Inhibition of protein synthesis by TOR inactivation revealed a conserved regulatory mechanism of the BiP chaperone in Chlamydomonas

1 Running head: TOR controls BiP phosphorylation in Chlamydomonas Corresponding author: José L. Crespo Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas (CSIC)-Universidad de Sevilla, 41092 Sevilla (Spain) Phone: +34 954489520 Fax: +34 954460065 Email: [email protected] Research area: Environmental Stress and Adaptation Plant Physiology Preview. Published on August 8, 2011, as DOI:10.1104/pp.111.179861 Copyright 2011 by the American Society of Plant Biologists 2 Title: Inhibition of protein synthesis by TOR inactivation revealed a conserved regulatory mechanism of the BiP chaperone in Chlamydomonas Sandra Díaz-Troya1, María Esther Pérez-Pérez1, Marta Pérez-Martín1, Suzette Moes2, Paul Jeno2, Francisco J. Florencio1 and José L. Crespo1* 1Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas (CSIC)-Universidad de Sevilla, 41092 Sevilla (Spain) 2Biozentrum, University of Basel, 4056 Basel (Switzerland) 3 FOOTNOTES This study was supported by the Spanish Ministry of Science and Innovation and Fondo Social Europeo (FSE) (grants BFU2009-07368 to JLC and BFU2010-15708 to FJF). Corresponding author: Jose L. Crespo ([email protected]) 4 ABSTRACT The TOR kinase integrates nutritional and stress signals to coordinately control cell growth in all eukaryotes. TOR associates to highly conserved proteins to constitute two distinct signaling complexes termed TORC1 and TORC2. Inactivation of TORC1 by rapamycin negatively regulates protein synthesis in most eukaryotes. Here we report that downregulation of TOR signaling by rapamycin in the model green alga Chlamydomonas reinhardtii resulted in pronounced phosphorylation of the endoplasmic reticulum chaperone BiP. Our results indicated that Chlamydomonas TOR regulates BiP phosphorylation through the control of protein synthesis since rapamycin and cycloheximide have similar effects on BiP modification and protein synthesis inhibition. Modification of BiP by phosphorylation was suppressed under conditions that require the chaperone activity of BiP such as heat shock stress or tunicamycin treatment, which inhibits N-linked glycosylation of nascent proteins in the endoplasmic reticulum. A phosphopeptide localized in the substrate-binding domain of BiP was identified in Chlamydomonas cells treated with rapamycin. This peptide contains a highly conserved threonine residue that might regulate BiP function as demonstrated by yeast functional assays. Thus, our study has revealed a regulatory mechanism of BiP in Chlamydomonas by phosphorylation/dephosphorylation events and assigns a role to the TOR pathway in the control of BiP modification. 5 INTRODUCTION The abundance and quality of nutrients are key factors that determine cell growth (increase in cell mass) in all living organisms. In eukaryotes, the availability of nutrients as well as other environmental cues is transmitted to the cell growth machinery through a number of important signalling proteins including the target of rapamycin (TOR) kinase (for reviews see (Crespo and Hall, 2002; De Virgilio and Loewith, 2006; Wullschleger et al., 2006; Ma and Blenis, 2009; Sengupta et al., 2010)). TOR is a Ser/Thr kinase that associates to other proteins to form two distinct multiprotein complexes, termed TORC1 and TORC2, which modulate two major signalling branches in eukaryotic cells (Hara et al., 2002; Kim et al., 2002; Loewith et al., 2002; Wedaman et al., 2003; Jacinto et al., 2004). TOR and other core components of these two complexes are evolutionarily conserved from lower to higher eukaryotes, indicating that these proteins must play central roles in nutrient signalling (Wullschleger et al., 2006). The TOR pathway stimulates cell growth by promoting anabolic processes, including translation and ribosome biogenesis, and inhibiting catabolic processes such as autophagy (Crespo and Hall, 2002). Control of protein synthesis is one of the bestcharacterized processes downstream of the TOR pathway. In yeast and mammals, TORC1 controls protein synthesis at different levels by positively regulating translation initiation and ribosome biogenesis (De Virgilio and Loewith, 2006; Ma and Blenis, 2009). The function of a number of translation factors, including eIF4E, eIF4G and eIF2 are regulated by TORC1 signalling (Beretta et al., 1996; Redpath et al., 1996; Raught et al., 2000) whereas key proteins in promoting translation such as the mammalian S6K or yeast SCH9 AGC (protein kinase A/protein kinase G/protein kinase C-family) kinases are direct