Julien Le Kernec
email: julien.le-kernec@lip6.fr

Martin Klepal

Vratislave 3d Sokol

Directional

# -Martin Klepal

3D directional coupler for impulse UWB

Introduction

The AWS Group was developing a UWB radar and UWB transceiver for indoor people location and tracking. A radar concept has been developed. The radar is composed of a pulse generator, a channel model, a low-noise amplifier, a matched filter, two pulse shapers, a flip-flop circuit and an integrator. This poster will focus on the design of the directional coupler. This component will provide a separation between the pulse generator and the Low Noise Amplifier. This enables the radar system to connect on the same antenna.

Conclusions

•Unsatisfactory experimental results

•New simulations with the SMA connectors and the extra microstrip length •Results matching the measurements for reflections •the substrate probably has a higher loss tangent of dielectric than its model

•Explains the higher transmission losses of the real directional coupler •An improved model of the substrate has to be incorporated to the model and some modifications to decrease overall losses, insertion losses and increase isolation before this 3D architecture can be considered viable. Vertically Installed Planar Circuit •Reduced the effective dielectric constant for even and odd mode from 2 to less than 0.2.

Stripline

•VIP was not feasible because the gap was too wide.

•Reduced effective dielectric constant for even and odd mode from 1.9 to less than 0.15.

Final Layout

•stronger coupling to compensate for the precision of the manufacturing •all the transitions were designed to reduce coupling and transmission losses.

Final

  parameters: simulated ≠ theoretical •Stronger transmission losses •Stronger reflections •Strong isolations •Uneven Band pass ripple •Cause: •frequency dispersion of the effective permittivity •Two propagation medium (air+substrate) •Effects: even and odd mode differences in •Impedances •phase velocities


Gyorgy Lur

Mark W Sherwood

Etsuko Ebisui

Lee Haynes

Stefan Feske

Robert Sutton

Robert D Burgoyne

Katsuhiko Mikoshiba

Ole H Petersenǁ

Alexei V Tepikin
email: a.tepikin@liv.ac.uk

IP 3 receptors and Orai channels in pancreatic acinar cells: co-localisation and its consequences

Keywords: Orai1, store-operated Ca 2+ entry, IP 3 receptors, pancreatic acinar cells, acetylcholine, Ca 2+ signalling

Orai1 proteins have been recently identified as subunits of store-operated Ca 2+ entry (SOCE) channels. In primary isolated pancreatic acinar cells, Orai1 showed remarkable co-localisation and co-immunoprecipitation with all 3 subtypes of IP 3 receptors. The colocalization between Orai1 and IP 3 Rs was restricted to the apical part of pancreatic acinar cells. Neither co-localization nor co-immunoprecipitation was affected by the Ca 2+ store depletion. Importantly we also characterised Orai1 in basal and lateral membranes of pancreatic acinar cells. The basal and lateral membranes of pancreatic acinar cells have been previously shown to accumulate STIM1 puncta as a result of store depletion. We therefore conclude that these polarized secretory cells contain two pools of Orai1 -apical that interact with IP 3 Rs and baso-lateral that interact with STIM1 following the Ca 2+ store depletion. Experiments on IP 3 Rs knockout animals demonstrated that the apical Orai1 localization does not require IP 3 Rs and that IP 3 Rs are not necessary for the activation of SOCE. However, the IP 3 -releasing secretagogue acetylcholine (ACh) produced a negative modulatory effect on SOCE, suggesting that activated IP 3 Rs could have an inhibitory effect on this Ca 2+ entry mechanism.

Introduction

Pancreatic acinar cells (PAC) are structurally and functionally polarised with secretory granules located in the apical region, whilst the basal and lateral parts contain welldeveloped rough endoplasmic reticulum (ER). Thin projections of ER are also present in the apical region [START_REF] Park | The endoplasmic reticulum as one continuous Ca(2+) pool: visualization of rapid Ca(2+) movements and equilibration[END_REF]. Important secretagogues such as acetylcholine (ACh) and cholecystokinin (CCK) utilise IP 3 and Ca 2+ signalling cascades to regulate secretion in these cells [START_REF] Petersen | Polarized calcium signaling in exocrine gland cells[END_REF]. The substantial Ca 2+ extrusion by the plasma membrane Ca 2+ ATPases (PMCAs) in PAC [START_REF] Tepikin | Pulsatile Ca2+ extrusion from single pancreatic acinar cells during receptor-activated cytosolic Ca2+ spiking[END_REF] necessitates a well-developed SOCE mechanism. IP 3 R2 and IP 3 R3 were shown to be the functional IP 3 Rs in PAC [START_REF] Futatsugi | IP3 receptor types 2 and 3 mediate exocrine secretion underlying energy metabolism[END_REF]. Local apical Ca 2+ transients can be triggered by IP 3 [START_REF] Ito | Micromolar and submicromolar Ca2+ spikes regulating distinct cellular functions in pancreatic acinar cells[END_REF][START_REF] Thorn | Local and global cytosolic Ca2+ oscillations in exocrine cells evoked by agonists and inositol trisphosphate[END_REF]. All 3 types of IP 3 Rs are found in the apical part of the cell [START_REF] Lee | Polarized expression of Ca2+ channels in pancreatic and salivary gland cells. Correlation with initiation and propagation of [Ca2+]i waves[END_REF][START_REF] Nathanson | Localization of the type 3 inositol 1,4,5-trisphosphate receptor in the Ca2+ wave trigger zone of pancreatic acinar cells[END_REF][START_REF] Yule | Evidence that zymogen granules are not a physiologically relevant calcium pool. Defining the distribution of inositol 1,4,5-trisphosphate receptors in pancreatic acinar cells[END_REF]. The role of IP 3 Rs in the activation of SOCE has been the subject of much debate [START_REF] Parekh | Store-operated calcium channels[END_REF]. The original conformational coupling hypothesis suggested that IP 3 Rs in the store activate Ca 2+ entry channels [START_REF] Berridge | Capacitative calcium entry[END_REF][START_REF] Irvine | Quantal' Ca2+ release and the control of Ca2+ entry by inositol phosphates--a possible mechanism[END_REF]. It was later found that STIM proteins serve as the Ca 2+ sensors in the store; the depletion of ER Ca 2+ results in the translocation of STIM to the plasma membrane, where it interacts with and activates Orai channels [START_REF] Feske | A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function[END_REF][START_REF] Liou | STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx[END_REF][START_REF] Luik | The elementary unit of store-operated Ca2+ entry: local activation of CRAC channels by STIM1 at ERplasma membrane junctions[END_REF][START_REF] Roos | STIM1, an essential and conserved component of store-operated Ca2+ channel function[END_REF]. The notion of conformational coupling was therefore confirmed, however with STIM rather than IP 3 R as the primary ER Ca 2+ sensor. This does not exclude that IP 3 Rs could play some regulatory role in SOCE, particularly considering a recent report describing an interaction between IP 3 Rs and Orai1 [START_REF] Woodard | TRPC3 regulates agonist-stimulated Ca2+ mobilization by mediating the interaction between type I inositol 1,4,5-trisphosphate receptor, RACK1, and Orai1[END_REF]. In PAC STIM1 was found to form puncta in the basal and lateral subplasmalemmal regions, where it was also shown to co-localise with Orai1 [START_REF] Lur | Ribosome-free terminals of rough ER allow formation of STIM1 puncta and segregation of STIM1 from IP(3) receptors[END_REF]. The basolateral SOCE in PAC is therefore mediated by STIM1 interacting with Orai1. Surprisingly, the highest density of Orai1 was found in the apical region -away from its activator STIM1 [START_REF] Lur | Ribosome-free terminals of rough ER allow formation of STIM1 puncta and segregation of STIM1 from IP(3) receptors[END_REF], but in the area populated with IP 3 Rs [START_REF] Lee | Polarized expression of Ca2+ channels in pancreatic and salivary gland cells. Correlation with initiation and propagation of [Ca2+]i waves[END_REF][START_REF] Nathanson | Localization of the type 3 inositol 1,4,5-trisphosphate receptor in the Ca2+ wave trigger zone of pancreatic acinar cells[END_REF][START_REF] Yule | Evidence that zymogen granules are not a physiologically relevant calcium pool. Defining the distribution of inositol 1,4,5-trisphosphate receptors in pancreatic acinar cells[END_REF]. This surprising finding led us to examine the relative positioning of the two proteins, which were found to be closely co-localised. In the second part of the study we probed the functional consequences of this co-localisation.

Materials and Methods.

Chemicals: All salts as well as Acetylcholine (ACh), goat serum, BSA and PBS were obtained from SIGMA (Gillingham, UK). Collagenase was from Worthington (Lorne Laboratories, Reading, UK). Thapsigargin and caffeine were from Calbiochem (San Diego, CA, USA). Protease inhibitor cocktail was from Roche Diagnostics (Mannheim, Germany). Protein G sepharose beads were from GE Healthcare (Uppsala, Sweden). Clean Blot reagent was from Pierce (Rockford, IL, USA). Fura-2 AM and Fluo-4 AM were from Invitrogen (Paisley, UK).

Animals and cell isolation. All animal experiments were conducted in accordance with the Animals (Scientific Procedure) Act of 1986. PAC were isolated from the pancreata of CD1 or BL6 (wild type or specified knockout) mice using collagenase digestion as described previously [START_REF] Tepikin | Pulsatile Ca2+ extrusion from single pancreatic acinar cells during receptor-activated cytosolic Ca2+ spiking[END_REF].

Immunofluorescence. Freshly isolated PAC were fixed in methanol for 10 minutes at -20°C. Nonspecific antibody binding was blocked for 1 hour in 10% goat serum and 1% M. Furuse from Kobe University). Appropriate secondary antibodies conjugated to Alexa 488, Alexa 594 and/or Alexa 647 (Invitrogen, Paisley, UK) were applied for 30 minutes and cover slips were mounted on microscope slides with Prolong Gold (Invitrogen, Paisley, UK). All fluorescent secondary antibodies, used in the study, were tested on PAC fixed with the same method but without the application of a primary antibody. None of these secondary antibodies produced any nonspecific staining in PAC. Cells were viewed on a Leica TCS SP2 AOBS inverted confocal microscope (Leica Microsystems, Mannheim, Germany) equipped with a ×63 oil immersion objective (N.A. 1.4). Optical sections were spaced by 0.5-1μm. Linear adjustments of contrast and brightness were applied if necessary in Leica Application Suite.

