Alignment of Conduits for the Nascent Polypeptide Chain in the
Contents
1. Three regulators of Ca levels are ino sitol 1 4 5 trisphosphate IP 11 cyclic adenosine 5 diphosphate ribose cADPR and nicotinic acid adenine dinucleotide phosphate NAADP 12 15 The re ceptor for IP is known 11 whereas those for cADPR and NAADP are not 15 A putative receptor for cADPR is the ryano dine receptor RyR 15 NAADP a me Y Wu J Kuzma E Mar chal R Foster N H Chua Laboratory of Plant Molecular Biology Rockefeller Uni versity 1230 York Avenue New York NY 10021 6399 USA R Graeff and H C Lee Department of Physiology Uni versity of Minnesota Minneapolis MN 55455 USA Present address Laboratoire de Physiologie Cellulaire V g tale Departement de Biologie Moleculaire et Cellu laire Universite Joseph Fourier et CEA Grenoble 17 rue des Martyrs F 38054 Grenoble Cedex 9 France To whom correspondence should be addressed E mail chua rockvax rockefeller edu tabolite of nicotinamide adenine dinucle otide phosphate NADP identified in vitro regulates a third Ca channel that appears to be distinct from the IP and cADPR receptors 14 cADPR can be produced by ADP ribosyl cyclase or by CD38 a lymphocyte protein both of which use nicotinamide adenine dinucle otide NAD as a precursor 16 Both IP and cADPR elicit Ca release from beet storage root vacuoles 17 and a RyR like activity sensitive to cADPR has been detected in beet microsomes2. 18 Here we demonstrate that cADPR is a like ly in vivo intermediate of ABA signal trans duction that exerts its effects by way of intracellular Ca release We used microinjection to screen for compounds that may be involved in ABA responses We studied the Arabidopsis genes rd29A also termed ti78 and cor78 5 a desiccation responsive gene 3 and kin2 also termed cor6 6 5 a cold in ducible gene 2 Both genes are rapidly induced by ABA without requiring new protein synthesis We microinjected 7 to 10 day old etio lated hypocotyls of the phytochrome defi cient tomato mutant aurea 19 20 with rd29A GUS kin2 GUS 21 and potential agonists and antagonists of ABA signal SCIENCE VOL 278 e 19 DECEMBER 1997 www sciencemag org
3. analyzed by su crose density gradient centrifugation followed by SDS PAGE analysis and immunoblotting of gradi ent fractions Lanes 1 to 6 represent top to bottom fractions probed with a peptide antibody to Sec618 For the ribosomes the Coomassie stained proteins were quantitated with NIH image aro units arbitrary units C Binding of the Sec61 complex to ribosomes is saturable In creasing amounts of the Sec61 complex O wg of Sec61a in lane 1 to 0 5 wg of Sec61a in lane 6 were incubated with a fixed amount of ribosomes attachment of the Sec61 oligomer to the ribosome does not appear to form the tight seal that was implicated in nascent chain fluorescence quenching experiments Seal forming proteins could be missing or more likely the signal sequence may be necessary for seal formation 2 3 8 22 The central pore of the Sec61 oligomer aligns precisely with an opening in the large ribosomal subunit that represents the exit of a tunnel Fig 3B This tunnel runs from Fig 2 A Cryo electron micrograph showing a field of yeast ribosome Sec61 complexes Scale bar 200 A B Aver aged projections of the ribosome Sec61 com plex obtained by classifi cation Particles marked with arrowheads show the ribosome in side view with the Sec61 complex visible as a 100 A long mass lying paral lel to the ribosome sur face Scale bar 200 A the interface canyon 12 to the lower por tion of the large subunit Fi