substrates of TORC1 (Pearson et al., 1995; Urban et al., 2007). Under favourable growth conditions, yeast TORC1 phosphorylates several 6 serine and threonine residues in the carboxy terminus of SCH9, and phosphorylated SCH9 participates in the activation of all three RNA polymerases (Urban et al., 2007). In mammalian cells, phosphorylated S6K promotes phosphorylation of the ribosomal protein S6, which in turn results in the up-regulation of translation initiation (Chung et al., 1992). In addition to activating protein synthesis, TORC1 inhibits autophagy when nutrients are available (Noda and Ohsumi, 1998). Autophagy is a membrane-trafficking process by which starved cells degrade and recycle cytosolic proteins and organelles. During this catabolic process, cytosolic components are nonselectively enclosed within a double-membrane structure (autophagosome) and delivered to the vacuole/lysosome for degradation (Xie and Klionsky, 2007; Nakatogawa et al., 2009). TORC1 has been identified as an essential component in transmitting nutrient starvation signals to the autophagic machinery in yeast and higher eukaryotes. TORC1 negatively controls autophagy via inhibition of the evolutionarily conserved protein kinase ATG1 that mediates an early activation step in the autophagic process (reviewed in (Diaz-Troya et al., 2008)). The TOR kinase has been described in plants and algae, indicating that this signalling pathway is also conserved in photosynthetic organisms (Menand et al., 2002; Crespo et al., 2005). In both systems, TOR has been involved in cell growth control, since disruption of the AtTOR gene in Arabidopsis thaliana is lethal (Menand et al., 2002), and rapamycin-mediated inactivation of CrTOR in Chlamydomonas reinhardtii results in strong inhibition of cell growth (Crespo et al., 2005). Components of TORC1 signalling are conserved in Arabidopsis and Chlamydomonas. AtRaptor1 and AtRaptor2 have been described as homologues of KOG1/raptor in Arabidopsis (Anderson et al., 2005; Deprost et al., 2005), and direct interaction of AtRaptor1 with AtTOR has been demonstrated, indicating that TORC1 might be conserved in plants 7 (Mahfouz et al., 2006). Besides KOG1/raptor and TOR proteins, two putative LST8 homologues can be identified in the Arabidopsis genome (Moreau, 2010; Dobrenel et al., 2011). In Chlamydomonas, it has been demonstrated that CrTOR localizes in highmolecular-mass complexes that include a highly conserved LST8 homologue (Diaz- Troya et al., 2008). These findings indicate that TORC1 might be functionally and structurally conserved in photosynthetic eukaryotes. However, no obvious homologues exist for TORC2-specific partners in plants or algae (Moreau, 2010; Pérez-Pérez, 2010; Dobrenel et al., 2011). Dissection of the TOR pathway in plants has been hampered by the natural resistance of these organisms to rapamycin likely due to the inability of plant FKBP12 to bind to this drug (Xu et al., 1998). Nevertheless, the generation of inducible RNAi lines that allow conditional silencing of the AtTOR gene (Deprost et al., 2007) or rapamycin-sensitive plants (Sormani et al., 2007; Leiber et al., 2010) have importantly contributed to understand how TOR controls plant cell growth. The finding that silencing of AtTOR expression arrests plant growth and reduces mRNA translation (Deprost et al., 2007) strongly suggests that control of protein synthesis by TOR is conserved in plants. Unlike plants, Chlamydomonas is sensitive to rapamycin and inactivation of TOR signalling by this drug leads to cell growth inhibition (Crespo et al., 2005) and autophagy activation (Perez-Perez and Crespo, 2010; Perez-Perez et al., 2010), demonstrating that TOR controls this degradative process in photosynthetic organisms. Recently, it has also been shown that TOR inhibits autophagy in plants (Liu and Bassham, 2010). Like in other systems, the identification of components of the TOR pathway in plants and algae will be essential to understand how TOR controls cell growth in these organisms. 8 In this study we show that the endoplasmic reticulum (ER) chaperone BiP becomes phosphorylated in Chlamydomonas cells treated with rapamycin. BiP phosphorylation also occurred in response to inhibition of protein synthesis, whereas ER stress induced by tunicamycin or heat shock resulted in the complete dephosphorylation of the protein. Our findings indicate that a rapamycin-sensitive TOR pathway controls the phosphorylation state of BiP in Chlamydomonas and reveals a conserved regulatory mechanism of this protein by phosphorylation/dephosphorylation events. 