Ca 2+ imaging. Freshly isolated PAC were loaded with 2.5μM Fura-2 AM or 2.5μM Fluo-4 AM for 30 minutes at room temperature. Fluo-4 labelling was used for experiments involving caffeine. Standard Na-HEPES based extracellular solution contained (in mM): NaCl: 140; KCl: 4.7; Hepes: 10; MgCl 2 : 1; glucose: 10; CaCl 2 : 1; pH 7.4. In specific experiments the Ca 2+ concentration in this solution was modified (i.e. reduced to nominally Ca 2+ free or increased to 2mM). For Fura-2 imaging we utilised Till Photonics Imaging System (TILL Photonics GmbH, Germany) or a BSI Olympus Collaboration Centre Imaging System (RIKEN, Saitama, Japan). Fura-2 was excited at 340nm and 380nm, emission was collected using 510nm high pass filter. Experiments with Fluo-4 loaded cells were conducted on the Till Photonics imaging system. Fluo-4 labelling was used for experiments involving caffeine because of the strong effect of caffeine on the fluorescence of Fura-2. Fluo-4 was excited by at 470nm and emission was collected through a 510nm high pass filter.

Co-immunoprecipitation and Western blotting: PAC were lysed in IP buffer containing 50mM Tris, 150mM NaCl, 1% Triton X-100, 0.25% sodium-deoxy-cholate, 0.2% sodium-dodecil-sulphate, 2mM EDTA, 2x protease inhibitor cocktail. In each condition lysate containing 600μg protein was added to 2μg of IP 3 receptor antibody (described above) and mixed with 20μl protein G sepharose beads in a total volume of 1ml of IP buffer for 2 hours at 4°C. Proteins were eluted, separated on a 4-12% Tris-Glycine gradient gel and transferred onto nitrocellulose membranes (VWR). Following blocking in 5% milk for 1 hour, membranes were probed with Orai1 antibodies (Alomone Labs, Israel) and IP 3 receptor antibodies or actin antibodies (SIGMA, Gillingham, UK). Following staining with Clean Blot, bands were visualized using ECL western blotting substrate in a BioRad gel documentation system. Bands were quantified using ImageJ gel quantification plug-in.

Orai1 distribution in pancreatic acinar cells lacking IP 3 receptors.

To find out if the IP 3 Rs are required for the apical positioning of Orai1, we imaged the distribution of Orai1 in PAC from IP 3 R2 knockout (KO) mice, from IP 3 R2/3 double KO mice [START_REF] Futatsugi | IP3 receptor types 2 and 3 mediate exocrine secretion underlying energy metabolism[END_REF] and from IP 3 R1 KO mice [START_REF] Matsumoto | Ataxia and epileptic seizures in mice lacking type 1 inositol 1,4,5-trisphosphate receptor[END_REF] produced in K. Mikoshiba`s laboratory. The apical membrane region of PAC is enriched with occludin and zona occludens 1 (ZO1) (S Fig 3). As the apical pole cannot be visualized using IP 3 R antibodies in the cells from the double KO animals, we used tight junction markers -occludin antibodies and/or ZO1 antibodies -to reveal the apical regions in clusters of acinar cells (both antibodies give very similar staining (S In these experiments thapsigargin (TG) was used to deplete Ca 2+ stores in cells maintained in nominally Ca 2+ -free extracellular solution. Addition of Ca 2+ to the extracellular solution resulted in SOCE-mediated increase in cytosolic Ca 2+ ([Ca 2+ ]c) followed by formation of an elevated [Ca 2+ ]c plateau (Fig 4). Addition of IP 3 -producing secretagogue ACh reversibly decreased the amplitude of the plateau (Fig 4A ,n=465). Using the Mn quench technique [START_REF] Barrow | ATP depletion inhibits Ca2+ release, influx and extrusion in pancreatic acinar cells but not pathological Ca2+ responses induced by bile[END_REF] we also observed a small (13 ± 2 %) but statistically significant inhibition of the influx by 300nM ACh (S Fig 5, n=145 cells in ACh -treated group and n=139 cells in control group). Caffeine (10mM), which in PAC very efficiently blocks IP 3 -induced Ca 2+ responses [START_REF] Toescu | Caffeine inhibits the agonist-evoked cytosolic Ca2+ signal in mouse pancreatic acinar cells by blocking inositol trisphosphate production[END_REF] has no effect on its own (Fig 4B, n= 169), but it partially reversed the ACh-induced reduction of the plateau (Fig 4C ,n=168). These experiments suggest that activation of IP 3 Rs has a mild inhibitory rather than stimulatory action on SOCE. Caffeine efficiently quenches fura-2, because of this property the experiments described above were conducted using single wavelength indicator fluo-4. We further tested the effects of ACh on TG-induced [Ca 2+ ]c plateau using the ratiometric probe fura-2. The results were similar to that observed using fluo-4 i.e. 300nM ACh (but not 50nM ACh) reversibly decreased TG-induced [Ca 2+ ]c plateau (Fig 5A andB, n= 61 for 50nM ACh and n=62 for 300nM ACh). Similar results were found in experiments on PAC from IP 3 R2 KO mice (Fig 5C andD, n=47 for 50nM ACh and n= 33 for 300nM ACh) and from IP 3 R2/3 double KO mice (Fig 5E andF, n= 46 for 50nM ACh and n= 31 for 300nM ACh) although the recovery phase (on removal of ACh) was less clear in experiments on KO and double KO mice. To assess SOCE more directly and provide an internal control for each experiment we used a two pulse protocol, where cellular Ca 2+ stores were depleted using TG in nominally Ca 2+ -free external solution and then two short pulses of extracellular calcium (2mM) were applied (Fig 6A). The first pulse (after the TG-induced Ca 2+ store depletion) was applied in agonist-free extracellular medium, whilst the second pulse was applied in the presence of ACh (Fig 6A ) or in agonist-free extracellular medium (control, not shown). The changes in the Fura-2 340nm/380nm ratio were differentiated (the procedure is illustrated on the insert in Fig 6A ) and the maximal rate determined. Considering the relatively slow Ca 2+ extrusion by PMCA at or near the resting [Ca 2+ ]c [START_REF] Tepikin | Pulsatile Ca2+ extrusion from single pancreatic acinar cells during receptor-activated cytosolic Ca2+ spiking[END_REF] and taking into account that the maximal rates of changes were also observed at close to resting [Ca 2+ ]c, we can assume that the maximal derivative reflects the maximal SOCE rate. The maximal SOCE rate during the second pulse was normalized to that recorded during the first pulse, in order to provide an internal control for every cell in each experiment (Fig 6A andB

Discussion

PAC structurally satisfy the requirements for IP 3 R and SOC channel interaction (see Fig 1) suggested as the basis for the original conformational-coupling theory of SOCE [START_REF] Berridge | Capacitative calcium entry[END_REF][START_REF] Irvine | Quantal' Ca2+ release and the control of Ca2+ entry by inositol phosphates--a possible mechanism[END_REF]. We were not able to detect SOCE up-regulation in response to stimulation with the IP 3 -producing agonist ACh. The inhibition of IP 3 Rs with caffeine also had no affect on SOCE. In these respects the study yielded important negative results. This finding is in line with conclusions from the study by Woodard and colleagues [START_REF] Woodard | TRPC3 regulates agonist-stimulated Ca2+ mobilization by mediating the interaction between type I inositol 1,4,5-trisphosphate receptor, RACK1, and Orai1[END_REF], which highlighted the importance of the interaction between IP 3 Rs and Orai1 for different Ca 2+ signalling processes, but indicated that the disruption of the interaction between IP 3 R1 and Orai1 does not prevent TG-induced SOCE and has no significant effect on this process.

The ACh application experiments suggest that IP 3 Rs could negatively regulate SOCE. This negative modulation could offer some protection against Ca 2+ overload induced by IP 3 -producing secretagogues. The effect of ACh is however moderate and SOCE develops efficiently in the acinar cells lacking both functional types of IP 3 Rs.

In our study the highest density of Orai1 was observed in the apical part of the cell containing IP 3 Rs, occludin and ZO-1. Importantly, we also found Orai1 along the lateral and basal membranes -far beyond the region containing tight junctions and IP 3 Rs (Fig 1, 2, 3 and Movie S1). In this basolateral region, Orai1 was shown to co-localise with STIM1 and form Orai1 and STIM1 puncta following ER store depletion [START_REF] Lur | Ribosome-free terminals of rough ER allow formation of STIM1 puncta and segregation of STIM1 from IP(3) receptors[END_REF]. It is possible that two mechanisms of SOCE operate in PAC; baso-lateral SOCE mediated by STIM1 and Orai1 proteins will have a reliable Ca 2+ source delivered by the interstitial fluid, whilst apical SOCE could help to re-capture Ca 2+ transported paracellularly [START_REF] Jansen | Role of calcium in exocrine pancreatic secreation. VI. Characteristics of the paracellular pathway for divalent cations[END_REF], extruded by PMCA (which are active in the apical region [START_REF] Belan | Localization of Ca2+ extrusion sites in pancreatic acinar cells[END_REF][START_REF] Lee | Polarized expression of Ca2+ pumps in pancreatic and salivary gland cells. Role in initiation and propagation of [Ca2+]i waves[END_REF]) and exocytosed with the content of secretory granules. It is conceivable that apical Orai1 could play a role in preventing the build-up of Ca 2+ in pancreatic ducts, and consequently, pancreatic stone formation in these structures. The close co-positioning of IP 3 Rs and Orai1 in the apical region suggests that if activated the apical Orai1 could efficiently re-load strategically important (IP 3 R containing) Ca 2+ stores. The ability of the apical Orai1 to participate in SOCE, the putative mechanism of activation and the physiological function of the apical Orai1 will need to be determined in a separate study. While this paper was in revision a study was published by J. Hong and colleagues [START_REF] Hong | Polarized but Differential Localization and Recruitment of STIM1, Orai1 and TRPC Channels in Secretory Cells[END_REF] indicating that Orai1 is localised in the apical part of acinar cells were it co-localised with IP 3 R3. The important difference between this study and our paper is that we observed a substantial presence of Orai1 in the plasma membrane regions outside the apical pole. Indeed our confocal images ( [START_REF] Lur | Ribosome-free terminals of rough ER allow formation of STIM1 puncta and segregation of STIM1 from IP(3) receptors[END_REF]. We therefore consider that the apical function of Orai1 is likely to be STIM1-independent. It is also important to note that the previous electron microscopy investigation of the sub-plasmalemmal ER and the plasma membrane in pancreatic acinar cells, revealed ribosome-free rough ER junctions decorated with STIM1 in basal and lateral sub-plasmalemmal regions, but not in the apical pole of the cell [START_REF] Lur | Ribosome-free terminals of rough ER allow formation of STIM1 puncta and segregation of STIM1 from IP(3) receptors[END_REF]. It is therefore difficult to reconcile some of the conclusions (specifically the role of STIM1 in activating apical Orai1 channels) of Hong's paper and our study.

