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First published online May 23, 2008; 10.1105/tpc.108.058123

The Plant Cell 20:1346-1362 (2008)
© 2008 American Society of Plant Biologists

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Dual Fatty Acyl Modification Determines the Localization and Plasma Membrane Targeting of CBL/CIPK Ca2+ Signaling Complexes in Arabidopsis[W]

Oliver Batistica, Nadav Sorekb, Stefanie Schültkea, Shaul Yalovskyb and Jörg Kudlaa,1

a Institut für Botanik, Universität Münster, 48149 Münster, Germany
b Department of Plant Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel

1 Address correspondence to jkudla{at}uni-muenster.de.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 METHODS
 REFERENCES
 
Arabidopsis thaliana calcineurin B–like proteins (CBLs) interact specifically with a group of CBL-interacting protein kinases (CIPKs). CBL/CIPK complexes phosphorylate target proteins at the plasma membrane. Here, we report that dual lipid modification is required for CBL1 function and for localization of this calcium sensor at the plasma membrane. First, myristoylation targets CBL1 to the endoplasmic reticulum. Second, S-acylation is crucial for endoplasmic reticulum-to-plasma membrane trafficking via a novel cellular targeting pathway that is insensitive to brefeldin A. We found that a 12–amino acid peptide of CBL1 is sufficient to mediate dual lipid modification and to confer plasma membrane targeting. Moreover, the lipid modification status of the calcium sensor moiety determines the cellular localization of preassembled CBL/CIPK complexes. Our findings demonstrate the importance of S-acylation for regulating the spatial accuracy of Ca2+-decoding proteins and suggest a novel mechanism that enables the functional specificity of calcium sensor/kinase complexes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 METHODS
 REFERENCES
 
Calcium signaling has a pivotal role in the regulation of almost every biological process in eukaryotic organisms (Berridge et al., 1998Go). Proteins that are regulated by fluctuations in cellular Ca2+ concentrations have evolved to mediate Ca2+-dependent stimulus–response coupling (Berridge et al., 2003Go). Such calcium sensor proteins, like calcineurin, calmodulin, and neuronal calcium sensors in animals and calcineurin B–like proteins (CBLs) and calmodulin in plants, sense and forward local changes in Ca2+ concentration to their target proteins (Luan et al., 2002Go; Batistic and Kudla, 2004Go). Although these Ca2+ binding proteins do not have an enzymatic activity of their own, binding of Ca2+ ions results in an increased affinity for and subsequent activation or deactivation of interacting proteins (Harmon et al., 2000Go; Sanders et al., 2002Go).

Cellular signaling networks are spatially organized by the specific localization of their protein constituents to distinct protein scaffolds, membrane systems, or microdomains. For calcium binding proteins, several mechanisms have been reported that contribute to their association with cellular membranes. Membrane binding can occur via transmembrane domains, electrostatic interaction, or lipid modification of the proteins. Several calcium sensor proteins, like calcineurin B and the group of the neuronal calcium sensor proteins, undergo lipid modification by myristoylation at their N termini (Burgoyne and Weiss, 2001Go).

The membrane association of myristoylated proteins often requires additional factors that allow closer contact with the plasma membrane. Such additional factors that can modulate the membrane association may include a polybasic domain, prenylation of the protein at the C terminus, or S-acylation of the protein (Greaves and Chamberlain, 2007Go). S-Acylation, more commonly referred to as palmitoylation, involves the reversible thioesterification of fatty acids, usually palmitic or stearic acid, to Cys thiols of proteins and serves to tether proteins to the cytoplasmic surfaces of cellular membranes (Huang and El-Husseini, 2005Go). Palmitoyl modifications are reversible, and rapid cycling between modified forms has been shown to regulate, for example, the activity of plasma membrane–localized H- and N-Ras (Rocks et al., 2005Go) and Arabidopsis thaliana Rho-like GTPase ROP6 (Sorek et al., 2007Go). Moreover, S-acylation regulates the localization of many key players in cellular signaling, including many G proteins, the {alpha} and β subunits of heterotrimeric G proteins, nonreceptor Tyr kinases like Fyn, Lck, and Yes, as well as Rho-like GTPases (Bijlmakers and Marsh, 2003Go; Sorek et al., 2007Go). However, with the exception of a flagellar calcium binding protein (FCaBP) from Trypanosoma cruzi (Godsel and Engman, 1999Go) and animal K+ channel–interacting proteins (KChIPs) 2 and 3 (Burgoyne, 2007Go), S-acylation has not been implicated in the regulation of cellular calcium sensor proteins.

CBLs, which are most similar to calcineurin B and neuronal calcium sensor proteins, have been recognized as fulfilling critical functions in diverse Ca2+-dependent processes in plants (Luan et al., 2002Go; Batistic and Kudla, 2004Go; Hedrich and Kudla, 2006Go). Analyses of the crystal structures of CBL4/SOS3 and CBL2 in complex with Ca2+ revealed how these proteins are able to sense Ca2+ (Nagae et al., 2003Go; Sanchez-Barrena et al., 2005Go). Increases in cellular Ca2+ lead to changes in the global conformation of the protein that increase the hydrophobic character of the macromolecule. Hydrophobic interactions mediate the binding of CBL proteins to the NAF domain of their target kinases, which, because of this specific interaction, have been designated the CBL-interacting protein kinases (CIPKs) (Albrecht et al., 2001Go; Kolukisaoglu et al., 2004Go; Sanchez-Barrena et al., 2007Go).