9 RESULTS Rapamycin treatment induces threonine phosphorylation of a 70 kDa protein In order to identify proteins regulated by TOR in Chlamydomonas and whose phosphorylation state is under the control of this signalling pathway, we performed western blot analysis of total extracts from Chlamydomonas cells treated with rapamycin using anti-phosphoserine (anti-P-Ser) and anti-phosphothreonine (anti-P- Thr) antibodies. Whereas no significant difference was observed in extracts from untreated cells compared to rapamycin-treated cells with the anti-P-Ser antibody, levels of Thr phosphorylation of a 70-kDa protein were increased, as detected with an anti-P- Thr antibody (Figure 1A). In order to resolve the 70 kDa molecular mass range, we conducted two-dimensional (2D) gel electrophoresis analysis of Chlamydomonas cells treated with rapamycin. Total extracts obtained from rapamycin-treated or untreated cells were subjected to isoelectric focusing followed by SDS-PAGE and examined by Coomassie staining and western blot with the anti-P-Thr antibody (Figure 1B). The comparative analysis of the different 2D gels obtained allowed us to identify a few spots in the 70 kDa region that become phosphorylated (Figures 1B and 1C). Three spots whose mobility was altered by rapamycin treatment were detected in untreated cells by Coomassie staining (Figure 1C). Accordingly, the signal obtained in this region with the anti-P-Thr antibody was more intense in rapamycin-treated cells, indicating the presence of phosphorylated proteins in at least some of these spots (Figure 1B). Proteins present in the selected spots from control and treated samples were identified by mass spectrometry. Most of the spots included a mixture of three proteins that belong to the same category, namely HSP70B and the BiP1/BiP2 chaperones (Suppl. Table 1). HSP70B and the two isoforms of the BiP chaperone are members of the HSP70 superfamily (Schroda, 2004) and have a molecular mass of about 72 kDa and 16 molecular mass (Figure 6B). It is well established that KAR2 is itself translocated into the ER in yeast via a mechanism that depends on KAR2 function, and mature and precursor forms of the protein can be detected under certain circumstances (Rose et al., 1989; Vogel et al., 1990). Therefore, the lower mobility band of BiP1 might very likely correspond to the precursor form of the protein that cannot be properly translocated into the ER. With the availability of a functional assay for Chlamydomonas BiP1, we next investigated whether the conserved Thr520 of this protein is involved in BiP function. Thr520 was replaced by Ala, Glu or Arg, and the point mutants (T520A, T520E and T520R, respectively) were expressed in yeast cells. Similar to wild-type BiP1, T520A and T520R proteins were able to promote growth of kar2-159 cells at 30ºC (Figure 6A). However, T520E mutation, which mimics a constitutive phosphorylation of the residue, could not fully complement BiP function at 30ºC, indicating that T520E mutation decreased BiP function (Figure 6A). None of the BiP1 mutants were able to promote growth at 37ºC (Figure 6A). Interestingly, all mutations in Thr520 resulted in a pronounced increase in the abundance of the precursor and mature forms of this protein in yeast cells compared to wild-type BiP1 (Figure 6B), which indicates that this residue is important for the stability and processing of BiP1 in yeast cells. Cellular relocalization of BiP upon ER stress induced by tunicamycin BiP has been localized in Chlamydomonas to discrete punctae all over the cell that are more abundant around the flagellar basal bodies ((Diaz-Troya et al., 2008) and Figure 7), a membrane-enriched region known as peri-basal body region where intraflagellar transport (IFT) particle polypeptides concentrate (Cole et al., 1998). We wanted to investigate whether phosphorylation/dephosphorylation events of BiP might 17 result in alterations in the cellular localization pattern of this protein. Compared to control cells, no difference was observed in cells where BiP phosphorylation was induced by rapamycin (Figure 7). However, tunicamycin-mediated dephosphorylation of BiP resulted in a relocalization of this protein in the cell, being accumulated in a central region of the cell near the nucleus instead of the distal peri-basal body region (Figure 7). The cellular distribution of BiP in cells subjected to heat shock did not significantly differ from untreated cells (data not shown). These results demonstrate that dephosphorylation and activation of BiP by a specific stress in the ER correlates with a mobilization and concentration of this protein to this cellular compartment, and may reflect the requirement of dephosphorylated BiP to overcome this stress. 