The current work revealed close positioning of IP 3 Rs and Orai1 channels in the apical pole of the PAC , documented the presence of Orai1 in the apical region of the cells lacking functional IP 3 Rs and concluded that, in spite of the remarkably close localisation of the two proteins, IP 3 Rs do not activate SOCE in the apical region of PAC.

Figure 1. Orai1 co-localisation and co-immunoprecipitation with IP 3 receptors in pancreatic acinar cells. (A)

Maximum projection of 20 optical sections spaced 1μm from each other in a PAC cluster. Orai1 (green) is present in both the basolateral (arrows) and apical (arrowheads) regions. In the apical pole Orai1 co-localises with IP 3 R3 (magenta). Inserts show a single confocal section from the same cluster in higher magnification (the arrow and arrowhead points to the same structures as in the main figure). Here and on (B) and (C) the scale bars correspond to 10μm in the projections and 5μm in the inserts. (B) Maximum projection of optical sections of a PAC cluster. IP 3 R2 (cyan) co-localises with IP 3 R3 (magenta) in the apical pole of the cells (arrowhead). Inserts display a single confocal section from the same cluster in higher magnification (the arrowhead points to the same structures as in the main figure). (C) Maximum projection of optical sections of a PAC cluster. The highest density of IP 3 R1 (yellow) is observed in the apical pole of the cells (arrowheads) where it co-localises with IP 3 R3 (magenta). Inserts display a single confocal section from the same cluster in higher magnification (the arrowhead points to the same structures as in the main figure). Note that a significant staining for IP 3 R1(unlike that for IP 3 R2 and IP 3 R3) was also found outside the apical region of the cell. (D) Co-immunoprecipitation of Orai1 with IP 3 R1 (left panel), IP 3 R2 (middle panel) (A) Confocal section from a cluster of PAC recorded 5μm from the cover slip. This section is below the apical regions of the cells and Orai1 (green) is clearly visible in the basal and lateral membranes (arrows). (B) Confocal section from the same cluster as in (A) but recorded 14μm from the top of the cover slip where IP 3 R3s (magenta) decorate the apical surfaces of the cells. Apical Orai1 present in this section (arrowhead) colocalises with the IP 3 R3s. (C) Confocal section of the same acinar cell cluster as in (A) and (B). Confocal section was positioned 23μm from the cover slip. IP 3 Rs are no longer visible as this section is above the apical regions of the cells; however Orai1 is still present in the lateral and basal membranes (arrows). Scale bars correspond to 5μm. (A) Intracellular Ca 2+ stores of Fura-2 -loaded PAC, isolated from wild type mice, were depleted during a 30 min preincubation in nominally Ca 2+ free extracellular solution containing 2µM thapsigargin (TG). The addition of 2mM Ca 2+ to the extracellular solution resulted in the formation of a [Ca 2+ ]c plateau. Subsequent ACh application triggered a small drop of the plateau that could be reversed by removal of ACh. (B) Bar graph shows the normalised (to the plateau level before ACh addition) plateau amplitude before (white bars) and following (striped bars) treatment with 50nM or 300nM ACh. Only the application of a large concentration of ACh (300nM) caused a significant reduction of the plateau amplitude in PAC from wild type mice (paired T-test, * indicates that p<0.05). (C) Intracellular Ca 2+ stores of Fura-2 loaded PAC, isolated from IP 3 R2 KO mice, were depleted with 2µM TG in nominally Ca 2+ free extracellular solution and subsequent addition of 2mM Ca 2+ resulted in the formation of a [Ca 2+ ]c plateau. Application of a large concentration of ACh resulted in a small decrease of the plateau. (D) Bar graph shows the normalised plateau amplitude, recorded from PAC from IP 3 R2 KO mice, before (white bars) and following (striped bars) treatment with 50nM or 300nM ACh. Only the application of a 300nM ACh caused a significant reduction of plateau amplitude in PAC from IP 3 R2 KO mice (paired T-test, * indicates that p<0.05). (E) Intracellular Ca 2+ stores of Fura-2 loaded PAC, isolated from IP 3 R2/3 KO mice, were depleted as described above and cytosolic Ca 2+ plateau was formed by introducing 2mM Ca 2+ to the extracellular solution. Subsequent addition of ACh resulted in a decrease of cytosolic Ca 2+ level. (F) Bar graph shows the normalised plateau amplitude, recorded from PAC from IP 3 R2/3 KO mice before (white bars) and following (striped bars) treatment with 50nM or 300nM ACh. Only the application of a 300nM ACh caused a small but statistically significant reduction of plateau amplitude (paired T-test, * indicates that p<0.05).

Biochemical

  Journal Immediate Publication. Published on 03 Mar 2011 as manuscript BJ20110083 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110083 A c c e p t e d M a n u s c r i p t 4 BSA prior to incubation with primary antibodies for 1 hour. IP 3 receptors were visualized by IP 3 R3 antibodies (BD Transduction Laboratories, Franklin Lakes, NJ, USA) or IP 3 R2 antibodies (rabbit polyclonal, raised against the C-terminal amino acids 2686-2702; a gift from Professor D. Yule, University of Rochester, USA) or IP 3 R1 antibodies (rabbit polyclonal, raised against C-terminal amino acids 2735-2749 of mouse IP 3 R1; a gift from Professor J. Parys from the University of Leuven). Orai1 channels were stained with an Orai1 antibody (rabbit polyclonal, raised against C terminal amino acids 278-294, produced by Dr. S. Feske) and tight junctions were visualized by occludin antibodies (Zymed Laboratories, Invitrogen, Carlsbad, CA, USA) or ZO1 antibodies (a gift from Dr.

Biochemical

  Journal Immediate Publication. Published on 03 Mar 2011 as manuscript BJ20110083 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110083 A c c e p t e d M a n u s c r i p t Results Orai1 co-localises with IP 3 Rs in the apical pole of pancreatic acinar cells. In the apical pole of PAC we found a striking co-localisation of endogenous Orai1 and IP 3 R3 (Fig 1A, n=7). IP 3 R2 and IP 3 R3 are also co-localised in this cellular region (Fig 1B, n=6). The highest density of IP 3 R1 was observed in the apical region where it co-localised with IP 3 R3 (Fig 1C, n= 6). We can therefore conclude that Orai1 closely colocalises with all types of IP 3 Rs in the apical pole of PAC. The apical localisation of Orai1 and its co-localisation with IP 3 R3 did not change in conditions when the cells were treated with the IP 3 -generating secretagogue ACh (S Fig 1A, n=3), the IP 3 R inhibitor caffeine (S Fig 1B, n=3), or the SERCA pump inhibitor thapsigargin (S Fig 1C, n=3). Orai1 co-immunoprecipitated with all types of IP 3 Rs (Fig 1D. n=6 for Orai1 and IP 3 R1; n=7 for Orai1 and IP 3 R2; n=16 for Orai1 and IP 3 R3). This co-immunoprecipitation also did not change significantly when the cells were treated with ACh, (Fig 1D. n=6 for Orai1 and IP 3 R1; n=7 for Orai1 and IP 3 R2; n=14 for Orai1 and IP 3 R3), caffeine (Fig 1D. n=3 for Orai1 and IP 3 R1; n=5 for Orai1 and IP 3 R2; n=4 for Orai1 and IP 3 R3;), or thapsigargin (TG, Fig 1D. n=3 for Orai1 and IP 3 R1; n=5 for Orai1 and IP 3 R2; n=4 for Orai1 and IP 3 R3). Importantly actin (which is present at high density in the apical region) was not co-immunoprecipitated with IP 3 receptors (S Fig 2, n=4). The observed coimmunopreciptation of Orai1 and IP 3 Rs does not of course guarantee that there is a direct interaction between these proteins but it is unlikely that the co-immunoprecipitation is due to the interaction of the proteins with actin. It is essential to note that unlike IP 3 Rs, Orai1 was observed not only in the apical region, but also on the basal and lateral membranes (shown by arrows on Fig 1A). This was particularly evident in the optical sections that were recorded below (Fig 2A section at +5µm) or above (see Fig 2C section at +23µm) the apical region of the cells. Orai1 was clearly present outside the apical region, but the intensity of its immunostaining increased substantially in the apical region decorated with IP 3 Rs (see Fig 2B section at +14µm) and in this region Orai1 was always found in the close vicinity of IP 3 Rs (Fig 1, Fig 2B section at +14µm and S Fig 1).

Fig 3 )

 3 and were co-localised with IP 3 Rs in the cells from the wild type animals (S Fig 4)). In the wild type animals Orai1 was mainly found co-localised with these proteins in the apical region, but was also present in the lateral and basal membranes (Fig3A andS Movie 1). The distribution of Orai1 in PAC of KO animals was similar to that observed in cells from the wild type animals (Fig 3). Prominent apical Biochemical Journal Immediate Publication. Published on 03 Mar 2011 as manuscript BJ20110083 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110083 A c c e p t e d M a n u s c r i p t Orai1 staining was present in cells lacking IP 3 R2 or both IP 3 R2 and IP 3 R3 or IP 3 R1 (Fig 3B-D; n=5 for both IP 3 R2 KO mice and for IP 3 R2/3 double KO mice, n=3 for IP 3 R1 KO mice). The basolateral presence of Orai1 was also unchanged in the cells from single and double IP 3 Rs KO animals (Fig 3). These experiments suggest that IP 3 Rs are not required for the targeting of Orai1 to basolateral or apical membrane regions of the PAC. Effects of the IP 3 -generating secretagogue ACh and the IP 3 R inhibitor caffeine on SOCE in PAC from the wild type and IP 3 Rs KO mice.