In Arabidopsis, 10 CBLs that specifically interact with distinct family members of the 25 CIPKs form a network-like signaling system for specific and synergistic stimulus–response coupling (Albrecht et al., 2001Go; Luan et al., 2002Go; Batistic and Kudla, 2004Go). Reverse genetics analyses have uncovered important functions of these proteins in abiotic stress responses and in the regulation of ion homeostasis (Albrecht et al., 2003Go; Zhu, 2003Go; Xu et al., 2006Go). The calcium sensors CBL1 and CBL9 have been shown to localize to the plasma membrane, where they form alternative complexes with their target kinases (D'Angelo et al., 2006Go; Cheong et al., 2007Go). Here, both calcium sensors function synergistically to activate the kinase CIPK23, which regulates the activity of the potassium channel Arabidopsis K+ Transporter1 (Xu et al., 2006Go). These findings suggest an important general function of CBL/CIPK complexes in regulating ion transport processes at the plasma membrane (Hedrich and Kudla, 2006Go). However, how this subcellular localization of CBL/CIPK complexes is regulated and how spatial specificity in this Ca2+ signaling system is achieved remain poorly understood. Four of the 10 CBL proteins harbor potential N-terminal myristoylation motifs that could promote the membrane association of these proteins. For CBL4/SOS3, it has been shown by complementation analysis of the respective mutants that myristoylation is important for CBL4 function, but why this lipid modification is required for CBL4 functionality remained a mystery (Ishitani et al., 2000Go).

Here, we report our analysis of the relevance of lipid modifications for the function and localization of the calcium sensor CBL1. Our biochemical, genetic, and cell biological studies indicate that both myristoylation and S-acylation are required for CBL1 function and for CBL1 localization at the plasma membrane. We also report that acylation not only stabilizes the plasma membrane binding of CBL1 but is also crucial for endoplasmic reticulum (ER)-to-plasma membrane trafficking, which appears to be insensitive to brefeldin A (BFA) and the secretion-associated and Ras-related protein1 (Sar1). Importantly, our data suggest that the lipid modification of the calcium sensor subunit determines the cellular localization and trafficking of preassembled CBL/CIPK complexes. Taken together, our results establish the importance of dual lipid modification for regulating spatial specificity in Ca2+-decoding signaling systems and thereby provide a mechanism for how calcium sensor/kinase complexes can contribute to regulating distinct physiological processes.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 METHODS
 REFERENCES
 
Myristoylation of CBLs in Vitro
Our previous analysis of the calcium sensors CBL1 and CBL9 revealed that both proteins localize to the plasma membrane (D'Angelo et al., 2006Go). In order to identify potential structural determinants for the cellular localization of both proteins, we performed a comparative amino acid analysis of all CBLs. This analysis identified CBL1, CBL4, CBL5, and CBL9 as harboring the MGXXX(S/T) consensus sequence for N-myristoylation (Figure 1A ). Moreover, we observed in these four CBL proteins a conserved Cys residue next to the Gly as a structural feature, suggesting a potential dual N-terminal lipid modification of these CBLs. Interestingly, in the amino acid sequence of CBL4/SOS3, we noticed in addition to the potential lipid modification residues an adjacent polybasic domain (Figure 1A) that could contribute to the membrane association of this protein and the interaction with phosphatidyl phosphoinositide lipids (Sciorra et al., 2002Go; Heo et al., 2006Go). This structural characteristic was not shared by CBL1 and CBL9, suggesting that the plasma membrane localization of these two proteins may be solely mediated by lipid modifications. Therefore, we used the CBL1 protein as a model to investigate the functional implications of potential lipid modifications.


Figure 1
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  		Figure 1. Myristoylation of CBLs in Vitro.

(A) Comparison of the N-terminal amino acids of the Arabidopsis CBL proteins. The N-terminal peptide sequences of CBL1, CBL2, CBL4, CBL5, and CBL9 were aligned with known myristoylated (human recoverin [Hs Rec] and yeast calcineurin B [Sc CNB]) and myristoylated–palmitoylated (At Ara6) proteins. CBL1, CBL4, CBL5, and CBL9 harbor a potentially myristoylated Gly residue adjacent to the first Met. In addition, these proteins contain a conserved Cys residue as a potential acceptor site of palmitoylation in the third position. By contrast, CBL2 harbors an extended N terminus containing adjacent Gly-Cys residues within the protein. Amino acids that are identical in at least two of the compared sequences are highlighted in black. The polybasic domain in CBL4 is labeled as underlined and by bold letters.

(B) Analysis of CBL1 myristoylation. CBL1 protein, mutated versions of CBL1 (G2A, C3S, and G2AC3S), CBL2 protein, as well as CBL1nCBL2 that contains the N terminus of CBL1 fused to CBL2 were generated by coupled in vitro transcription/translation in rabbit reticulocyte lysates. In vitro translation was performed in the presence of either [3H]myristic acid (top gel) or [35S]Met (bottom gel). The translated products were separated by SDS-PAGE and analyzed by fluorography. Only CBL1, CBLC3S, and the CBL1nCBL2 proteins were labeled by [3H]myristic acid, indicating effective myristoylation.