18 DISCUSSION We have previously reported that, unlike plants, growth of Chlamydomonas cells is sensitive to rapamycin, indicating that a rapamycin-sensitive TOR pathway operates in this unicellular green alga to govern cell growth (Crespo et al., 2005; Diaz- Troya et al., 2008). In this study we show that inactivation of TOR signalling by rapamycin caused inhibition of protein synthesis in Chlamydomonas (Figure 3B), which demonstrates that TOR promotes protein synthesis in this photosynthetic organism. Control of protein synthesis by TOR has been widely characterized in yeast and mammalian cells. In these systems, inhibition of TORC1 signalling by rapamycin resulted in a decrease in protein synthesis (Barbet et al., 1996; Beretta et al., 1996; Loewith et al., 2002). In Arabidopsis, inducible RNAi of the AtTOR gene or rapamycin treatment of plant lines expressing yeast FKBP12 caused a reduction in the abundance of high-molecular weight polysomes (Deprost et al., 2007; Sormani et al., 2007), indicating that TOR also controls protein translation in plants likely through a conserved TORC1 signaling branch since core components of this complex are conserved (Anderson et al., 2005; Deprost et al., 2005; Mahfouz et al., 2006). Our analysis of rapamycin-treated cells identified the ER chaperone BiP as a phosphoprotein whose phosphorylation state is regulated by TOR through the control of protein synthesis in Chlamydomonas. Inhibition of TOR signalling by rapamycin strongly induced phosphorylation of BiP on threonine residues (Figures 1 and 2) and a phosphopeptide in a regulatory region of BiP that contains a highly conserved threonine (Thr520) could be identified in rapamycin-treated cells (Figure 5). Although our study identified a single phosphopeptide in this chaperone, we cannot rule out that other residues might be phosphorylated as well. Actually, our results indicate that rapamycin further induced a basal, low level of phosphorylation in BiP, suggesting the presence of 19 additional phosphorylation sites (Figures 1 to 4). Post-translational modification of BiP, including phosphorylation and ADP-ribosylation, has been previously reported in mammalian cells (Carlsson and Lazarides, 1983; Welch et al., 1983; Hendershot et al., 1988; Freiden et al., 1992). Phosphoamino acid analysis of purified mammalian BiP demonstrated that this protein is predominantly phosphorylated on threonine residues with only a trace of detectable phosphoserine (Hendershot et al., 1988; Gaut, 1997). Moreover, in vivo threonine phosphorylation has been mapped to a region of the peptide-binding domain of mammalian BiP (Gaut, 1997) adjacent to the phosphopeptide identified in Chlamydomonas BiP (Figure 5). Modification of mammalian BiP by phosphorylation or ADP-ribosylation seems to be restricted to a pool of BiP that does not bind to substrate proteins (Hendershot et al., 1988; Freiden et al., 1992). Furthermore, it has been demonstrated that stresses which increase the need for active BiP in mammalian cells such as glucose starvation or tunicamycin treatment promote dephosphorylation of BiP and its association with proteins (Hendershot et al., 1988; Satoh et al., 1993). These findings led to the theory that modified BiP represents an inactive form of the protein, and that unmodified BiP is able to bind to proteins and promote their folding and assembly (Hendershot et al., 1988). It has been also proposed that post-translational modification of BiP might provide a storage pool of BiP that can be recruited back to the active form in response to need (Gething, 1999). The present study strongly suggests that regulation of BiP by phosphorylation is conserved in the photosynthetic alga Chlamydomonas, and assigns a novel role to the TOR pathway in the control of BiP phosphorylation. Similar to rapamycin, treatment of Chlamydomonas cells with cycloheximide resulted in increased phosphorylation of BiP (Figure 3). Both drugs have a negative effect on protein synthesis although with different efficiencies since cycloheximide is more effective than 20 rapamycin in translation inhibition (Figure 3). Interestingly, BiP phosphorylation occurred faster in response to cycloheximide since phosphorylated BiP was detected within one hour in cycloheximide-treated cells whereas rapamycin induced BiP phosphorylation after at least four hours (Figure 3). Thus our results establish a correlation between phosphorylation of BiP and protein synthesis through the TOR pathway in Chlamydomonas. Supporting the