  ). In wild type cells, a low concentration of ACh (50nM) slightly reduced the SOCE rate (by 10±3%, n=61; Fig 6B), whilst the treatment with 300nM ACh resulted in a reduction in the SOCE rate of 27±4% (n=62, Fig 6B). In similar experiments (S Fig 6) we tested the effect of caffeine on SOCE rate. Caffeine (10mM) alone did not affect SOCE rate (S Fig 6, n=48). Biochemical Journal Immediate Publication. Published on 03 Mar 2011 as manuscript BJ20110083 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110083 A c c e p t e d M a n u s c r i p tThe ability of ACh to inhibit SOCE was further tested using the two pulse protocol on the PAC from IP 3 R2 single KO and IP 3 R2/3 double KO mice (Fig 6C-F). These experiments showed that both concentrations of ACh induced small, but statistically significant, reductions in the SOCE rate in PAC from IP 3 R2 KO mice (Fig 6Cand 6D). Both concentrations reduced SOCE rate by 20±2%. These results were qualitatively similar to those obtained in cells from wild type animals. The Ca 2+ responses in cells from the IP 3 R2/3 double KO mice were different -the response to TG was drastically reduced in comparison with that in the wild type and the single KO mice (Fig 6E compare with 6A and 6C). This suggests that IP 3 Rs amplify TG response by Ca 2+ -induced Ca 2+ release and that this amplification mechanism is absent in IP 3 R2/3 double KO mice. The responses to the external Ca 2+ pulses in PAC from the double KO mice were, however, surprisingly robust (Fig6E). In fact the maximal SOCE rate during the first pulse was slightly higher in the cells from the double KO mice (26% higher) than the single KO mice. Using the two pulse protocol we have not observed changes in the SOCE rate upon application of 50nM or 300nM ACh in the cells from double KO animals (Fig 6F, n=67 for 50nM and n=76 for 300nM). The SOCE rate during the second pulse was however already reduced by approximately 20% in comparison with the first pulse even in control (no ACh) conditions (Fig 6F). It is therefore possible that we do not observe ACh-induced SOCE suppression in these experiments because the ACh simply cannot inhibit the Ca 2+ influx any further.

Biochemical

  Journal Immediate Publication. Published on 03 Mar 2011 as manuscript BJ20110083 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110083 A c c e p t e d M a n u s c r i p t

  Fig 1, S Fig 1, Fig 3, S Movie 1 and particularly Fig 2) clearly reveal substantial Orai1 staining outside the plasma membrane regions decorated with IP 3 Rs, occludin or ZO-1. Also contrary to conclusions from Hong's study (but see Fig 5A from Hong's paper), in our previous work we have not observed apical localisation of STIM1 in cells with depleted ER Ca 2+ stores

Biochemical

  Journal Immediate Publication. Published on 03 Mar 2011 as manuscript BJ20110083 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110083 A c c e p t e d M a n u s c r i p t or with IP 3 R3 (right panel) in PAC lysates. The first lane in both panels corresponds to beads that were not bound to anti-IP 3 R antibodies. Western blots show the IP 3 Rs and Orai1 eluted from sepharose beads decorated with the corresponding IP3Rs. Bar graphs show the quantification of Western blots.

Figure 2 .

 2 Figure 2. Orai1 only co-localises with IP 3 receptors in the apical region of the acinar cells: apical and basolateral Orai1.

Figure 3 .

 3 Figure 3. Orai1 is present in the apical pole of acinar cells lacking IP 3 receptors. (A) Confocal section of an acinar cell cluster isolated from wild type mouse. Orai1 (green) is visible in basolateral membranes (arrows) and in the apical pole (arrowheads) where it co-localises with tight junctions (red) marking the apical membrane. (B) Confocal section of a cluster of acinar cells isolated from IP 3 R2 KO mice. Orai1 is apparent in both apical (arrowhead) and basolateral membranes (arrows), its distribution is similar to that in the cells from wild type animals. (C) Confocal section of an acinar cell cluster isolated from mice lacking both IP 3 R2 and IP 3 R3 (IP 3 R2/3 KO). Orai1 is present in basolateral membranes (arrow) as well as in the apical pole (arrowhead) where it co-localises with tight junction markers. (D) Confocal section of an acinar cell cluster isolated from IP 3 R1 KO mice. Orai1 staining is visible in the apical pole (arrowhead) and in basolateral membranes (arrow). Scale bars represent 5μm.

Figure 4 .

 4 Figure 4. Effect of ACh and caffeine on the cytosolic Ca 2+ plateau in pancreatic acinar cells. (A) TG (2μM) induced depletion of the ER Ca 2+ stores in cells placed in the nominally Ca 2+ free extracellular solution. Subsequent addition of 2mM Ca 2+ to the extracellular solution resulted in the increase of [Ca 2+ ]c. ACh application (following the formation of the plateau) caused a decrease in the level of cytosolic Ca 2+ . Removal of ACh resulted in partial restoration of the amplitude of [Ca 2+ ]c. (B) Application of caffeine (10mM) did not affect the [Ca 2+ ]c plateau. Bar graph (see insert) shows the quantification of the amplitude of the [Ca 2+ ]c plateau before and after addition of caffeine. (C) Caffeine treatment partially reverses the effect of ACh on [Ca 2+ ]c plateau. (D) Bar graph shows the quantification of the results illustrated on panels A-C. The amplitude of the [Ca 2+ ]c plateau was measured before treatment (white bar), following the addition of ACh (striped bar), following the removal of ACh from the extracellular solution (cross-hatched

Figure 5 .

 5 Figure 5. Effect of ACh on the cytosolic Ca 2+ plateau in acinar cells isolated from wild type and IP 3 R KO mice.

Figure 6 . 1 Biochemical 2 BiochemicalFigure 4 Biochemical

 6124 Figure6. Effects of ACh on the rate of SOCE in pancreatic acinar cells isolated from wild type and IP 3 R KO mice. (A) Fura-2 ratio changes upon TG treatment and pulses of external Ca 2+ in the absence and presence of 300nM ACh in PAC from the wild type animals. To assess the rate of Ca 2+ influx the rising phase (induced by a short pulse of 2mM Ca 2+ ) of the curve was differentiated and the maximum rate of influx was estimated as the maximal value of the derivative (see insert). The same procedure was repeated for the second pulse of external Ca 2+ (delivered in the presence or absence of ACh). (B) Summary of the effects of 50nM ACh and 300nM ACh on SOCE rate in the cells from wild type animals. Here, as well as in (D) and (F), the statistical significance was probed by ANOVA test; symbol * indicates p<0.05. (C) Fura-2 ratio changes upon TG treatment and pulses of external Ca 2+ in the absence and presence of 300nM ACh in PAC from IP 3 R2 KO mice. (D) Summary of the effects of 50nM and 300nM ACh on SOCE rate in the cells from IP 3 R2 KO mice. (E) Fura-2 ratio changes upon TG treatment and pulses of external Ca 2+ in the absence and presence of 300nM ACh in PAC from

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2011 The Authors Journal compilation © 2011 Portland Press Limited

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

Acknowledgements

The support from RIKEN BSI-Olympus Collaboration Centre is gratefully acknowledged. Authors declare no conflict of interest, except S.F. who is a scientific cofounder of CalciMedica.

Funding

The work was supported by Medical Research Council (UK) grants G0700167 and G19/22, Wellcome Trust grant 080906/Z/06/Z, National Institute for Health Research (UK) grant to the Liverpool NIHR Pancreas Biomedical Research Unit. K. M. was supported by Japan Science and Technology Agency (Japan) grant (Ca 2+ oscillations project). S. F. was supported by National Institute of Health (US) grant AI066128. M.S. is a RIKEN Foreign Postdoctoral Research Fellow.


Keywords: G proteins, dimeric GTPases, GDI function, protein translocation, substrate-based regulation Biochemical

GTPases act as molecular switches to control many cellular processes, including signaling, protein translation and targeting. Switch activity can be regulated by external effector proteins or intrinsic properties, such as dimerization. The recognition and translocation of pre-proteins into chloroplasts (via the Translocator of the outer/inner envelope membrane of chloroplasts; TOC/TIC) is controlled by two homologous receptor GTPases, Toc33 and Toc159, whose reversible dimerization is proposed to regulate translocation of incoming proteins in a GTP dependent manner. Toc33 is a homodimerizing GTPase. Functional analysis suggests that homodimerization is a key step in the translocation process, the molecular functions of which as well as the elements regulating this event are largely unknown. Here, we show that homodimerization reduces the rate of nucleotide exchange, which is consistent with the observed orientation of the monomers in the crystal structure. Pre-protein binding induces a dissociation of the Toc33 homodimer and results in the exchange of GDP for GTP. Thus, homodimerization does not serve to activate the GTPase activity as manifold discussed but to control the nucleotide loading state. We discuss this novel regulatory mode and its impact on the current models of chloroplasts protein import.

INTRODUCTION

In eukaryotic cells, protein targeting and translocation are highly ordered and regulated processes. The targeting of proteins to the appropriate membrane is defined by specific signals within their amino acid sequence. Sequential transfer of an incoming protein across a membrane is achieved by transient protein-protein interactions within the multicomponent translocation machineries (translocons). The initial steps of protein translocation across the membrane of the endoplasmic reticulum, the prokaryotic plasma membrane and the outer chloroplast membrane are regulated by dimeric GTPases [Fig. 1; e.g. [START_REF] Schnell | Protein translocons: multifunctional mediators of protein translocation across membranes[END_REF][START_REF] Wickner | Protein translocation across biological membranes[END_REF]. In the last decade, functional and structural analyses have significantly advanced our understanding of the GTPases controlling the targeting of proteins to the endoplasmic reticulum or the bacterial plasma membrane [START_REF] Egea | Targeting proteins to membranes: structure of the signal recognition particle[END_REF][START_REF] Rapoport | Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes[END_REF]. However the transfer of precursor proteins across the chloroplasts membranes is not yet entirely understood [START_REF] Becker | Preprotein recognition by the Toc complex[END_REF]. It is widely accepted that the recognition of chloroplast precursor proteins with a typical N-terminal extension, called transit peptide, is performed by two GTP-dependent receptors, namely Toc159 and Toc33 [START_REF] Oreb | Policing Tic 'n' Toc, the doorway to chloroplasts[END_REF][START_REF] Kessler | Chloroplast protein import: solve the GTPase riddle for entry[END_REF]. These are thought to cooperatively regulate the insertion of the precursor protein into Toc75, the translocation pore of the outer envelope [START_REF] Jarvis | Targeting of nucleus-encoded proteins to chloroplasts in plants[END_REF][START_REF] Sommer | Molecular interactions within the plant TOC complex[END_REF]. However, the individual role of both receptors as well as the sequence of events during the translocation process is not well understood.