 
To investigate the myristoylation of CBL1 in vitro, we synthesized different versions of CBL1 using a coupled in vitro transcription/translation system from rabbit reticulocyte lysates. We analyzed the translation products of a wild-type CBL1 cDNA as well as of CBL1 cDNAs encoding a G2A mutation (CBL1G2A) that prevents myristoylation, a C3S mutation (CBL1C3S) that abolishes acylation at this position, and a construct combining both mutations (CBL1G2AC3S). In addition, we investigated CBL2 as a control and a construct in which the first 12 amino acids of the CBL2 protein were replaced by the N terminus of CBL1 (CBL1nCBL2) (see Supplemental Figure 1A online). Faithful expression of all CBL proteins was confirmed by control translations in the presence of [35S]Met, which revealed a similar synthesis of all proteins resulting in single polypeptide bands of ~25 kD (Figure 1B). We next performed in vitro transcriptions/translations with supplementation of [3H]myristic acid as a substrate of N-myristoyltransferase, which resulted in the incorporation of radiolabel into the CBL1 wild-type protein, indicating the myristoylation of this protein at the G2 position (Figure 1B). By contrast, for a CBL1 protein in which the myristate-acceptor Gly was mutated (CBL1G2A), no incorporation of myristate was detectable. Similarly, the CBL1G2AC3S mutant protein was not radioactively labeled. However, a protein carrying only the C3S mutation was myristoylated to a similar extent as the wild-type CBL1, indicating that myristoylation of the G2 residue in CBL1 can proceed independently of a potential S-acylation of the adjacent Cys. In vitro transcription/translation of CBL2, which harbors an internal GC motif, did not result in detectable protein labeling, thereby verifying the specificity of the observed in vitro myristoylation to CBL1. Remarkably, the CBL1nCBL2 fusion protein was myristoylated with similar efficiency as the wild-type CBL1 construct. This result revealed that the first 12 amino acids from CBL1 were sufficient for efficient recognition and modification by the N-myristoyltransferase (Figure 1B). Moreover, we analyzed the in vitro myristoylation of wild-type constructs for CBL4, CBL5, and CBL9 (see Supplemental Figure 1B online) and observed myristoylation of all three proteins. These data suggest that these CBL proteins are subject to myristoylation and that the first 12 amino acids of CBL1 are required and sufficient to allow this lipid modification.

CBL1 Is Acylated at Its N Terminus
We next addressed whether CBL1 could be lipid-modified by S-acylation. In this analysis, we wanted to exclude any possible interference of potential additional S-acylation modifications that can also occur at internal amino acid residues of proteins (Greaves and Chamberlain, 2007Go; Linder and Deschenes, 2007Go). Since we had established that the first 12 amino acids of CBL1 are sufficient for lipid modification, we fused this peptide as well as mutated versions of this peptide (G2A, C3S, and G2AC3S) to the N terminus of green fluorescent protein (GFP), a protein that is known not to be a substrate for S-acylation. To distinguish constructs that harbor only the N-terminal 12 amino acids of CBL1 from fusion proteins containing full-length CBL1, all CBL1 peptide fusion proteins containing only the N-terminal 12 amino acids are designated CBL1n-XXX. As a control, we included wild-type GFP. To investigate the in vivo acylation of CBL1n:GFP, we employed an assay that is based on the in planta expression of recombinant proteins. Subsequent separation by long-range SDS-PAGE and protein detection by protein gel blot analysis allowed the detection of lipid modifications through their influence on protein mobility. The reversible character of S-acylation could further be used to distinguish the identity of the lipid modification. Since the S-acyl group is linked via a thioester linkage to the Cys residue, a treatment with a reducing agent like DTT or hydroxylamine leads to the removal of the acyl group and therefore results in a faster migration of the deacylated protein (Fukata et al., 2004Go).

All constructs were transiently expressed by Agrobacterium tumefaciens–mediated infiltration in Nicotiana benthamiana leaves. Proteins were extracted, subsequently separated by long-range SDS-PAGE, and detected by protein gel blot analysis with GFP-specific antibodies. In these experiments, CBL1n:GFP exhibited the slowest mobility, which reflected the lipid modification of the wild-type peptide (Figure 2A ). By contrast, all GFP fusion proteins carrying mutations in one or both lipid modification sites of the CBL1n peptide migrated faster than the CBL1n:GFP protein, which suggests an altered lipid modification pattern. Notably, the G2A and the G2AC3S mutant proteins migrated faster than CBL1C3Sn:GFP (Figure 2A). This observation suggests the abolishment of both myristoylation and acylation in these fusion proteins, while the C3S mutation still allowed for myristoylation to occur.


Figure 2
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  		Figure 2. Analysis of Protein Lipid Modification by DTT Cleavage of Thioester Bonds.

Proteins were transiently expressed in N. benthamiana leaves and, after protein extraction, separated by high-resolution SDS-PAGE. CBL1nGFP fusion proteins were detected with a GFP-specific antibody after protein gel blot transfer.

(A) CBL1nGFP displayed the slowest mobility compared with non-lipid-modified mutant proteins or GFP wild-type protein.

(B) Treatment with DTT results in a faster migrating protein band of CBL1nGFP (asterisk). This protein migrates at a rate similar to the nonacylatable CBL1nC3SGFP, due to the removal of the thioester bound lipid group.

 
To further corroborate the S-acylation of CBL1n:GFP, we compared protein extracts treated with 200 mM DTT or a buffer control prior to separation by long-range SDS-PAGE. As depicted in Figure 2B, reducing the thioester linkage accelerated the migration of only the wild-type CBL1n:GFP protein. Proteins containing the CBL1G2A and CBL1G2AC3S substitutions did not exhibit any difference in mobility after DTT treatment, indicating that these proteins were not modified by acylation. Comparison of the mobility of the CBL1C3S and the CBL1G2A fusion proteins revealed a faster migration of the CBL1G2A fusion protein in both the DTT-treated and nontreated samples. This difference most likely reflects the stable myristoylation of the CBL1C3S protein, which cannot be removed by treatment with DTT. These data support the results of the in vitro myristoylation analyses, which indicated that myristoylation of the G2 residue occurs independently of acylation of the C3 residue. Moreover, these data suggest that myristoylation is a prerequisite for the subsequent acylation of CBL1. These results provide further evidence for the myristoylation and acylation of CBL1. However, the conclusive interpretation of these results is somewhat limited due to technical constraints, like small protein mobility shifts that may be due to conformational changes induced by DTT.