model that unmodified BiP is active (Hendershot et al., 1988), we found in Chlamydomonas that stress conditions that require the chaperone activity of BiP such as heat shock or inhibition of protein glycosylation in the ER by tunicamycin, triggered the fast dephosphorylation of BiP (Figure 4). Moreover, complete BiP dephosphorylation in response to heat shock or tunicamycin occurred even in cells where BiP phosphorylation was previously induced by inhibition of TOR signalling, emphasising the linkage between dephosphorylation and activation of BiP. Our results also demonstrated that the cellular localization of BiP is regulated by the phosphorylation state of the protein. Tunicamycin-induced dephosphorylation and activation of BiP clearly modified the cellular distribution of this chaperone in the cell as it accumulated in a central region of the cell near the nucleus where ER and Golgi compartments likely localize (Figure 7). Our findings in Chlamydomonas indicated that modification of BiP by phosphorylation/dephosphorylation events may play a regulatory role in the function of this chaperone, and that this molecular mechanism of regulation proposed in mammals (Hendershot et al., 1988; Freiden et al., 1992; Satoh et al., 1993) is conserved in photosynthetic eukaryotes. In vivo phosphorylation of BiP has been described in maize (Fontes et al., 1991) and spinach (Anderson et al., 1994), although to our knowledge a link between the phosphorylation state of the protein and its activity has not been shown in plants. 21 Yeast complementation assays performed with Chlamydomonas BiP1 indicated that this protein is able to replace only partially endogenous KAR2 function (Figure 6) despite the high identity between both proteins. Functional complementation of kar2 mutants has been achieved with mammalian and plant BiP proteins (Normington et al., 1989; Denecke et al., 1991). However, neither cytosolic nor mitochondrial yeast HSP70s are able to substitute functionally for KAR2 (Brodsky et al., 1993; Brodsky et al., 1998) likely due to the inability of these proteins to productively interact with proteins such as cochaperones or components of the ER translocation machine. The partial complementation of kar2-159 mutant cells by Chlamydomonas BiP1 might be due to an ineffective interaction of this protein to substrates or specific partners. Accordingly, most Chlamydomonas BiP1 was detected in yeast extracts as a precursor form, indicating that, unlike endogenous KAR2 (Rose et al., 1989; Vogel et al., 1990), this protein is not efficiently translocated into the ER lumen. Mutation of the conserved Thr520 in BiP1 led to a reduction in protein function (Figure 6A). These results suggest that this residue plays a role in the control of BiP1 activity in yeast cells and are in agreement with the finding that threonine phosphorylation inhibits BiP activity in mammals (Hendershot et al., 1988; Satoh et al., 1993; Gaut, 1997). Given that phosphorylation maps to the peptide-binding domain, this modification might interfere with substrate binding and/or interaction with partners such as cochaperones, as proposed for mammalian BiP (Gaut, 1997). The finding that Thr520 mutation in Chlamydomonas BiP1 stimulated maturation of this protein in yeast cells indicated that this residue is indeed important for the regulation of BiP1. Biochemical fractionation and indirect immunofluorescence microscopy studies revealed that TOR complexes in Chlamydomonas might associate, at least in part, with membranes from the ER (Diaz-Troya et al., 2008). Actually, the cellular distribution of 22 Chlamydomonas TOR and BiP proteins was coincident in some aspects. Both proteins localize to discrete bodies and accumulate in the peri-basal body region ((Diaz-Troya et al., 2008) and Figure 7). The common distribution of TOR and ER proteins may reflect functional interactions between the TOR pathway and this cellular compartment in Chlamydomonas. Several lines of evidence have recently associated TOR signalling and ER stress in mammals. Loss of the tuberous sclerosis (TSC) tumor suppressor complex, an upstream negative regulator of mTORC1 (Garami et al., 2003; Saucedo et al., 2003; Tee et al., 2003; Zhang et al., 2003), causes increased translation in certain mouse or human tumors due to the up-regulation of mTORC1 signalling, which in turn induces ER stress (Ozcan et al., 2008). This finding also indicates that mTORC1 genetically functions upstream of ER stress (Ozcan et al., 2008). Interestingly, it has been reported that the accumulation of misfolded proteins in mice induces mTORC1 signalling, which