The crystal structures of the GTPase (short G) domains of Toc33 from Arabidopsis thaliana (atToc33) and its orthologue Toc34 from Pisum sativum (psToc34) revealed a homodimeric interaction [START_REF] Koenig | The GTPase cycle of the chloroplast import receptors Toc33/Toc34: implications from monomeric and dimeric structures[END_REF][START_REF] Sun | Crystal structure of pea Toc34, a novel GTPase of the chloroplast protein translocon[END_REF]. Indeed, homodimerization of Toc33 at the chloroplast membrane was demonstrated by copper induced crosslinking [START_REF] Lee | Toc receptor dimerization participates in the initiation of membrane translocation during protein import into chloroplasts[END_REF]. Moreover, mutations reducing the affinity of homodimerization affect the import efficiency in A. thaliana plants [START_REF] Aronsson | Nucleotide binding and dimerization at the chloroplast pre-protein import receptor, atToc33, are not essential in vivo but do increase import efficiency[END_REF]. This suggests that Toc33 is a GTPase regulated by dimerization [START_REF] Gasper | It takes two to tango: regulation of G proteins by dimerization[END_REF]. The structural observations led to the proposal that GTP-regulated dimerization constitutes an element of the GTPase regulation within the translocon. Alternatively, it was proposed that the Toc33 homodimer mimics a heterodimeric GTPase complex between Toc33 and the highly homologous G domain of Toc159, a complex thought to be vital for translocation. Thus, the precise role of the Toc33 dimerization in the translocation reaction has not yet been defined.

One potential role of dimerization is to provide a reciprocal GAP function either in the homo-or the heterodimeric context. This hypothesis was based on the geometry of the active site, as a conserved arginine residue from one protomer was inserted into the active site of the other at a position near the β-phosphate of GDP. The positioning of this arginine residue is reminiscent of the interaction seen in GTPase/GAP complexes [e.g. Ras-like GTPases; 15]. However, experimental evidence indicates that the hydrolysis rate of GTP does not increase significantly at concentrations favoring the homodimeric state [START_REF] Koenig | On the significance of Toc-GTPase homodimers[END_REF][START_REF] Reddick | In vitro comparative kinetic analysis of the chloroplast Toc GTPases[END_REF]. It was speculated that a third molecule might be required as a "coGAP" or, alternatively, only the Toc33-Toc159 heterodimer might have the function of a mutual GAP complex [START_REF] Jarvis | Targeting of nucleus-encoded proteins to chloroplasts in plants[END_REF]. The larger G domain Toc159 receptor possesses an arginine residue at a similar position as Toc33 and additional insertions that may harbor the further needed catalytic amino acids [START_REF] Jarvis | Targeting of nucleus-encoded proteins to chloroplasts in plants[END_REF]. This concept would be consistent with the notion that both receptors preferably interact in the presence of GTP and precursor proteins [START_REF] Becker | Preprotein recognition by the Toc complex[END_REF].

Independent of a putative GAP function, Toc33 forms homodimers in the GDP loaded state in vitro [START_REF] Koenig | On the significance of Toc-GTPase homodimers[END_REF] (see below). Furthermore, a stable interaction between Toc33 and a precursor protein in the context of the TOC complex could be detected by labeltransfer experiments only in the presence of GDP, but not GTP [START_REF] Kouranov | Analysis of the interactions of preproteins with the import machinery over the course of protein import into chloroplasts[END_REF]. In turn, reports have described an influence of the precursor protein on GTP hydrolysis of Toc33 [START_REF] Reddick | In vitro comparative kinetic analysis of the chloroplast Toc GTPases[END_REF] A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. [START_REF] Jelic | Two Toc34 homologues with different properties[END_REF][START_REF] Schleiff | Structural and guanosine triphosphate/diphosphate requirements for transit peptide recognition by the cytosolic domain of the chloroplast outer envelope receptor[END_REF][START_REF] Sveshnikova | Toc34 is a preprotein receptor regulated by GTP and phosphorylation[END_REF][START_REF] Jelic | The chloroplast import receptor Toc34 functions as preprotein-regulated GTPase[END_REF], an observation which cannot be reconciled directly with the GDP dependence of the label transfer. This prompted us to investigate a possible physiological function of Toc33 homodimers with respect to nucleotide exchange and precursor protein recognition.

For the biochemical analyses in this study, Toc33 was expressed in E. coli as a truncated version (atToc33 C ), lacking the C-terminal membrane anchor. The truncated protein dimerizes in solution with low affinity [START_REF] Oreb | Phosphorylation regulates the assembly of chloroplast import machinery[END_REF], and thereby represents a suitable model to analyze dynamic interactions expected within the TOC complex. We demonstrate that the dissociation of GDP, and therefore nucleotide exchange is inhibited in the homodimeric state of atToc33 C . Dissociation of the homodimer is induced by the recognition of a precursor protein, which promotes binding of GTP. It was recently hypothesized that this might be required for a subsequent interaction of Toc33 with Toc159 [START_REF] Sommer | Molecular interactions within the plant TOC complex[END_REF]. Thus, the homodimeric conformation could reflect an inactive state of the translocon, preventing the initiation of GTP dependent downstream interactions that lead to pre-protein translocation across the outer membrane. By this we provide an example of GTPase regulation by dimerization which is distinct from the model presented for usual GADs (G proteins activated by nucleotide-dependent dimerization) [START_REF] Gasper | It takes two to tango: regulation of G proteins by dimerization[END_REF]. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

EXPERIMENTAL General

Protein expression and purification [START_REF] Jelic | Two Toc34 homologues with different properties[END_REF] was previously described. EDA-GTP-ATTO 550 (2'/3'-O-(2-Aminoethyl-carbamoyl)-Guanosine-5'-triphosphate, triethylammonium salt) and mantGDP (2'/3'-O-(N-Methyl-anthraniloyl)-guanosine-5'-diphosphate, triethylammonium salt) were obtained from Jena Bioscience (Jena, Germany). All calculations were preformed with SigmaPlot (SPSS Inc.). Peptides were synthesized in the department of Peptide and Protein Chemistry at the Charité, Berlin. The sequences used are: B1 (derived from Nicotiana tabacum pSSU) -MVAPFTGLKSAA-S(PO 3 )-FPVSRKQNLDITSC [START_REF] Schleiff | Structural and guanosine triphosphate/diphosphate requirements for transit peptide recognition by the cytosolic domain of the chloroplast outer envelope receptor[END_REF]; control peptide: C -SKSMTEIEVTDVDMPC. Analytical ultracentrifugation of psToc34 was previously described [START_REF] Koenig | The GTPase cycle of the chloroplast import receptors Toc33/Toc34: implications from monomeric and dimeric structures[END_REF].

Nucleotide exchange on TOC GTPases

One hundred µM recombinant atToc33 C or atToc33 C -G1m, containing the triple mutation [G45R/K49N/S50R, see 24], were incubated on ice for 60 min in the presence of 1 mM GDP in HMK buffer (50 mM Hepes-KOH, pH 7.5, 2 mM MgCl2, 40 mM KAc). Free nucleotides were depleted by gel filtration [START_REF] Chen | Insertion of the 34-kDa chloroplast protein import component, IAP34, into the chloroplast outer membrane is dependent on its intrinsic GTPbinding capacity[END_REF]. The protein was then incubated at room temperature for 30 min to ensure complete hydrolysis of residual protein-bound GTP. The reactions were cooled to 0 o C and incubated with 10 µM [α-32 P]GTP (150 mCi/µmol) in the presence or absence of 5 µM of urea-denatured (6M) recombinant pSSU. All samples contained a final urea concentration (<100 mM) to control for any effects of urea on intrinsic GTPase activity. A fraction of each reaction was blotted onto nitrocellulose membrane at the times indicated; the membrane was washed three times with 10 ml ice-cold HMK buffer containing 0.3% Tween-20 and subsequently air-dried. Bound [α-32 P]GTP was detected and quantified using a phosphor imager (Fuji Fla-5000) and Multi Gauge V2.02 software.

Fluorescence GTP binding measurements

Binding of EDA-GTP-ATTO-550 to atToc33 C was determined in 20 mM Tris/HCl pH 8.0, 100 mM NaCl and 1 mM MgCl 2 with the indicated protein concentrations. Indicated amounts of GTP were produced by mixing GTP and EDA-GTP-ATTO-550 in a ratio of 1mM to 300 µM. Fluorescence was excited by 554 nm and recorded at 576 nm. GTP binding by the monomeric species was analyzed by

= + * [ ] [E1] and = / [E2]
with kapparent as the apparent rate constant of GTP binding at a given GTP concentration, kdiss the dissociation rate of GTP, kass the association rate of GTP, [S] the GTP concentration and KD the equilibrium dissociation constant.

Analysis of dimerization by size exclusion chromatography

For molecular weight determination, 180 µl of purified protein at indicated concentrations and in the presence of indicated concentrations of peptide was loaded onto a Superdex75 HR 10/300 gel filtration column (GE-Healthcare), equilibrated with 50 mM Tris/HCl at pH 8, containing 100 mM NaCl, 10 mM imidazole, 10 mM arginine, 5% glycerol and 1 mM MgCl 2 . Monomeric and dimeric species were analyzed by fitting two independent Gaussian distributions to the elution profile as described before [START_REF] Koenig | The GTPase cycle of the chloroplast import receptors Toc33/Toc34: implications from monomeric and dimeric structures[END_REF][START_REF] Oreb | Phosphorylation regulates the assembly of chloroplast import machinery[END_REF].