4. correlation coefficient Both re constructions proved to be indistinguishable within the measured resolution range The final resolution estimated with the Fourier shell correlation with a cutoff value at 0 5 B Bottcher S A Wynne R A Crowther Nature 386 88 1997 was 26 A 22 R Gilmore and G Blobel Cell 42 497 1985 23 In trying to gauge the correct threshold value we were led by two criteria i We observed the structure as the threshold was increased There is normally a olateau a range of threshold values within which the appearance or the volume encompassed varies only slightly ii Three dimensional connectivity must not be violated which means that in this case we could not choose a threshold within the plateau defined above that makes the connecting rod dis appear Thus the plateau was further narrowed These criteria were applied separately in the prepa ration of the 3D representations of the ribosome and the channel Because of the residual uncertainty in the molecular boundaries the measurements for pore size and the distance between channel and ribosome have an uncertainty of 25 24 We thank A Fischer for the purification of the Sec61 complex members of the Blobel lab and R Agrawal for discussions S Darst and A Malhotra for discus sions Support with the electron microscopy and assistance with the image processing A Heagle for help with the illustrations and the National Cen
5. structure with an outer diameter of 95 A an inner diameter ranging from 15 to 35 A depending on depth and an overall thickness of 40 A supported by a single stem attached to the base of the large ribosomal subunit A single site of rigid attachment may facilitate later al opening of the channel at the opposite site to allow the release of nascent trans membrane segments into the lipid bilayer The surface of the Sec61 oligomer facing the ribosome is parallel to the surface of the large subunit The distance between these surfaces ranges from 15 to 20 A Hence the Fig 1 Purification and binding of the trimeric Sec61 complex to ribosomes A Purification of the Sec61 complex Crude nuclear envelopes from Sec63prA cells were extracted with digito nin and the digitonin extract was incubated with lgG Sepharose The trimeric Sec61 complex was eluted with Triton X 100 and analyzed by SDS PAGE followed by Coomassie blue staining Lane 1 crude nuclear envelopes 1 30 000 lane 2 digitonin extract 1 30 000 lane 3 IgG Sepha rose flow through 1 30 000 and lane 4 Triton X 100 eluate 1 10 Ten to 15 micrograms of Sec61a by comparison with a bovine serum al bumin standard were typically purified from 80 g of packed cells Molecular mass is given in kilodal tons at the left B The Sec61 complex binds to ribosomes The Sec61 complex 0 5 wg was in cubated with and without ribosomes 0 5 Asso and the incubation mixture was
6. the ribosome tunnel The arrow indicates the stem connecting the ribosome with the Sec61 oligomer the space between the two ribosomal subunits is indicated by an asterisk The ribosomal tunnel and its alignment with the Sec61 pore is indicated by a broken yellow line Scale bar 100 A B t Fig 4 Three closeup views of the Sec61 oligomer A Surface facing the ribosome There is a vestibule diameter of 35 A formed by the funnel like structure and the pore diameter of 15 A B Surface facing away from the ribosome C View of the side opposite the attachment site The ribosome would be located underneath the channel The wall opposite the attachment site is thinner and more irregular Arrows indicate the attachment site Scale bar 50 A www sciencemag org SCIENCE VOL 278 e 19 DECEMBER 1997 modate two trimeric Sec61 complexes The reasons for the asymmetric appearance of the ribosome bound Sec61 oligomer are presently not clear It is likely that the Sec 61 oligomer bound to the ribosome represents the PCC in its inactive and closed conformation Future structural analyses of an in vitro assembled complex containing a ribosome a nascent chain and the Sec61 complex will lead to information regarding the ac tive state of the PCC REFERENCES AND NOTES 1 G Blobel and B Dobberstein J Cell Biol 67 835 1975 2 S M Simon and G Blobel Cell 65 371 1991 ibid 69 677 1992 K S Crowl