is directly regulated by chaperone availability (Qian et al., 2010). These studies have revealed the important role that control of protein synthesis by TORC1 plays on the regulation of ER homeostasis. Components of TORC1 signaling controlling protein synthesis such as raptor or the S6 kinase are conserved in plants and seem to participate in the general response of the plant to osmotic stress signals (Mahfouz et al., 2006; Deprost et al., 2007). However, it remains to be explored if, like in Chlamydomonas, TOR may function in ER stress through the control of protein synthesis. In conclusion, our studies show that the ER chaperone BiP can be regulated by reversible phosphorylation in Chlamydomonas and connect TOR signalling to this process. A better understanding of BiP regulation by phosphorylation/ dephosphorylation events will come with the identification of kinases and phosphatases 23 involved in the post-translational modification of this chaperone and their possible control by TOR and/or other signalling pathways. 24 MATERIALS AND METHODS Strains and Growth Conditions Chlamydomonas reinhardtii wild-type strain 6C+ was obtained from the laboratory of J.-D. Rochaix (University of Geneva), and rapamycin-insensitive rap2 strain has been previously described (Crespo et al., 2005). Cells were grown as described by Harris (1989) under continuous illumination at 25ºC. If required, Trisacetate phosphate medium (TAP) was solidified with 1.2% Bacto agar (Difco). When specified, cells were treated with 500 nM rapamycin (LC Labs) from a 4 mM stock in 90% ethanol-10% Tween20, 5 μg/ml tunicamycin (Calbiochem) from a 5 mg/ml stock in dimethylformamide, or 10 μg/ml cycloheximide (Sigma) from a 3 mg/ml stock in water. Saccharomyces cerevisiae wild-type (MS10) and kar2-159 were obtained from the laboratory of M. D. Rose. YJL034W (KAR2) heterozygous mutant was obtained from the EUROSCARF mutant collection. Yeast cells were grown in rich or synthetic medium as previously described (Sherman, 1991). Protein preparation Chlamydomonas cells from liquid cultures were collected by centrifugation (3000 x g, 5 min), washed once in lysis buffer (50 mM Tris-HCl (pH 7.5), 10 mM NaF, 10 mM NaN3, 10 mM p-Nitrophenylphosphate, 10 mM NaPPi, 10 mM betaglycerophosphate), and resuspended in a minimal volume of the same solution supplemented with Protease Inhibitor Cocktail (Sigma). Cells were lysed by two cycles of slow freezing to -80ºC followed by thawing to room temperature. The soluble cell extract was separated from the insoluble fraction by centrifugation (15700 x g, 15 min) at 4ºC, and stored at -80ºC until they were analyzed. Yeast extracts from logarithmically growing cells were prepared in lysis buffer (PBS buffer, 1 mM PMSF, 25 0.5% Tween20, 10 mM NaF, 10 mM NaN3, 10 mM p-Nitrophenylphosphate, 10 mM NaPPi, 10 mM beta-glycerophosphate) by vortexing them 10 times for 1 min each time with glass beads (Sigma-Aldrich). Crude extracts were cleared by centrifugation at 15,000g for 15 min at 4_C. Proteins were quantitated with the Coomassie dye binding method (BioRad). 2D gel electrophoresis For 2D electrophoresis, proteins from soluble cell extract samples were precipitated with 2D Clean-Up Kit (GE Healthcare), resuspended in rehydration solution, and quantitated with RC DC Protein Assay (BioRad) according to the manufacturers’ instruction. Isoelectric focusing (IEF) for the first dimension was carried out by rehydrating 11 cm long pH 3-10NL or pH 4-7 Immobiline DryStrips (GE Healthcare) with 200 μ l of DeStreak Rehydration Solution (GE Healthcare) supplemented with 0,5% Bio-Lyte 3/10 Ampholytes (BioRad) containing 150 μ g of protein. Following overnight active rehydration (50V, 20°C), IEF was performed for 15kVh (for pH 3-10NL strips) or 20kVh (for pH 4-7 strips) at 20°C using the Protean IEF Cell (Bio-Rad). 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B, Soluble extracts of Chlamydomonas cells treated (rap) or not (control) with 500 nM rapamycin for 8 hours were resolved by 2D electrophoresis as described in Experimental Procedures. Gels were stained with Coomassie Brilliant Blue (CBB) or processed for immunodetection with the phosphothreonine (P-Thr) antibody. C, Enlarged images of box sections in 2D gels shown in panel B. Arrow indicates spots whose mobility differs in untreated and rapamycin-treated cells. Numbers refer to spots where proteins have been identified by mass spectrometry (see Suppl. Table I for details). Figure 2. Rapamycin induces threonine phosphorylation of the BiP chaperone in its peptide-binding domain. A, Phosphorylation time course of the 70 kDa protein following rapamycin treatment. 