To establish in vivo S-acylation of CBL1 unambiguously, we determined the fatty acyl modification of CBL1 by gas chromatography–mass spectrometry (GC-MS) analysis. To this end, we expressed CBL1n:GFP and CBL1nC3S:GFP transiently in N. benthamiana leaves. The expression of both fusion proteins was monitored by detecting GFP fluorescence microscopically. The high expression levels of CBL1n:GFP and CBL1nC3S:GFP and their acidic pI values enabled the purification of both proteins to a high degree of homogeneity using only three steps: centrifugal separation of soluble and insoluble membrane fractions, differential ammonium sulfate precipitation, and anion-exchange chromatography. The purified proteins were detected with anti-GFP monoclonal antibodies (Figure 3A ).


Figure 3
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  		Figure 3. In Vivo S-Acylation of CBL1n.

(A) SDS-PAGE of CBL1n:GFP and CBL1nC3S:GFP before (Total) and after purification by differential ammonium sulfate precipitation (AS) and DEAE–cellulose ion-exchange chromatography (DEAE). The degree of protein purification was evaluated by staining gels with Coomassie blue. GFP fusion proteins were identified by immunoblots decorated with anti-GFP antibody ({alpha}GFP).

(B) Initial standards of ethyl palmitate (32 min) and ethyl stearate (34.3 min) derivatives of palmitate and stearate formed by hydrogenation.

(C) CBL1n:GFP is S-acylated by both palmitate and stearate.

(D) No acylation signal is detectable for CBL1nC3S:GFP.

(E) and (F) MS chromatograms of ethyl palmitate (E) and ethyl stearate (F) standards (top panels) and of ethyl palmitate and ethyl stearate released from CBL1n:GFP (bottom panels).

 
Prior to lipid extraction, the purified proteins were subjected to three successive washes in large volumes of pentane to remove any noncovalently bound lipids. The lipids were then released from the purified proteins by hydrogenation. Hydrogenation reactions with CBL1nC3S:GFP were used as negative controls. Indeed, no lipids were extracted from CBL1nC3S:GFP (Figure 3D), and the minor peaks detected along the chromatogram are all recognized column contaminants. To monitor the acyl lipids, the data were examined after GC-MS in the single-ion mode for ion 101, which is typical of acyl lipids (Figures 3E and 3F). Commercially available palmitic and stearic acids were used as internal standards (Figures 3B, 3E, and 3F) for the identification of lipids released from proteins. As a result of hydrogenation, the eluted compounds by GC were ethyl palmitate (32 min), a derivative of palmitate, and ethyl stearate (34.3 min), a derivative of stearic acid (Figures 3B and 3C). The identity of the acyl lipids was further verified by the existence of the molecular ions 284 and 312 for ethyl palmitate and ethyl stearate, respectively (Figures 3E and 3F). To determine the exact site of acylation, we investigated CBL1nC3S:GFP (Figure 3D), which was not S-acylated in our analysis, and found that C3 is the site for CBL1 S-acylation. In a previous work (Sorek et al., 2007Go), we showed that palmitate (C16) does not accumulate as a result of stearate (C18) breakdown. Therefore, these results indicate that CBL1 is S-acylated in vivo on C3 by either palmitate or stearate. Taken together, these results unambiguously prove S-acyl modification of CBL1 in vivo and establish the N-terminal 12–amino acid peptide as being sufficient for dual lipid modification of this protein in planta.

Dual Lipid Modification by S-Acylation and Myristoylation Is Required for CBL1 Function
We next sought to investigate the relevance of both modifications for the functionality of this calcium sensor protein. Our earlier reverse genetics analysis of a cbl1 mutant had established that loss of CBL1 function renders plants hypersensitive to salt stress (Albrecht et al., 2003Go). Therefore, we pursued a complementation approach based on the salt-sensitive phenotype of the cbl1 mutant. To this end, we transformed cDNAs encoding the CBL1 wild-type sequence as well as cDNAs encoding the G2A, C3S, and G2AC3S substitutions under the control of the constitutive 35S promoter into the cbl1 mutant. In addition, we transformed this mutant with constructs expressing CBL2 and the chimeric CBL1nCBL2 construct, which had been shown to represent a substrate for dual lipid modification (Figure 1). For each construct, three independent homozygous F2 lines were generated and initially analyzed. The presence of the transgenes and their expression levels were verified by genomic PCR and RT-PCR, respectively (see Supplemental Figure 1C online). Survival rates of the seedlings were scored after 8 d of exposure to salt stress. All analyzed transgenic lines exhibited a similar phenotype. Therefore, one representative line for each construct was chosen for detailed analyses, which were performed in triplicate.

As presented in Figure 4 and in Supplemental Figure 1D online, only expression of the CBL1 wild-type protein in the cbl1 mutant background was able to complement the salt sensitivity of the cbl1 knockout (51.7 ± 6.3% survival compared with 7.5 ± 2.5% survival for cbl1 seedlings). Consistent with our earlier analysis of CBL1 overexpression lines, we did not observe complete restoration of salt tolerance to wild-type levels (92.5 ± 2.5% survival) (Figure 4) due to toxicity effects of CBL1 overexpression that negatively affect the expression of functional CBL1 protein in transgenes (Albrecht et al., 2003Go).


Figure 4
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  		Figure 4. Both G2A and C3S Mutations Abolish CBL1 Function in Plant Salt Tolerance.