7. 12 GenBank accession number X73298 13 B Baur K Fisher K Winter K J Dietz Plant Physiol 106 1225 1994 14 M Wada personal communication 15 Mutant cDNA sequences were obtained by reverse transcription followed by PCR amplification with total RNAs and NPH1 specific primers PCR products were purified with a QIAGEN PCR purification kit and sequenced directly Both strands of the entire cDNA sequence for each mutant were examined for muta tions and each lesion was confirmed by two inde pendent PCR experiments 16 J P Khurana and K L Poff Planta 178 400 1989 17 Because noh1 2 is in the Estland ecotype we also sequenced an Estland wild type cDNA for comparison 18 An additional allele noh7 3 is identical in sequence to nph1 1 19 S Hill S Austin T Eydmann T Jones R Dixon Proc Natl Acad Sci U S A 93 2143 1996 20 S Bibikov R Biran K E Rudd J S Parkinson J Bacteriol 179 4075 1997 A Rebbapragada et al Proc Natl Acad Sci U S A 94 10541 1997 21 W A Catterall Science 242 50 1988 J Tytgat K Nakazawa A Gross P Hess J Biol Chem 268 23777 1998 22 X Li J Xu M Li J Biol Chem 272 705 1997 23 M Ahmad and A R Cashmore Nature 366 162 1998 C Lin M Ahmad J Chan A R Cashmore Plant Physiol 110 1047 1996 24 B Zhulin B L Taylor R Dixon Trends Biochem Sci 22 331 1997 Z J Huang Edery M Ros b
8. ER 3 An aqueous pore with a diameter of 40 to 60 A during cotranslational translo cation is suggested by similar experiments 4 The Sec61 trimeric complex is a strong candidate for the PCC of the ER in yeast and mammalian cells 5 6 The Sec61 complex provides the principal binding site for ribosomes at the ER during protein translocation 7 8 and together with oth er membrane proteins is associated with ribosomes after solubilization of rough mi crosomes with digitonin 6 A two dimen sional map of the purified Sec61 complex obtained by electron microscopy has re vealed a quasi pentagonal circular structure with a central depression 9 The three dimensional 3D structure of monomeric ribosomes is currently known at various resolutions for Escherichia coli 10 36 G Blanco M Drummond P Woodley C Kennedy ibid 9 869 1993 R Raina U K Bageshwar H K Das Mol Gen Genet 237 400 1998 37 D Siddavattam H D Steibl R Kreutzer W Kling muller Mol Gen Genet 249 629 1995 38 H Allmeier B Cresnar M Greck R Schmitt Gene 111 11 1992 39 R F Smith and T E Smith Protein Eng 5 35 1992 40 F Gropp and M C Betlach Proc Natl Acad Sci U S A 91 5475 1994 41 F Narberhaus H S Lee R A Schmitz L He S Kustu J Bacteriol 177 5078 1995 42 S K Crosthwaite J C Dunlap J J Loros Science 276 763 1997 43 We thank K Meyer and W Lukowit
9. ash Nature 364 259 1993 25 E Huala et al data not shown 26 Single letter abbreviations for the amino acid resi dues are as follows A Ala C Cys D Asp E Glu F Phe G Gly H His lle K Lys L Leu M Met N Asn P Pro Q Gln R Arg S Ser T Thr V Val W Trp and Y Tyr 2 K Meyer M P Leube E Grill Science 264 1452 1994 28 J J Kieber M Rothenberg G Roman K A Feld mann J R Ecker Cell 72 427 1993 29 F Kunst et al Nature 390 249 1997 30 P Ballario et al EMBO J 15 1650 1996 31 D Leong F Pfeifer H W Boyer M C Betlach J Bacteriol 170 4903 1988 32 J W Warmke and B Ganetzky Proc Natl Acad Sci U S A 91 3438 1994 33 J Ludwig et al EMBO J 13 4451 1994 34 T Kaneko et al DNA Res 3 109 1996 35 M H Drummond and J C Wooton Mol Microbiol 1 37 1987 Alignment of Conduits for the Nascent Polypeptide Chain in the Ribosome Sec61 Complex Roland Beckmann Doryen Bubeck Robert Grassucci Pawel Penczek Adriana Verschoor G nter Blobel Joachim Frank An oligomer of the Sec61 trimeric complex is thought to form the protein conducting channel for protein transport across the endoplasmic reticulum A purified yeast Sec61 complex bound to monomeric yeast ribosomes as an oligomer in a saturable fashion Cryo electron microscopy of the ribosome Sec61 complex and a three dimensional reconstruction showed that the S