500 nM rapamycin or drug vehicle were added to wildtype (wt) or rapamycin-insensitive (rap2) Chlamydomonas cells and samples were collected at the indicated times. Soluble extracts were fractionated by SDS-PAGE and analyzed by western blotting probing with phosphothreonine (P-Thr), BiP and HSP70B specific antibodies. B, 2D western blot analysis of BiP and HSP70B. Wild type Chlamydomonas cells were treated with 500 nM rapamycin for 12 hours (rap), heat shocked at 40ºC for 1 hour (HS) or remained untreated (control). Soluble extracts were 36 resolved by 2D electrophoresis as described in Experimental Procedures using pH 4-7 strips and gels were processed for immunodetection with phosphothreonine (P-Thr), BiP and HSP70B specific antibodies. Figure 3. Inhibition of protein synthesis by rapamycin or cycloheximide induces BiP phosphorylation. A, Time course of BiP phosphorylation following rapamycin and/or cycloheximide treatment. 500 mM rapamycin (rap), 10 μ g/ml cycloheximide (CHX) or both drugs simultaneously (CHX + rap) were added to wild-type Chlamydomonas cells and samples were taken at the indicated times. Soluble extracts were fractionated by SDS-PAGE and analyzed by western blot probing with P-Thr or BiP specific antibodies. B, Incorporation of [14C]arginine in Chlamydomonas cells. Wild-type (wt) and rapamycin-insensitive (rap2) Chlamydomonas cells were treated with 500nM rapamycin for 6 hours (rap), 10 μ g/ml cycloheximide for 30 min (CHX), or drug vehicle (C) and processed for the analysis of [14C]arginine incorporation as described in Experimental Procedures. Values are represented in relation to the [14C]arginine incorporation detected in control, untreated cells and correspond to six independent determinations (*p<0.05, **p<0.01). C, Effect of CHX and rapamycin on protein synthesis. Chlamydomonas cultures were treated with 500 nM rapamycin (triangles) or 10 μ g/ml CHX (circles) or drug vehicle (squares) and samples were taken at different times for protein synthesis determination. Data correspond to results from three independent experiments made in duplicate. Figure 4. BiP dephosphorylation by ER stress-inducing agents. BiP dephosphorylation by heat stress. Chlamydomonas cells pretreated (B) or not (A) with 500 nM rapamycin (rap) for 12 hours were subjected to heat stress at 40ºC and samples were taken at the 37 indicated times. Soluble extracts were fractionated by SDS-PAGE and analyzed by western blot probing with P-Thr and BiP specific antibodies. C, BiP dephosphorylation by tunicamycin treatment. Chlamydomonas cells were treated with 500 nM rapamycin (rap), 5 μ g/ml tunicamycin (tun), or first treated with rapamycin (rap, lane 8) and then with tunicamycin (rap tun, lane 9). Samples were taken at the indicated times and soluble extracts were processed and analyzed by western blot. Figure 5. Identification of a phosphopeptide in Chlamydomonas BiP. A, Schematic representation of the BiP protein and identification of a phosphopeptide in the peptidebinding domain. The scheme shows the ATPase and peptide-binding domains of BiP. The position of the phosphopeptide identified in Chlamydomonas is indicated by a red box whereas the grey box shows the position of the phosphopeptide identified in mammalian BiP (Gaut, 1997), which spans from Thr455 to Asp506 of Chlamydomonas BiP. B, Amino acid sequence alignment of the phosphopeptide identified in Chlamydomonas BiP (from Glu504 to Lys525) with the corresponding region of BiP proteins from other organisms. Cr, Chlamydomonas reinhardtii; Vc, Volvox carteri; Ot, Ostreococcus tauri; Ol, Ostreococcus lucimarinus; Msp, Micromonas sp. RCC299; Sl, Solanum lycopersicum; Nb, Nicotiana benthamiana; Vv, Vitis vinifera; Pt, Populus trichocarpa; Zm, Zea mays; Os, Oryza sativa; Gm, Glycine max; At, Arabidopsis thaliana; Pt, Physcomitrella patens; mBiP, Mus musculus. C, Modeling of Chlamydomonas BiP1 based on the crystal structure of mammalian 70 kDa heat shock protein (3c7nA). Conserved Thr520 was mapped onto the crystal structure. Structure prediction was performed using the SWISS-MODEL bioinformatics tool and image was generated by iMol software. ATPase and peptide-binding domains are shown in yellow and blue, respectively. 38 Figure 6. Chlamydomonas BiP1 partially complements KAR2 function in a kar2-159 ts mutant. A, MS10 (wt) and kar2-159 strains transformed with empty plasmid or plasmids expressing Chlamydomonas BiP1, T520A, T520E or T520R proteins were streaked for single colonies. Plates were incubated at the indicated temperatures for 3-4 days. B, MS10 (wt) and kar2-159 strain expressing the indicated Chlamydomonas BiP1 proteins were grown at the permissive temperature of 25ºC and expression of BiP1 and endogenous KAR2 proteins in these cells were detected by western blot. Precursor and mature forms of BiP1 are indicated. Cr, soluble extract from Chlamydomonas cells grown in TAP medium. 