The Arabidopsis cbl1 mutant was transformed with cDNAs for CBL1 (cbl1/CBL1), mutated versions of CBL1 (cbl1/CBL1G2A, cbl1/CBL1C3S, and cbl1/CBL1G2Ac3S), CBL2 (cbl1/CBL2), and CBL1nCBL2 (cbl1/CBL1nCBL2). Survival rates of T2 plants were determined after 8 d of cultivation on medium supplemented with 100 mM NaCl. Only significant numbers of wild-type plants and cbl1 plants expressing the normal CBL1 protein were able to survive this treatment. CBL1nCBL2, which is targeted to the plasma membrane and interacts with CIPK24, was not able to complement the salt-hypersensitive phenotype. Shown are mean values of the survival rate in percentage with SD (n = 3 experiments; 40 plants in each experiment).

 
Overexpression of any CBL1 mutant versions (G2A, C3S, or G2AC3S) in the cbl1 mutant background only slightly elevated the survival rate of these lines above the percentage observed for the cbl1 mutant (5.8 ± 1.4% survival for CBL1G2A, 18.3 ± 1.4% for CBL1C3S, and 17.5% for CBL1G2AC3S). Similarly, overexpression of the vacuolar membrane (tonoplast)–localized CBL2, which, like CBL1, interacts with the kinases CIPK1 and CIPK24 (Albrecht et al., 2001Go, 2003Go), did not efficiently complement the cbl1 phenotype (survival rate of 21.7 ± 2.9%). This result indicates a rather high functional specificity of the distinct CBL isoform in signal–response coupling. Interestingly, overexpression of the chimeric CBL1nCBL2 protein, which not only can interact with the salt tolerance–mediating kinase CIPK24 but also undergoes dual lipid modification and localizes to the plasma membrane (see below), only marginally increased the salt tolerance when compared with the cbl1 mutant (11.7 ± 1.4%) (Figure 4). To interpret the results of the complementation analysis, it needs to be considered that although all transgenic lines were selected for similar levels of transgene RNA expression, we reproducibly observed differences in the amount of protein accumulation. While the expression of the CBL2, CBL1C3S, and CBL1G2AC3S constructs was detectable by protein gel blot analysis, protein expression of the functional wild-type CBL1 construct was hardly detectable. This may point to a posttranscriptional regulation of CBL1 accumulation that interferes with overexpression. In addition, this may explain the partial rescue observed in the CBL2, CBL1C3S, and CBL1G2AC3S transgenic lines; perhaps these proteins are able to accumulate close enough to the plasma membrane to partially substitute for CBL1 function. In summary, these experiments support the importance of both myristoylation and acylation for the functioning of the calcium sensor CBL1 and provide evidence that the specific function of distinct CBLs is irreplaceable and requires precise targeting of the particular calcium sensor to the respective compartment.

Myristoyl and Acyl Modifications Are Necessary for Membrane Attachment of CBL1
We next investigated biochemically whether the lipid modification has an effect on membrane attachment. To this end, we expressed full-length CBL:GFP fusion proteins transiently in N. benthamiana leaves and prepared native protein extracts. In these analyses, we included GFP fusion proteins of CBL1, CBL1G2A, CBL1C3S, CBL1G2AC3S, CBL2, and CBL1nCBL2 (Figure 5 ). After biochemical separation of soluble and membrane fractions, CBL1:GFP fusion proteins were detected after SDS-PAGE and protein gel blotting with an anti-GFP antiserum.


Figure 5
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  		Figure 5. Both Myristoylation and Acylation Are Required for the Membrane Association of CBL1.

CBL:GFP fusion proteins were expressed in N. benthamiana leaves, isolated, and separated by centrifugation into soluble (S) and insoluble fractions containing membranes (P). Lipid modification of CBLs (or preventing the modification) resulted in different distribution of the proteins. Lane 1, negative control (A. tumefaciens expressing only the helper plasmid 19K); lane 2, CBL1:GFP; lane 3, CBL1G2A:GFP; lane 4, CBL1C3S:GFP; lane 5, CBL1G2AC3S:GFP; lane 6, CBL2:GFP; lane 7, CBL1nCBL2:GFP.

 
While CBL1, CBL2, and CBL2nCBL1 fusions were predominantly detected in insoluble pellet fractions, fusion proteins with disrupted acceptor sites for lipid modifications (i.e., CBL1G2A, CBL1C3S, and CBL1G2AC3S fusions) accumulated to a substantial extent in soluble fractions. This indicates that lipid modification of CBL1 is crucial for the attachment to cellular membranes.

Lipid Modification Determines the Localization of CBL1 in Planta
To assess further the significance of lipid modifications for the function of CBL1 in planta, we comparatively investigated the subcellular localization of the CBL1 wild-type protein and its mutant versions with substituted lipid modification residues by microscopic analysis of GFP fusion proteins that were transiently expressed in N. benthamiana leaves. The integrity of the investigated fusion proteins was confirmed by protein gel blot analysis (see Supplemental Figure 1E online). We investigated the distribution of GFP fluorescence in epidermal cells of intact leaves as well as in protoplasts (Figure 6 ). In these experiments, we observed a ring-like distribution of CBL1:GFP at the plasma membrane that was excluded from the cytoplasm and nucleoplasm in protoplasts. The nonmyristoylatable CBL1G2A:GFP and CBL1G2AC3S:GFP proteins exhibited a cytoplasmic and nucleoplasmic distribution. By contrast, in cells expressing CBL1C3S:GFP, fluorescence was detected not only in the cytoplasm and nucleus but also to a substantial extent at the nuclear envelope (indicated by the arrow in Figure 6) and at internal cellular structures that resembled the ER (Figure 6; see Supplemental Figure 2A online) . Subsequent colocalization analysis of CBL1C3S:GFP fluorescence with an ER-localized orange fluorescence protein containing a HDEL ER retention signal (see Supplemental Figure 2A online) clearly confirmed an ER localization. As this protein can undergo myristoylation but not acylation, these results imply that the CBL1 protein becomes myristoylated during its synthesis in the cytoplasm, that myristoylation is important for ER targeting of the protein, and that subsequent acylation at the ER enables further trafficking to the plasma membrane.