10. d 1 mM CaCl The ice aN O1 O CO O O 2125 identity of the proteins was confirmed by specific antibodies to Sec61a Sec61p and Sec61 B Sbh1p 17 14 S Panzner L Dreier E Hartmann S Kostka T A Rapoport Cell 81 561 1995 15 For the purification of ribosomes the yeast strain DF5 was grown in 3 5 liters of yeast extract pep tone and dextrose medium At an optical density of 600 nm OD o of 1 0 the cells were washed with water and incubated for 15 min at 25 C in 100 mM tris SO pH 9 4 and 10 mM DTT After homogeni zation by French press in buffer A 50 mM triethanol amine OAc pH 7 5 50 mM KOAc 5 mM MgCl 1 mM DTT and 0 5 mM PMSF the homogenate was centrifuged for 30 min at 100 000g at 4 C The su pernatant was layered over a continuous 10 to 40 sucrose gradient in buffer A After centrifugation for 4 5 hours at 200 000g at 4 C the monomeric ribo somes were pooled according to the Ass profile The ribosomes were pelleted by centrifugation for 4 5 hours at 145 000g at 4 C resuspended in water and frozen in liquid No 16 Purified Sec61 complex and ribosomes were incubat ed for 30 min on ice in a buffer containing 0 5 Triton X 100 100 mM KOAc 5 mM triethanolamine OAc OH 7 5 5 glycerol 1 5 mM Mg OAc s 0 5 mM DTT and 0 56 mM CaCl To separate unbound Sec61 complex from ribosome bound Sec61 com plex we carried out gradient centrifugation using a 10 to 50 sucrose ste
11. ec61 oligomer is attached to the large ribosomal subunit by a single connection Moreover a funnel shaped pore in the Sec61 oligomer aligned with the exit of a tunnel traversing the large ribosomal subunit strongly suggesting that both structures function together in the translocation of proteins across the endoplasmic reticulum membrane The existence of a protein conducting channel PCC for protein transport across the endoplasmic reticulum ER was pro posed in 1975 1 Electrophysiological ex periments in 1991 provided the first direct evidence for the existence of the PCC 2 Moreover fluorescently labeled nascent chains in membrane bound ribosomes re main in an aqueous environment sealed from the cytoplasm and accessible to fluo R Beckmann and G Blobel Howard Hughes Medical Institute Laboratory of Cell Biology Rockefeller Universi ty 1230 York Avenue New York NY 10021 USA D Bubeck R Grassucci A Verschoor Wadsworth Cen ter New York State Department of Health Empire State Plaza Albany NY 12201 0509 USA P Penczek and J Frank Wadsworth Center New York State Department of Health and Department of Biomed ical Sciences State University of New York at Albany Empire State Plaza Aloany NY 12201 0509 USA To whom correspondence should be addressed E mail beckmar rockvax rockefeller edu www sciencemag org SCIENCE VOL 278 e 19 DECEMBER 1997 rescence quenching from the lumen of the
12. ey G D Reinhart A E Johnson ibid 73 1101 1993 K S Crowley S Liao V E Worrel G D Reinhart A E Johnson ibid 78 461 1994 B D Hamman J C Chen E E Johnson A E Johnson ibid 89 535 1997 R J Deshaies and R Schekman J Cell Biol 105 633 1987 C J Stirling J Rothblatt M Hosobu chi R Deshaies R Schekman Mol Biol Cell3 129 1992 D G rlich S Prehn E Hartmann K U Kalies T A Rapoport Cell 71 489 1992 D G rlich and T A Rapoport ibid 75 615 1993 7 K U Kalies D G rlich T A Rapoport J Cell Biol 126 925 1994 B Jungnickel and T A Rapoport Cell 82 261 1995 D Hanein et al ibid 87 721 1996 J Frank et al Nature 376 441 1995 H Stark et al Structure 3 815 1995 11 A Verschoor S Srivastava R Grassucci J Frank J Cell Biol 133 495 1996 12 A Verschoor J R Warner S Srivastava R A Grassucci J Frank Nucleic Acids Res in press 13 A yeast strain Sec63prA was constructed in which the genomic copy of the gene encoding Sec63p was tagged by COOH terminal in frame integration of a DNA fragment encoding for the immunoglobulin G IgG binding domains of protein A The DNA frag ment encoding the protein A gene and adjacent HIS3 and URA3 markers was amplified by polymer ase chain reaction with specific primers for SEC63 and a template plasmid as described J D Aitchi son M P Rout M Mare