Nucleotide dissociation measurements atToc33

C was bound to Ni-NTA agarose and incubated over night with two column volumes of 1 mM GDP supplemented with 30 µM mantGDP in 50 mM Tris/HCl, pH 8.0, 300 mM NaCl, 1 mM MgCl 2 , 10 mM imidazol, 5% (v/v) glycerol at 4 °C under constant agitation. The matrix was washed two times with the nucleotide free buffer before the protein was eluted with buffer supplemented with 500 mM imidazol. The dissociation rate of mantGDP from the protein was determined using Cary Eclipse fluorescence spectrometer (Varian) at 22 °C. Unlabeled nucleotides and peptides were first added to the cuvette and the baseline was recorded for 1 min.

RESULTS

Pre-protein binding promotes nucleotide exchange on atToc33 C

The dimer interface of the TOC receptors is formed by several G-loops involved in nucleotide binding and a number of specific sequence elements [START_REF] Koenig | The GTPase cycle of the chloroplast import receptors Toc33/Toc34: implications from monomeric and dimeric structures[END_REF][START_REF] Sun | Crystal structure of pea Toc34, a novel GTPase of the chloroplast protein translocon[END_REF]. Thus upon Toc33 dimerization, GDP is bound within a cage, with no readily apparent route for nucleotide exchange [START_REF] Koenig | The GTPase cycle of the chloroplast import receptors Toc33/Toc34: implications from monomeric and dimeric structures[END_REF][START_REF] Sun | Crystal structure of pea Toc34, a novel GTPase of the chloroplast protein translocon[END_REF][START_REF] Koenig | On the significance of Toc-GTPase homodimers[END_REF]. Although it is unknown to date whether this structure represents only one example of a dynamic ensemble of possible modes of interactions occurring in the context of the TOC complex, this observation posed the question of whether nucleotide binding could be regulated by dimerization. To reconcile the preprotein binding induced stimulation of GTPase activity of Toc33 [START_REF] Becker | Preprotein recognition by the Toc complex[END_REF][START_REF] Jelic | Two Toc34 homologues with different properties[END_REF][START_REF] Schleiff | Structural and guanosine triphosphate/diphosphate requirements for transit peptide recognition by the cytosolic domain of the chloroplast outer envelope receptor[END_REF][START_REF] Sveshnikova | Toc34 is a preprotein receptor regulated by GTP and phosphorylation[END_REF] with relevance to dimerization, we analyzed whether the pre-protein stimulates GTPase activity indirectly by dimer disruption, thereby promoting nucleotide exchange. As a first step to test this possibility, we examined the effects of pre-protein binding on GDP/GTP exchange of atToc33 C . We pre-incubated atToc33 C in buffer to promote hydrolysis of any residual bound GTP and subsequently with GDP to ensure that the maximum amount of protein was in its GDP-bound form. To assess nucleotide exchange, we incubated GDP-loaded atToc33 C with [α-32 P]GTP in the presence or absence of a urea-denatured precursor of the small subunit of Rubisco (pSSU), a well studied TOC model substrate. The reaction was performed at 0 °C to minimize [α-32 P]GTP hydrolysis, and exchange was quantified by measuring protein-bound [α-32 P]GTP at various time points.

Low levels of [α-32 P]GTP binding to atToc33 C were detected in the absence of pSSU (Figure 1). In the presence of pSSU, the rate of nucleotide exchange increased significantly. The levels of bound [α-32 P]GTP in the presence of pSSU were 6-fold higher than in the absence of pSSU at the latest time point assayed (60 min). The same holds true for the rate of GTP binding, which is 10 fold lower in the absence of pSSU as determined by least square fit analysis using a single exponential equation (Figure 1). [α-32 P]GTP binding to pSSU alone or to atToc33 C -G1m, a receptor that lacks nucleotide binding, were undetectable, indicating that [α-32 P]GTP binding was specific to native atToc33 C . Thus, the previously reported stimulation of Toc33 GTP hydrolysis after pre-protein binding might be a result of an accelerated GDP/GTP exchange rates or an increased GTP affinity [START_REF] Becker | Preprotein recognition by the Toc complex[END_REF][START_REF] Jelic | Two Toc34 homologues with different properties[END_REF][START_REF] Schleiff | Structural and guanosine triphosphate/diphosphate requirements for transit peptide recognition by the cytosolic domain of the chloroplast outer envelope receptor[END_REF][START_REF] Sveshnikova | Toc34 is a preprotein receptor regulated by GTP and phosphorylation[END_REF][START_REF] Weibel | Dimerization of Toc-GTPases at the chloroplast protein import machinery[END_REF]. We therefore went on to dissect the influence of dimerization and precursor binding on the nucleotide binding of the Toc33 GTPase.

GTPase dimerization reduces the rate of nucleotide exchange First, we determined the apparent rate constants for association of purified atToc33 C (Figure 2a) with fluorescent EDA-GTP-ATTO at two different protein concentrations: 4 µM, containing predominantly monomeric receptor, and 200 µM with estimated 40% of the protein in the homodimeric conformation according to the established KD [START_REF] Oreb | Phosphorylation regulates the assembly of chloroplast import machinery[END_REF].

The apparent rates (kapparent in equation [E1,2]) were determined by a least square fit analysis to a single exponential function for the reaction with 4 µM and two independent exponential functions for the reaction with 200 µM protein (Figure 2b). For the latter we assumed that the dimeric and monomeric species bind GTP independently from each other, although co-operativity of the association of the two GTP molecules within the dimer cannot be excluded at this stage. The residuals of the least square fit analysis justify the usage of a double exponential (Figure 2b, inset). The apparent rates of GTP binding for the monomeric (Figure 2c, white circles for the 200 µM protein and black circles for the 4 µM protein) and dimeric atToc33 C (grey circles for 200 µM protein) are linearly dependent on the concentration of the ligand. Analysis of the plots by equation [E1,E2] revealed an association rate constant (kass) of 4.9 M -1 s -1 for the monomers and 1.3 M -1 s -1 for the dimeric species. Thus, the monomeric species has an approximately four-fold higher association rate constant for EDA-GTP-ATTO than the dimeric species (Figure 2c). Considering that recombinantly expressed atToc33 C is almost exclusively GDP loaded [START_REF] Koenig | The GTPase cycle of the chloroplast import receptors Toc33/Toc34: implications from monomeric and dimeric structures[END_REF] and that GDP release from GTPases is thought to be the rate limiting step for nucleotide exchange [e.g. 26], the observed kinetics of EDA-GTP-ATTO binding is therefore most likely dependent on the velocity of GDP dissociation.

To quantify this effect, we investigated the influence of dimerization on GDP dissociation from purified atToc33 C (Figure 2a). The receptor was preloaded with mantGDP according to the protocol described in the experimental section. mantGDP dissociation was determined by the decay of fluorescence. The rates obtained in multiple measurements were plotted against the concentration of atToc33 C (Figure 3a, black circles). The highest mantGDP dissociation rate is found at the lowest receptor concentration and the rate is reduced at higher protein concentrations. This suggests a dimerization-dependent reduction of the GDP dissociation rate.

An opportunity to corroborate this observation was offered by the distinct physicochemical properties of psToc34 C , the functional orthologue of atToc33 C from pea (see above). Heterologously produced psToc34 C has an equilibrium dissociation constant for dimerization of about 50 µM, which is nearly 8 fold lower than observed for atToc33 C [START_REF] Koenig | The GTPase cycle of the chloroplast import receptors Toc33/Toc34: implications from monomeric and dimeric structures[END_REF][START_REF] Reddick | In vitro comparative kinetic analysis of the chloroplast Toc GTPases[END_REF][START_REF] Oreb | Phosphorylation regulates the assembly of chloroplast import machinery[END_REF]. The reason for the discrepancy between the two proteins from different sources is, however, not yet understood. Nevertheless, here it is an advantage because we can use this information to confirm the relation between GDP release and dimerization. When mantGDP dissociation from purified psToc34 C (Figure 2a) was measured (Figure 3a, grey circles), the rates were reduced by increasing protein concentrations, consistently with the results obtained for atToc33 C . Comparison of this concentration-dependent effect between both proteins reveals a shift to lower concentrations for psToc34 C , which can be explained by the higher affinity of psToc34 C for homodimerization.

The suggested relation between GDP release and homodimerization was further substantiated by the analysis of dimerization inhibited variants of atToc33 and psToc34, atToc33R130A and psToc34R133A [START_REF] Schnell | Protein translocons: multifunctional mediators of protein translocation across membranes[END_REF][START_REF] Reddick | In vitro comparative kinetic analysis of the chloroplast Toc GTPases[END_REF][START_REF] Weibel | Dimerization of Toc-GTPases at the chloroplast protein import machinery[END_REF][START_REF] Yeh | Dimerization is important for the GTPase activity of chloroplast translocon components atToc33 and psToc159[END_REF], which are able to bind nucleotides [START_REF] Koenig | On the significance of Toc-GTPase homodimers[END_REF]. The GDP dissociation rate of these two proteins (Figure 2A) differs with respect to the source organism (Figure 3b), which parallels the observed distinct physicochemical properties of the native proteins (Figure 3a). However, the GDP dissociation rate of each of the mutants is independent of protein concentration in the range analyzed (Figure 3b). This observation supports the conclusion that dimerization reduces nucleotide release from the wild type protein. Following this notion, homodimer dissociation would be rate limiting for GDP dissociation.

To challenge this assumption by numerical analysis, a model representing a unidirectional reaction scheme was applied (equation [E3]). The model considers the transition of the nucleotide bound protein from its dimeric to its monomeric state and the subsequent dissociation of the nucleotide (excluding GDP dissociation from dimer).

