial functions are not as well characterized. However, DISCUSSION the observation that inhibitors of oxidative phosphorylation Based on protein synthesis studied in vitro and in vivo we or electron transport induce the heat shock response (29,30) have provided evidence that maize organelles (i.e. mitochon- suggests that changes in the mitochondrial respiratory metabdria, chloroplasts, and proplastids) are unable to respond to olism take place during heat shock. More recently, a decrease heat shock by synthesizing hsp. Our resultsshow that artifacts in the content of cellular ATP was observed following heat can be obtained when analyzing the products of protein syn- shock in Tetrahymena (31). Little is known about the mechthesis in vivo if the tissues utilized are not maintained under anism of recovery from heat stress in the chloroplasts. The sterile conditions. Also, the choice of appropriate techniques role of hsp in this mechanism is worth further study. If hsp when isolating plant organelles and the inclusion of controls are implicated, obviously these organelles may depend upon to show energy dependence for protein synthesis in organello hsp imported from the cytoplasm for their recovery. There is are very important due to theease of contamination by other evidence that in plants some small hsp (21-27-kDa class) are

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transported into chloroplasts during heat shock (32). Also in plants, the same hsp class has been located inthe mitochondrial fraction followingheat shock (33, 34). More studies are needed to explain whatis the molecular basis forthe absence of hsp synthesis in plastids and mitochondria. Acknowledgments-We gratefully acknowledge the help of Dr. Kathy Newton, Department of Biological Sciences, University of Missouri at Columbia, for her advice on isolation of mitochondria from maize tissues and in whose laboratory we performed our first experiment on the effect of heat shock on mitochondrial protein synthesis in vitro. We also thank Antonio Reinero, Department of Biology, Washington University, for advice on chloroplast isolation.

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