Figure 6
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  		Figure 6. Localization of CBL:GFP Fusion Proteins in Plant Cells.

Binary plasmids expressing the GFP fusion proteins indicated at left were expressed in N. benthamiana leaves by A. tumefaciens–mediated infiltration. The right panels depict the fluorescence in intact leaf epidermal cells (together with bright-field images on the left), and the left panels show the localization in protoplasts prepared from the same leaf (together with bright-field images on the left).

 
Because we had observed a membrane association of CBL2 in our biochemical analysis, we were also interested in determining the subcellular localization of this protein. In contrast with CBL1, investigation of CBL2 localization revealed an exclusive distribution of fluorescence at the tonoplast (Figure 6; see Supplemental Figure 3 online).

The ascertained localization of CBL1 at the plasma membrane and CBL2 at the tonoplast enabled us to investigate whether the lipid modifications of the N-terminal 12 amino acids of CBL1 determine protein localization exclusively. To this end, we first examined the distribution of GFP fusion proteins with CBL1nCBL2 in leaves to investigate whether the N terminus of CBL1, added to CBL2, is sufficient to target the resulting hybrid protein. In addition, we analyzed the localization of G2A, C3S, and G2AC3S mutants of CBL1nCBL2. The CBL1nCBL2:GFP fusion protein was detected exclusively at the plasma membrane (Figure 7 ). All mutant versions of the CBL1nCBL2 hybrid protein displayed exactly the same cellular fluorescence pattern as the respective mutant CBL1:GFP fusion proteins. These results unambiguously establish the N-terminal peptide of CBL1 and its lipid modification status as dominant determinants for the cellular localization of the affected protein. To further assess the importance and specificity of S-acylation for CBL1 localization, we investigated the localization of CBL1:GFP fusion proteins after the application of the palmitoyl inhibitor 2-bromopalmitate (Lavy et al., 2002Go). In these experiments, application of 2-bromopalmitate resulted in an accumulation of CBL1:GFP at internal membranes that was not observed after the application of 10% DMSO as a control (see Supplemental Figure 4A online). These results further suggest that specific modification by S-acylation is essential for CBL1 plasma membrane targeting. In an additional set of experiments, we added the N terminus of CBL1 and its mutated versions to GFP as a general soluble and noncalcium binding protein (see Supplemental Figure 4B online). The resulting fusion proteins displayed the same cellular distribution as observed for the respective CBL1 and CBL1nCBL2:GFP fusion proteins.


Figure 7
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  		Figure 7. Cellular Localization of CBL1nCBL2:GFP and Derived Mutated Versions (G2A, C3S, and G2AC3S) in N. benthamiana Leaves.

Binary plasmids expressing the GFP fusion proteins indicated at left were expressed in N. benthamiana leaves. The right panels depict fluorescence patterns of intact leaf epidermal cells (together with bright-field images on the left), and the left panels show the localization of GFP fusion proteins in protoplasts prepared from the same leaves (together with bright-field images on the left).

 
Taken together, these data confirm that both lipid modifications are important for plasma membrane targeting in planta and furthermore corroborate that myristoylation is important for ER targeting, which serves as the starting point to route the protein to the plasma membrane. Moreover, these and biochemical separation results (see Supplemental Figure 2B online) indicate that calcium binding of CBL1 is not required for membrane association and thereby suggest that the localization of CBL1 is not regulated by a calcium-myristoyl-palmitoyl switch mechanism. To further corroborate this conclusion, we investigated the membrane targeting of CBL1:GFP in the presence of EDTA or calcium and did not observe any differences in localization (see Supplemental Figure 2B online).

BFA and Sar1 Do Not Affect Acylation and Plasma Membrane Targeting of CBL1
Until now, the biosynthesis and targeting of only a few myristoylated and palmitoylated proteins, like Lck, Fyn, and heterotrimeric G-protein subunits, have been analyzed in detail (Bijlmakers and Marsh, 2003Go). In these studies, BFA, a noncompetitive inhibitor of ARF-GTP exchange factors, has been shown to inhibit the transport of several proteins, such as Lck, to the plasma membrane. Moreover, BFA can also inhibit the palmitoylation of proteins, as observed for GAP43 (Bijlmakers and Marsh, 2003Go). Since BFA interferes with post-ER transport processes, a BFA-independent targeting of CBL1 to the plasma membrane would be indicative of S-acylation of this protein at the ER.

We compared the localization of CBL1:GFP and CBL1nCBL2:GFP in transiently transformed N. benthamiana leaves that were treated with BFA or with DMSO as a solvent control. As a control for the efficiency of the BFA treatment, we analyzed the BFA-sensitive localization of TM23:GFP, a modified GFP containing a transmembrane domain (Brandizzi et al., 2002Go).