13. g 3D At the threshold level chosen 23 a small seg ment of the tunnel is blocked because of the limiting resolution but it appeared to be open at increased threshold levels 17 A similar effect has been observed in the re construction of the 70S E coli ribosome 10 These tunnels seen in cryo electron microscopy maps of the ribosome from E coli 10 and Saccharomyces cerevisiae 12 A 123 4 B Gradient fraction 1 2 3 4 5 6 o7 i SS oo Sec61 complex ammm Anti Sec61p 66 s re Sec61 complex 7 45 Mme Sec61c ribosomes au AmiSELEIR 30 e i 12 35 i h 8 Protein 20 Ribosomes 6 arb units 14 2 Sec61B y C Unbound Bound l D 1234567 23 456 5 eeererr 0 15 Sec610 k a a a a Y E 30 20 a 14 0 125 Asgo and analyzed by density gradient centrifugation followed by SDS PAGE and SYPRO Red staining Asterisks denote Sec61a D Quantitation of data in C The amount of bound Sec61a was calculated according to the calibration shown in the inset 2124 D a 14 0 10 512 o g 10 5 8 fe 8 6 N D e 5 4 20 05 3 5 i T 01 03 05 Amount of Sec610 ug Amount of Sec61a bound to 0 1 0 2 Amount of Sec61 ug 0 3 0 4 0 5 SCIENCE VOL 278 e 19 DECEMBER 1997 www sciencemag org have been proposed as exit pathways of the nascent polypeptide chain although the ev idence for that is indirect 20 The precise a
14. lignment between the pore of the Sec6l oligomer and the tunnel Fig 3 C and D provides strong support for this hypothesis Conversely this structural arrangement also implies that the Sec61 oligomer indeed constitutes the PCC Detailed analysis of the Sec61 oligomer Fig 4 A to C shows that the azimuthal distribution of mass is irregular with the bulk of the mass lying on the side attached to the stem Again as speculated above the thinner wall opposite the attachment stem might facilitate lateral opening of the chan nel There is a further asymmetry in the Sec61 oligomer the pore is funnel shaped with a diameter of 15 A at the lumenal site of the ER widening toward the ribosome so that a small vestibule with a diameter of 35 A is formed Consistent with the esti mate from kinetic data the measured vol ume of the Sec61 oligomer would accom B 40S D J 60S 60S A Fig 3 Three dimensional reconstruction of the ribosome Sec61 complex in a surface representation A Front view with the Sec61 oligomer shown in red B Front view with the Sec61 oligomer shown as transparent to demonstrate the alignment of the Sec61 oligomer pore with the tunnel exit of the large ribosomal subunit indicated with the yellow arrow C Side view with the Sec61 oligomer shown in red D Ribosome Sec61 complex lying in the same orientation as in C cut along a plane that cross sections the pore of the Sec61 oligomer and
15. lli G Blobel R Wozniak J Cell Biol 131 1133 1995 A crude nuclear pellet was prepared from a 36 liter culture as described C Strambio de Castillia G Blobel M P Rout ibid p 19 Thirty milliliters of crude nuclei one fifth of the preparation was extracted by incubation with de oxyribonuclease 20 pg ml and Heparin 1 mg ml in buffer E 10 mM bis tris Cl OH 6 5 1 mM MgCl 10 mM KOAc 1 mM dithiothreitol DTT and 0 5 mM phenylmethylsulfonyl fluoride PMSF for 20 min at 25 C and 40 min on ice The crude nuclear enve lopes were sedimented for 40 min at 145 000g at 4 C and extracted with 40 ml of buffer S 3 digito nin 0 4 M sucrose 10 mM triethanolamine OAc pH 7 5 750 mM KOAc 1 5 mM Mg OAc s 0 5 mM EDTA and 1 mM DTT for 30 min on ice After pel leting by centrifugation of