50 μg of total protein were loaded per lane. Figure 7. Relocalization of BiP in tunicamycin-treated cells. Wild-type Chlamydomonas cells growing exponentially in acetate medium were treated with 500 nM rapamycin (rap) or 5 μg/ml tunicamycin (tun) for 8 h. Cells were collected and processed for IF microscopy analysis. The signal recognized by the BiP antibody is shown in green. DNA staining is shown in blue (DAPI). A B C control rap CBB P-Thr IEF pH 3-10 NL SDS-PAGE control rap 123 1’2’3’ 4’5’ 98 64 50 36 kDa 98 64 50 36 kDa 04812 04812 time (h) time (h) P-Ser P-Thr * Figure 1. Rapamycin treatment induces alterations on the threonine phosphorylation pattern in Chlamydomonas soluble extract. A, Western blot analysis of Chlamydomonas soluble extracts with phosphoserine (P-Ser) and phosphothreonine (P-Thr) antibodies. Log-phase wild-type cells were treated with 500 nM rapamycin (rap) or drug vehicle and samples were taken at the indicated times. Soluble extracts were fractionated by SDS-PAGE and analyzed by western blot. The asterisk denotes the 70 kDa band detected with the P-Thr antibody. B, Soluble extracts of Chlamydomonas cells treated (rap) or not (control) with 500 nM rapamycin for 8 hours were resolved by 2D electrophoresis as described in Experimental Procedures. Gels were stained with Coomassie Brilliant Blue (CBB) or processed for immunodetection with the phospho-threonine (P-Thr) antibody. C, Enlarged images of box sections in 2D gels shown in panel B. Arrow indicates spots whose mobility differs in untreated and rapamycin-treated cells. Numbers refer to spots where proteins have been identified by mass spectrometry (see Suppl. Table I for details). 02468 02468time (h) wt rap2 P-Thr BiP HSP70B control rap HS P-Thr BiP HSP70B A BIEF PAGE Figure 2. Rapamycin induces threonine phosphorylation of the BiP chaperone in its peptide-binding domain. A, Phosphorylation time course of the 70 kDa protein following rapamycin treatment. 500 nM rapamycin or drug vehicle were added to wild-type (wt) or rapamycin-insensitive (rap2) Chlamydomonas cells and samples were collected at the indicated times. Soluble extracts were fractionated by SDS-PAGE and analyzed by western blotting probing with phosphothreonine (P-Thr), BiP and HSP70B specific antibodies. B, 2D western blot analysis of BiP and HSP70B. Wild type Chlamydomonas cells were treated with 500 nM rapamycin for 12 hours (rap), heat shocked at 40ºC for 1 hour (HS) or remained untreated (control). Soluble extracts were resolved by 2D electrophoresis as described in Experimental Procedures using pH 4-7 strips and gels were processed for immunodetection with phosphothreonine (P-Thr), BiP and HSP70B specific antibodies. control rap CHX CHX + rap 01 84time (h) 0 20 40 60 80 100 C rap CHX C rap CHX wt rap2 protein synthesis (%) AB *** ** Figure 3. Inhibition of protein synthesis by rapamycin or cycloheximide induces BiP phosphorylation. A, Time course of BiP phosphorylation following rapamycin and/or cycloheximide treatment. 500 mM rapamycin (rap), 10 μg/ml cycloheximide (CHX) or both drugs simultaneously (CHX + rap) were added to wild-type Chlamydomonas cells and samples were taken at the indicated times. Soluble extracts were fractionated by SDS-PAGE and analyzed by western blot probing with P-Thr or BiP specific antibodies. B, Incorporation of [14C]arginine in Chlamydomonas cells. Wild-type (wt) and rapamycin-insensitive (rap2) Chlamydomonas cells were treated with 500nM rapamycin for 6 hours (rap), 10 μg/ml cycloheximide for 30 min (CHX), or drug vehicle (C) and processed for the analysis of [14C]arginine incorporation as described in Experimental Procedures. Values are represented in relation to the [14C]arginine incorporation detected in control, untreated cells and correspond to six independent determinations (*p<0.05, **p<0.01). C, Effect of CHX and rapamycin on protein synthesis. Chlamydomonas cultures were treated with 500 nM rapamycin (triangles) or 10 μg/ml CHX (circles) or drug vehicle (squares) and samples were taken at different times for protein synthesis determination. Data correspond to results from three independent experiments made in duplicate. 0 1 2 3 4 5 6 7 8 120 100 80 60 40 20 0 protein synthesis (%) time (h) C P-Thr BiP P-Thr BiP P-Thr BiP P-Thr BiP