Surprisingly, while the localization TM23:GFP was strongly affected by BFA, leading to a complete relocation of fluorescence from the plasma membrane to internal compartments, the plasma membrane targeting of CBL1:GFP and CBL1nCBL2:GFP was not inhibited by BFA (Figure 8A ). Therefore, we additionally investigated the localization of the CBL1nGFP fusion protein and did not observe an effect of BFA on the steady state plasma membrane localization (see Supplemental Figure 5A online). Consequently, these data not only establish that ER-to-plasma membrane trafficking of CBL1 relies on a BFA-insensitive pathway but also indicate that the N-terminal peptide of CBL1 represents a sufficient and specific signal for this intracellular protein-sorting pathway. Moreover, these findings suggest that acylation of CBL1 is not impaired by BFA.


Figure 8
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  		Figure 8. Effects of BFA and Nt Sar1H74L on CBL1 Localization.

(A) The constructs indicated at left were transiently expressed in N. benthamiana leaves. Two days after infiltration, BFA (100 µM) was applied to the leaves and incubated for 16 h. While BFA relocated the normally plasma membrane–localized GFP:TM23 protein to BFA compartments (arrows), the plasma membrane localization of CBL1:GFP and CBL1nCBL2:GFP was not affected.

(B) Coexpression of Nt Sar1H74L also affects the localization of GFP:TM23, resulting in fluorescence around the nucleus (arrow). Instead, targeting of CBL1:GFP was not affected when Nt Sar1H74L was coexpressed.

 
In addition, we coexpressed a dominant negative mutant of the tobacco (Nicotiana tabacum) Sar1 protein (H74L), which has been reported to block vesicle trafficking between the ER and the Golgi (Andreeva et al., 2000Go). As shown in Figure 8B, targeting of GFP:TM23 was severely affected when Nt Sar1H74L was coexpressed, leading to strong fluorescence, especially surrounding the nucleus, which we identified as representing the ER. By contrast, targeting of CBL1 appeared not to be affected when Nt Sar1H74L was coexpressed (in any of our repeated investigations). These findings support the conclusion that CBL1 is targeted to the plasma membrane via a novel transport system.

CBL1-Dependent Membrane Targeting of the Kinase CIPK1 Requires Dual Lipid Modification of the Calcium Sensor Protein
To investigate whether targeting of CIPK1 to the plasma membrane depends on the lipid modifications of CBL1, we employed the yeast Ras recruitment system (Broder et al., 1998Go) and introduced wild-type and mutated versions (G2A, C3S, and G2AC3S) of CBL1 into yeast and coexpressed these proteins with CIPK1. These plasmid combinations were tested for complementation of the temperature-sensitive phenotype of cdc25, which is indicative of an interaction between CIPK1 and the respective version of CBL1 at the plasma membrane (Figure 9A ). While coexpressing wild-type CBL1 with CIPK1 complemented temperature sensitivity, none of the mutated CBL1 proteins (G2A, C3S, or G2AC3S) was able to restore the growth of the cdc25 mutant at restrictive temperatures. These results clearly indicate that both myristoylation and acylation of the CBL1 protein are required to target the CBL1/CIPK1 complex to the plasma membrane in yeast.


Figure 9
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  		Figure 9. Complementation of the Yeast cdc25-2 Mutant through the Interaction of CBL1 with CIPK1 at the Plasma Membrane.

The yeast strain cdc25-2 containing the plasmid combinations indicated at left (CBL in pVT-U plasmid and RAS:CIPK1 in pADNS plasmid) was grown in selective medium and spotted for 3 d at permissive (24°C; left panels) or restrictive (35°C; right panels) temperatures. Decreasing cell densities in the dilution series are illustrated by narrowing triangles.

(A) Cells expressing both CBL1 and the RAS:CIPK1 fusion protein were able to grow at 35°C, indicating interaction between the two proteins at the plasma membrane. No plasma membrane targeting was observed when CBL1G2A, CBL1C3S, or CBL1G2AC3S was analyzed in combination with CIPK1.

(B) While the combination of CBL2 with CIPK1 did not restore yeast growth at the restrictive temperature, the fusion protein CBL1nCBL2 containing the lipid modification sites of CBL1 was able to target CIPK1 to the membrane.

 
As shown in Figure 9B, tonoplast-bound CBL2 was not able to complement the cdc25 mutation. In agreement with the results of our in planta localization studies (Figure 6), coexpression of CIPK1 and the chimeric protein CBL1nCBL2 resulted in the partial complementation of cdc25. These results provide additional support for the conclusion that the first 12 amino acids of CBL1 are sufficient for plasma membrane targeting of the protein (and the CBL1/CIPK1 complex) and that this N terminus undergoes dual lipid modification.

In Planta Localization of the CBL1/CIPK1 Complex
To directly investigate the influence of CBL1 lipid modification on CBL1/CIPK1 complex formation and localization in vivo, we used bimolecular fluorescence complementation (Walter et al., 2004Go). To this end, cDNAs encoding wild-type CBL1 and mutated versions of CBL1 (G2A and C3S) were cloned into pSPYCE-35S vectors and a cDNA encoding CIPK1 was cloned into the pSPYNE-35S vector. The respective combinations of plasmids were introduced into N. benthamiana leaves, and fluorescence was analyzed after 3 d of incubation. In these assays, CBL1/CIPK1 complexes were almost exclusively detected at the plasma membrane, with weak residual fluorescence found at the nuclear envelope (Figure 10A ). We also observed specific fluorescence patterns resulting from the formation of CBL1G2A/CIPK1 and CBL1C3S/CIPK1 complexes, thereby indicating that the in planta interaction of CBL1 with its target kinase CIPK1 does not require lipid modification of the calcium sensor protein. However, CBL1G2A/CIPK1 complexes exhibited a cytoplasmic–nucleoplasmic distribution, while CBL1C3S/CIPK1 complexes were detected at the nuclear envelope and the ER (Figure 10A). Importantly, the localization of the respective CBL/CIPK complexes was identical to the localization observed for the respective wild-type and mutated CBL1:GFP fusions (Figure 6). Taken together, this analysis suggests that in plant cells the localization and targeting of preassembled calcium sensor/protein kinase complexes are exclusively regulated by the lipid modification status of the calcium sensor moiety. In addition, we investigated whether CIPK1 can interact with CBL2 at the tonoplast. Indeed, using CBL2-SPYCE together with CIPK1-SPYNE, we observed fluorescence at the tonoplast (Figure 10B). However, when CBL1nCBL2 was used as the interacting partner of CIPK1, this interaction was detected at the plasma membrane, corroborating the dominant targeting function of the N-terminal CBL1 peptide in plant cells.