insoluble material for 35 min at 145 000g at 4 C the extract was diluted with 1 volume of buffer D 0 4 M sucrose triethanol amine OAc pH 7 5 1 5 mM Mg OAc gt s 1 mM PMSF and 1 mM DTT and incubated overnight with 1 ml of lgG Sepharose Cappel Durham NC at 4 C The column was washed with 10 volumes of buffer W 1 digitonin 100 mM KOAc Azolectin 0 5 mg ml 10 mM triethanolamine OAc pH 7 5 10 glyc erol 3 MM Mg OAc s5 0 5 mM DTT and 1 mM CaCl and the trimeric Sec61 complex was eluted with buffer T 1 Triton X 100 200 mM KOAc 10 mM triethanolamine OAc pH 7 5 10 glycerol 3 mM Mg OAc 0 5 mM DTT an
16. nti Sec61B Fig 1B and Sec6la anti Sec6la 17 In agreement with the known salt sensitivity of the Sec61 ribosome interaction there was no binding at 1 M KOAc OAc acetate 17 Incubation of a fixed amount of ribosomes with increasing amounts of Sec61 complex resulted in saturation of ribosome binding sites Fig 1 C and D On the basis of the amount of Sec6la and ribosomes we esti mate that at saturation two to four Sec61 trimers were bound per ribosome and that the dissociation constant K4 is about 10 nM The ribosome Sec61 complex formed under saturating conditions was examined by cryo electron microscopy 18 In the 2123 electron micrographs Fig 2A the ribo some Sec61 complex appears in random orientations Fig 2B This distribution al lowed an artifact free 3D reconstruction by means of a 3D projection alignment proce dure 19 with an existing reconstruction of the ribosome from yeast 12 as a reference In side views of the ribosome marked by arrows in Fig 2B an 100 A long ellip soidal mass of density appears at the surface of the large subunit This location on the ribosome is the same as the site where in projection the exiting polypeptide chain was located on both eubacterial and eukary otic ribosomes by immuno electron micros copy 20 In the resulting reconstruction Fig 3 which has a resolution of 26 A 21 the Sec61 complex appears as a slightly pen tagonally shaped toroidal
17. o scopy 58 251 1994 20 C Bernabeu E M Tobin A Fowler Jabin J A Lake J Cell Biol 96 1471 1983 21 Micrographs were checked for drift astigmatism and presence of Thon rings by optical diffraction Scan ning was done with a step size of 25 um correspond ing to 4 78 A on the object scale on a Perkin Elmer PDS 1010 A microdensitometer Particles were se lected by an automated selection procedure that dif fered from the one previously described K R Lata P Penczek J Frank Ultramicroscopy 58 381 1995 in that the particle candidates were compared directly with the reference set of 87 quasi evenly spaced pro jections 79 of an existing reconstruction of the ribo some from yeast 72 A total of 13 178 particles were picked The reconstruction was done with two inde pendent approaches to obtain the orientations of the projections In the first approach an existing recon struction of the ribosome from yeast 72 was used as a reference in the 3D projection alignment procedure 79 In the second approach an initial reconstruction was obtained with the simultaneous minimization technique P Penczek J Zhu J Frank Ultramicros copy 68 205 1996 In both cases four steps of the 3D projection alignment procedure 79 were ap plied with a 2 angular interval In each step the 2126 refined 3D structure was calculated with 70 of the best matching particles on the basis of the value of the cross