This analysis reveals that both the rate constants for GDP dissociation from the monomeric receptor (k2, [E3]) and dissociation of the homodimer (k1, [E3]) are not altered by varying protein concentration (values listed in Table 1). Furthermore, the GDP dissociation rate constant determined by this approach resembles that of the monomeric species. In addition, the rate constant for atToc33 C homodimer dissociation is consistent with previous results [START_REF] Jelic | Two Toc34 homologues with different properties[END_REF]. By comparison of the rate constant for dimer and nucleotide dissociation we can conclude that dissociation of the homodimer is the rate limiting step for GDP dissociation. In other words, the dimeric state prevents exchange of GDP by GTP, which is consistent with the occlusion of the nucleotide-binding pocket as observed in dimeric receptor structures [START_REF] Koenig | The GTPase cycle of the chloroplast import receptors Toc33/Toc34: implications from monomeric and dimeric structures[END_REF][START_REF] Sun | Crystal structure of pea Toc34, a novel GTPase of the chloroplast protein translocon[END_REF].

Precursor recognition disturbs dimerization and induces nucleotide release

It has been shown that the transit peptide promotes an interaction between Toc33 and Toc159 when the receptors are in their GMP-PNP-bound forms [START_REF] Becker | Preprotein recognition by the Toc complex[END_REF]. In addition, our data indicate that pre-protein binding modulates receptor GTPase activity by stimulating nucleotide exchange [Fig. 1; e.g. [START_REF] Jelic | Two Toc34 homologues with different properties[END_REF]. This prompted us to analyze the influence of peptides representing the pSSU transit sequence on dimerization and nucleotide exchange by atToc33 C or psToc34 C . For that purpose, a 26 amino acid peptide with a phophoserine at position 13 exhibiting the highest affinity for psToc34 C in previous studies [B1 ; 20] was chosen. The same peptide was also used to study the interactions between Toc33 and Toc159 [5; therein referred to as B2].

Dimerization of 170µM psToc34 C and 500µM atToc33 C was analyzed by size exclusion chromatography [START_REF] Koenig | On the significance of Toc-GTPase homodimers[END_REF][START_REF] Oreb | Phosphorylation regulates the assembly of chloroplast import machinery[END_REF] in the absence or presence of B1 peptide. The different concentrations were chosen to warrant a comparable amount of dimeric species for the two proteins with different dissociations constants for homodimerization (see above). We observed a peptide concentration dependent destabilization of the atToc33 C and psToc34 C homodimer (Figure 4a, black circles, grey triangles). Remarkably, the concentration of the peptide at which 50% of the destabilization is observed (~200 µM) is comparable to the previously observed K D for the interaction between B1 and psToc34 C [START_REF] Schleiff | Structural and guanosine triphosphate/diphosphate requirements for transit peptide recognition by the cytosolic domain of the chloroplast outer envelope receptor[END_REF]. In contrast to the influence observed for B1 on homodimerization, addition of a control (C, sequence given in experimental) peptide at the highest concentration used for B1 did not result in disturbance of the homodimer (Figure 4a, white circles).

Guided by this observation we next analyzed the influence of the peptide on GDP dissociation from the receptor. We observed a rather slow dissociation of mantGDP from the monomeric atToc33 C (11 µM, according to the established KD) when no competing nucleotide was present (Figure 4b, black). The dissociation rate of the labeled nucleotide was increased in the presence of excessive unlabelled nucleotide due to the repressed rebinding of mantGDP (Figure 4b, black vs. grey). Addition of either control peptide (C) or B1 peptide to atToc33 C in the presence of excessive unlabelled nucleotide did not alter the rate or amount of mantGDP released (Figure 4b, green & red). Therefore, we can conclude that the peptide does not influence the release of mantGDP from the monomeric receptor.

Next we determined the mantGDP release from atToc33 C at a concentration of 330µM containing about 45% of the receptor in a dimeric conformation (according to the established KD). Again, addition of unlabelled nucleotide (Figure 4c, black vs. grey) enhanced the dissociation rate due to the repressed rebinding of mantGDP. Addition of the control peptide did not further influence the release, neither in terms of the rate nor in terms of the amount of released mantGDP (Figure 4c, grey vs. green). In contrast, addition of the peptide B1 resulted in an increase of the total amount of released nucleotide in the course of the measurement (Figure 4c, grey vs. red). This increase of GDP release in the presence of B1 can be explained by its ability to enhance the dissociation rate of the homodimer (Figure 4a) , thereby abrogating its rate-limiting role on nucleotide exchange. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

To verify the influence of the peptide on nucleotide exchange we have added the peptide near the plateau phase of mantGDP release from atToc33 C after about 600 sec (Figure 4d). Addition of the control peptide (C) or buffer (Bu) did not alter the equilibrium, but once again B1 induced a significant release of mantGDP. This confirms that dimerization and, in consequence, nucleotide exchange are influenced by the transit peptide.

DISCUSSION

In the past several properties have been attributed to the homodimerization of Toc33, which is of functional relevance as documented in vivo and in vitro [START_REF] Lee | Toc receptor dimerization participates in the initiation of membrane translocation during protein import into chloroplasts[END_REF][START_REF] Aronsson | Nucleotide binding and dimerization at the chloroplast pre-protein import receptor, atToc33, are not essential in vivo but do increase import efficiency[END_REF]. In contrast to GADs described so far [START_REF] Gasper | It takes two to tango: regulation of G proteins by dimerization[END_REF], homodimerization was not found to be GTP dependent and does not directly contribute to GTP hydrolysis [e.g. [START_REF] Koenig | The GTPase cycle of the chloroplast import receptors Toc33/Toc34: implications from monomeric and dimeric structures[END_REF][START_REF] Reddick | In vitro comparative kinetic analysis of the chloroplast Toc GTPases[END_REF] raising the question by which functional mechanism homodimerization contributes to the regulation of protein translocation. Our data indicate that dimerization decelerates the rate of GTP binding (Fig. 2) and GDP release (Fig. 3) by capturing the nucleotide in a cage at the dimer interface. In the case of monomeric Ras-like GTPases, an analogous function is fulfilled by GDP dissociation inhibitors [GDI, e.g. [START_REF] Bos | GEFs and GAPs: critical elements in the control of small G proteins[END_REF][START_REF] Bourne | The GTPase superfamily: conserved structure and molecular mechanism[END_REF][START_REF] Zheng | Interactions among proteins involved in bud-site selection and bud-site assembly in Saccharomyces cerevisiae[END_REF], which have to be replaced by a GDI-displacement factor [GDF; e.g. [START_REF] Ingmundson | Legionella pneumophila proteins that regulate Rab1 membrane cycling[END_REF][START_REF] Machner | A bifunctional bacterial protein links GDI displacement to Rab1 activation[END_REF] prior to nucleotide exchange. Thus, a function comparable to that of a GDF is accomplished by the receptor substrate, the transit peptide of the pre-protein. The peptide alters the kinetic properties of homodimerization and the affinity of this reaction results in an enhanced GDP release (Fig. 3). The transit peptide does not induce the release of the bound GDP from the monomeric receptor directly and thereby does not function as a classical nucleotide exchange factor (GEF). Thus, one has to conclude that the influence of the transit peptide on nucleotide exchange appears to result from an indirect effect on Toc33 dimerization as determined by size exclusion chromatography. However, this technique only senses the influence of the substrate on the dissociation rate (kdiss in equation E2) of the homodimer. Conclusions regarding the equilibrium dissociation constant (KD, see [E2]) cannot be drawn because the monomers are immediately removed from the monomer/dimer equilibrium in the course of the chromatographic separation process. Indeed, addition of the transit peptide did not influence the dissociation constant determined by analytical ultracentrifugation (not shown). Thereby, the association rate constant appears to be altered to the same extent as the dissociation rate constant [E2]. A similar observation was made for the SRP-RNA, which kinetically controls the dimerization between Ffh and FtsY. Here the association rate (kass) is accelerated to the same extent as the rate of dimer dissociation (kdiss) resulting in the same equilibrium dissociation constant (KD) as in the absence of RNA [START_REF] Neher | SRP RNA controls a conformational switch regulating the SRP-SRP receptor interaction[END_REF]. Analagously, the transit peptide seems not to influence the affinity of heterodimerization but to lower the energetic barrier of dissociation. Expanding the current categorization of dimeric GTPases [START_REF] Gasper | It takes two to tango: regulation of G proteins by dimerization[END_REF], we propose considering Toc33 as GID (G protein inhibited by nucleotidedependent dimerization).

How can these observations be reconciled with current models of TOC receptor function? In vitro experiments suggest that Toc33 dimerization is not nucleotide dependent [START_REF] Sommer | Molecular interactions within the plant TOC complex[END_REF], although a slight preference for homodimerization in the GDP loaded state exists [START_REF] Koenig | The GTPase cycle of the chloroplast import receptors Toc33/Toc34: implications from monomeric and dimeric structures[END_REF]. Initial recognition of precursor proteins by a GDP loaded Toc33 is concordant with chemical crosslinking experiments, where -in the context of the TOC complex -an interaction between Toc33 and the transit peptide was only observed in the presence of GDP, while GTP promotes its transfer to Toc159 [START_REF] Becker | Preprotein recognition by the Toc complex[END_REF][START_REF] Kouranov | Analysis of the interactions of preproteins with the import machinery over the course of protein import into chloroplasts[END_REF][START_REF] Oreb | Phosphorylation regulates the assembly of chloroplast import machinery[END_REF]. Albeit speculative, precursor binding by the homodimeric receptor would enhance the association and dissociation rate of dimerization, resulting in nucleotide exchange driven by a high intracellular GTP to GDP ratio [START_REF] Meyer | Analysis of the nucleotide pool during growth of suspension cultured cells of Nicotiana tabacum by high performance liquid chromatography[END_REF]. This in turn would promote heterodimerization with GTP loaded Toc159 [START_REF] Becker | Preprotein recognition by the Toc complex[END_REF], and subsequent GTP hydrolysis would ensure its dissociation again. The switch from homodimeric to the heterodimeric state and GTP hydrolysis are envisioned to initiate pre-protein translocation [START_REF] Schleiff | A GTP-driven motor moves proteins across the outer envelope of chloroplasts[END_REF] and reformation of the Toc33 GDP dimer.