18. p gradient in 0 5 Triton X 100 100 mM KOAc 10 mM triethanolamine OAc 1 mM DTT and 3 mM Mg OAc After centrifugation for 60 min at 240 000g at 4 C six fractions were collected manually For saturation assays the first two fractions were pooled as the unbound fraction and the follow ing two as the bound fraction Fractions were ana lyzed by SDS polyacrylamide gel electrophoresis PAGE and stained with SYPRO Red The amount of protein was quantitated with the STORM system red fluorescence and NIH image For immunoblotting proteins were precipitated separated on 10 to 20 SDS PAGE gradient gels transferred to a nitrocellu lose membrane incubated consecutively with anti Sec61 or anti Sec61B and horseradish peroxidase conjugated donkey antibodies to rabbit and detected by ECL as described Amersham 17 R Beckmann et al data not shown 18 Incubation to form the ribosome Sec61 complex was performed as described 76 and the mixture was diluted with 4 volumes of water immediately before it was applied to the grid Grids for cryo electron microscopy were prepared as described T Wagenknecht R Grassucci J Frank J Mol Biol 199 137 1988 J Dubochet et al Q Rev Biophys 21 129 1988 Micrographs were recorded under low dose conditions on a Philios EM 420 with 1 5 um defocus and magnification of 52 200 2 as checked by a tobacco mosaic virus standard 19 P A Penczek R Grassucci J Frank Ultramicr
19. ter for Supercomputer Applications University of Illinois at Urbana Champaign for computing support Sup ported by grants from NIH RO1 GM29169 and NSF BIR 9219048 to J F and a fellowship of the Deutsche Foschungsgemeinschaft to R B 26 September 1997 accepted 17 November 1997 Abscisic Acid Signaling Through Cyclic ADP Ribose in Plants Yan Wu Jennifer Kuzma Eric Marechal Richard Graeff Hon Cheung Lee Randy Foster Nam Hai Chuat Abscisic acid ABA is the primary hormone that mediates plant responses to stresses such as cold drought and salinity Single cell microinjection experiments in tomato were used to identify possible intermediates involved in ABA signal transduction Cyclic ADP ribose CADPR was identified as a signaling molecule in the ABA response and was shown to exert its effects by way of calcium Bioassay experiments showed that the amounts of cADPR in Arabidopsis thaliana plants increased in response to ABA treatment and before ABA induced gene expression Plants endure environmental challenges such as drought salinity or cold by adjust ing rather than escaping These responses are mediated by ABA 1 which through unknown signals affects the regulation of many genes 2 7 One signaling interme diary is calcium Ca 8 ABA mediated increases in guard cell Ca levels lead to stomatal closure 9 Ca can also induce the expression of an ABA responsive gene in maize protoplasts 10
20. z for providing the genomic library J R Ecker for providing the cDNA and YAC libraries C Somerville for assistance with the AFLP technique and P Reymond for many help ful comments on the manuscript This work was funded by NSF grants MCB 9219256 and IBN 9601164 This paper is Carnegie Institution of Washington Department of Plant Biology publica tion 1367 2 September 1997 accepted 5 November 1997 wheat germ 11 and yeast 12 Among the structural features recognized is a tunnel that traverses the large ribosomal subunit and has been considered a candidate for the nascent chain conduit Here we present a 3D reconstruction of the ribosome Sec61 complex For purification of the trimeric Sec61 complex 13 containing the Sec6la Sec61B and Sec6ly subunits Sec6lp Sbhlp and Ssslp a heptameric complex 14 was isolated first with protein A tagged Sec63 protein followed by elution of the trimeric Sec61 complex with Triton X 100 Fig LA To determine whether the trimeric Sec61 complex could bind to ribo somes 15 in a membrane free system we incubated the purified Sec61 complex with ribosomes and analyzed the incubation mix ture by sucrose density gradient centrifuga tion 16 The Sec61 complex incubated without ribosomes remained in the top frac tion of the gradient In the presence of ribosomes however the Sec61 complex migrated with ribosomes as determined by immunoblotting 16 with antibodies to Sec61B a
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