The data presented herein, the structural interpretations given earlier [START_REF] Lee | Toc receptor dimerization participates in the initiation of membrane translocation during protein import into chloroplasts[END_REF][START_REF] Scheffzek | Crystal structure of the nuclear Ras-related protein Ran in its GDP-bound form[END_REF] and other experimental findings [e.g. 17 and references therein] all suggest that the Toc33 dimer exists in a GDP loaded ground state. However, a different scenario in planta cannot be ruled out, e.g. a mixed GTP/GDP loaded dimer. Our current hypothesis of the cooperative mechanism for Toc33 and Toc159 in precursor transfer proposes that the Toc33 homodimer is disrupted by the pre-protein to allow one Toc33 monomer to interact with Toc159. The role of the second Toc33 monomer is unclear, but this mechanism could account for the molar excess of Toc33 relative to Toc159 within TOC complexes [START_REF] Chen | Precursor binding to an 880-kDa Toc complex as an early step during active import of protein into chloroplasts[END_REF][START_REF] Kikuchi | Characterization of the preprotein translocon at the outer envelope membrane of chloroplasts by blue native PAGE[END_REF][START_REF] Schleiff | Characterization of the translocon of the outer envelope of chloroplasts[END_REF]. Hence, an initial understanding of the molecular events of the TOC translocon emerges, and this model can now be tested by in vivo experiments. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

TABLES

FIGURE LEGENDS

Figure 1 The precursor pSSU stimulates GTP binding by Toc33. GTP binding to 100µM atToc33 C (circles), 100µM atToc33 C in the presence of pSSU (triangles), 100µM atToc33 C -G1m mutant (G45R/K49N/S50R; squares), 100µM atToc33 C -G1m in the presence of pSSU (diamonds) or pSSU (hexagons) was determined. Recombinant protein was incubated at 25°C for 30 min to ensure complete hydrolysis of bound GTP. The reaction was cooled to 0°C and incubated with [α-32 P]GTP (150 mCi/μmol) for 0, 15, 30 or 60 minutes. Subsequently, a fraction of the incubation was blotted onto cellulose membrane, washed to remove unbound nucleotide, and bound [α- 32 (a) The rates of mantGDP dissociation determined by single exponential analysis are shown for atToc33 C (black circles, left axis) and psToc34 C (grey circles, right axis). The black and the grey arrow indicate the concentration corresponding to the K D for dimerization of atToc33 C and psToc34 C , respectively. (b) The rates of mantGDP dissociation determined by single exponential analysis are shown for atToc33 C (R130A) (black triangles, on y-axis 10 2 ) and psToc34 C (R133A) (grey triangles, on y-axis 10 3 ). The average value is indicated as dashed line for atToc33 C (R130A) and psToc34 C (R133A), respectively. The dissociation rates of the atToc33 C or psToc34 C dimer and of GDP according to equation [E3] were calculated and are listed in Table 1. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  The measurement was initiated by the addition of mantGDP loaded protein. The fluorescence was excited at 355 nm and recorded at 448 nm for typically 10 minutes. Dissociation of GDP was analyzed by a single exponential equation. The dissociation rate (k diss ) can be determined based on two mutually exclusive assumptions: (i) GDP can dissociate from both monomers and dimers with different rates or (ii) GDP dissociation occurs only in the monomeric state, implicating that the dissociation rate of the dimer determines the dissociation rate of GDP. Assuming that dissociation of the (homo-)dimer ( ) is slower than dissociation of GDP one can write: → → + From this reaction scheme one can approximate [ ] = * - * * ( -) * ( -) [E3], with being the amount of atToc33 C without nucleotide, being the dissociation rate of the dimer, the dissociation rate of GDP and the starting concentration of the dimer. Biochemical Journal Immediate Publication. Published on 24 Mar 2011 as manuscript BJ20110246 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110246A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

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Biochemical

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Biochemical

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Biochemical

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Biochemical

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  ] b (3.4±0.1) * 10 -3 dimer [E2] b (4.5±0.5) * 10 -4 psToc34 C R133A GDP a (4.1±0.1) * 10 -3 *Constants are calculated from results presented in Fig. 2A and C for wild type considering only the low receptor concentrations (a) and Fig. 2B and D according to the indicated equation (b). Biochemical Journal Immediate Publication. Published on 24 Mar 2011 as manuscript BJ20110246 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110246

  FIGURE LEGENDSFigure1The precursor pSSU stimulates GTP binding by Toc33. GTP binding to 100µM atToc33 C (circles), 100µM atToc33 C in the presence of pSSU (triangles), 100µM atToc33 C -G1m mutant (G45R/K49N/S50R; squares), 100µM atToc33 C -G1m in the presence of pSSU (diamonds) or pSSU (hexagons) was determined. Recombinant protein was incubated at 25°C for 30 min to ensure complete hydrolysis of bound GTP. The reaction was cooled to 0°C and incubated with [α-32 P]GTP (150 mCi/μmol) for 0, 15, 30 or 60 minutes. Subsequently, a fraction of the incubation was blotted onto cellulose membrane, washed to remove unbound nucleotide, and bound [α-32 P]GTP was measured by phosphor imaging. The values are expressed in arbitrary units and the error bars reflect the standard deviation of three independent experiments. The lines represent the least square fit analysis to a single exponential function.

Figure 2 GTP

 2 Figure 2 GTP association depends on the protein concentration.(a) Shown is an example of the isolated proteins (5µg) used in this study separated on SDS-PAGE and stained by Coomassie-Blue. The migration of the molecular weight standards is indicated on the left (in kDa). (b) Binding of 2 mM GTP supplemented with EDA-GTP-ATTO-550 to 4 µM or 200 µM atToc33 C (concentration is indicated) was followed by fluorescence intensity increase (grey lines). The kinetics of GTP association was analyzed by a single exponential function (black line for 4µM) or two independent exponential functions (black line for 200µM, assuming two independent rate constants for the monomeric and dimeric receptor species) yielding P values < 0.0001. The inset shows the residuals for the analysis of the binding curve to atToc33 C at 200µM using the single exponential (black line) or double exponential (grey line) equation. (c) Rate constants for GTP binding were determined from kinetic experiments at 4 µM (white circles) or 200 µM atToc33 C (black circles for monomeric and grey circles for dimeric species) as shown in (b). Lines show the least square fit analysis to [E1,E2] for the monomeric (m) and dimeric (d) rate constants. The inset shows the logarithmic representation to visualize the distribution of the spots at low GTP concentrations. Dotted line in the inset indicates the region of extrapolation. For better readability the error bars are not shown which are smaller than 10% of the value shown.

Figure 3

 3 Figure 3 Dimerization reduces GDP dissociation.(a) The rates of mantGDP dissociation determined by single exponential analysis are shown for atToc33 C (black circles, left axis) and psToc34 C (grey circles, right axis). The black and the grey arrow indicate the concentration corresponding to the K D for dimerization of atToc33 C and psToc34 C , respectively. (b) The rates of mantGDP dissociation determined by single exponential analysis are shown for atToc33 C (R130A) (black triangles, on y-axis 10 2 ) and psToc34 C (R133A) (grey triangles, on y-axis 10 3 ). The average value is indicated as dashed line for atToc33 C (R130A) and psToc34 C (R133A), respectively. The dissociation rates of the atToc33 C or psToc34 C dimer and of GDP according to equation [E3] were calculated and are listed in Table1.

Biochemical

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Figure 4 Figure 1 ,Figure 2 ,

 412 Figure 4 Influence of a transit peptide sequence on dimerization and nucleotide release. (a) atToc33 C (180 µl of 500 µM concentration, black circles) or psToc34 C (180 µl of 170 µM concentration, grey triangles) were subjected to size exclusion chromatography in the absence or presence of B1 peptide at indicated concentrations. For control, atToc33 C (180 µl of 500 µM concentration) was incubated with the control peptide C at indicated concentration (white circles). The absorption profiles were analyzed by two Gaussian distributions, the dimeric fraction was calculated from their area. The decrease of dimeric population was analyzed by a hyperbolic function. (b, c) The dissociation of mantGDP from 11 µM (a) or 330 µM atToc33 C (b) was determined by fluorescence (in arbitrary units) in absence (-, black) or presence of 1 mM GDP (Bu, GDP, grey). In addition to GDP, 1 mM control peptide (C, green) or B1 peptide (B1, red) were added. (d) The dissociation of mantGDP from 600 µM atToc33 C was determined by fluorescence and identical volumes of buffer (Bu, grey), control peptide (C, final concentration 1 mM) or B1 peptide (B1, final concentration 1 mM) were added at the time point indicated.

Figure 3 ,Figure 4 ,

 34 Figure 3, Oreb et al.

atToc33

  ΔC + C psToc34 ΔC + B1 atToc33 ΔC + B1 Biochemical Journal Immediate Publication. Published on 24 Mar 2011 as manuscript BJ20110246 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110246 A c c e p t e d M a n u s c r i p t

  Biochemical Journal Immediate Publication. Published on 24 Mar 2011 as manuscript BJ20110246 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110246

  Biochemical Journal Immediate Publication. Published on 24 Mar 2011 as manuscript BJ20110246 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110246

Table 1 . The dissociation rate constants for GDP release and dimer dissociation. Protein Rate constant k diss (sec

 1

	-1 )*

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

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ACKNOWLEDGEMENTS

We thank Giesela Englich (Goethe University, Frankfurt) for constant technical assistance. Further, we thank Thorsten Hugel (Technical University, Munich) for constant support and critical discussions and Orla Slattery (Goethe University, Frankfurt) for helpful comments on the manuscript.

FUNDING

This work was supported by grants from the Deutsche Forschungsgemeinschaft in the frame of the SFB-807 (P17, to AH, MSS and ES), from the Center of Excellence "Macromolecular Complexes" Frankfurt (to PK and ES), from the Volkswagenstiftung (to MO, and ES) and from the interdisciplinary PhD program "Molecular Machines: Mechanisms and Functional Interconnections" of the Land Baden-Württemberg (to PK and IS). Biochemical Journal Immediate Publication. Published on 24 Mar 2011 as manuscript BJ20110246 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110246

A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

AUTHOR CONTRIBUTION

Mislav Oreb, Anja Höfle, Patrick Koenig, Fei Wang and Maik S. Sommer conducted the experiments presented. Mislav Oreb, Irmgard Sinning, Ivo Tews, Danny J. Schnell and Enrico Schleiff were involved in the experimental design and data analysis. All authors were involved in manuscript preparation, which was drafted by Enrico Schleiff.