Figure 10
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  		Figure 10. Localization of CBL/CIPK1 Complexes in Plant Cells.

(A) Investigation of the interaction of CBL1, CBL1G2A, and CBL1C3S with CIPK1 by bimolecular fluorescence complementation. Plasmid combinations are indicated at left.

(B) Formation and localization of CBL2/CIPK1 and CBL1nCBL2/CIPK1 complexes as revealed by bimolecular fluorescence complementation. Plasmid combinations are indicated at left.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
METHODS
 REFERENCES
 
CBLs determine the cellular localization of their interacting protein kinases (D'Angelo et al., 2006Go; Xu et al., 2006Go; Cheong et al., 2007Go). The resulting CBL/CIPK complexes exert important functions at the plasma membrane by regulating the activity of ion channels and H+-ATPases (Xu et al., 2006Go; Fuglsang et al., 2007Go). The aims of this study were to investigate the potential dual lipid modification of CBL proteins by myristoylation and acylation and to unravel the influence of these lipid modifications on the functional regulation of processes decoding calcium signals. Our studies identify myristoylation and S-acylation by palmitic and stearic acids as essential modifications for calcium sensor function and report novel steps in the plasma membrane transport of acylated CBL proteins and in the membrane targeting of CBL/CIPK complexes. Importantly, our observation that the lipid modification status of the CBL protein determines the targeting of preassembled CBL/CIPK complexes provides a novel mechanism for regulating and directing signal transduction processes.

We show that the CBL1 protein undergoes myristoylation at the G2 position and that this modification occurs independently of the acylation at the adjacent C3 position. Identical results were obtained when all four plasma membrane–localized CBLs, CBL1, CBL4, CBL5, and CBL9, were investigated in in vitro myristoylation assays, indicating that myristoylation represents a general feature of plasma membrane–localized CBLs. Our analysis also revealed that the first 12 amino acids of CBL1, when fused to CBL2, are sufficient to provide a substrate for N-myristoyltransferase. This observation enabled the investigation of CBL1n:GFP acylation by DTT-mediated cleavage of the thioester-linked acyl groups from CBL1. The results of this experiment suggest that CBL1 is indeed subject to acylation at the C3 position and that myristoylation at G2 represents an essential prerequisite for subsequent acylation. We subsequently determined the fatty acyl modification of CBL1n:GFP by GC-MS analysis. This investigation unequivocally established the modification of this protein by palmitic and stearic acids. A similar dual fatty acyl modification was reported previously for the Arabidopsis Rho-related GTPase, ROP6 (Sorek et al., 2007Go). Although the S-acylation modification of very few plant proteins has been determined experimentally, it is tempting to speculate that such dual palmitoyl–stearyl modification represents a general feature of plant proteins. Due to the design of our assays, these results of course do not exclude potential additional acylation of the native CBL1 protein at amino acid residues located C terminal to the peptide investigated. Previous analysis of CBL4/SOS3 revealed N-terminal myristoylation of this protein but did not address a potential modification by S-acylation (Ishitani et al., 2000Go). With the exception of the flagellar calcium binding protein FCaBP from T. cruzi (Godsel and Engman, 1999Go), none of the calcium binding proteins belonging to the group of recoverin- or calcineurin-like calcium sensor proteins appears to be modified by dual lipid modification via myristoylation and S-acylation. Consequently, this unique and reversible pattern of lipid modification of CBLs may allow for novel, CBL-specific functional implications in the execution of calcium-decoding functions by CBL proteins.

We employed the salt-sensitive phenotype of the cbl1 loss-of-function mutant in a complementation approach to investigate the importance of myristoylation and acylation for CBL1 function. Our finding that both a G2A and a C3S mutation interfere with CBL1 function in salt tolerance establishes the importance of both lipid modifications for proper CBL1 function. Also of interest is our observation that the expression of CBL1nCBL2 in the cbl1 mutant did not complement its salt-sensitive phenotype. In yeast two-hybrid analyses, both CBL1 and CBL2 interact with CIPK24/SOS2, the CBL-interacting protein kinase mediating salt tolerance in Arabidopsis (Albrecht et al., 2003Go; Kolukisaoglu et al., 2004Go). Replacement of the N terminus of CBL2 with the N terminus of CBL1 redirects the localization of the resulting fusion protein to the plasma membrane (Figure 8), where CBL1 usually exerts its function. The failure of this fusion protein to functionally compensate for the loss of CBL1 function, therefore, suggests that, in addition to subcellular localization and specificity in CIPK interaction, factors that are inherent to the particular version of the CBL protein determine the functional specificity of the resulting CBL/CIPK complex toward its substrate(s). Such a functional specificity determinant may lie in the differences in the EF hand composition of CBL1 and CBL2, which could lead to distinct Ca2+ binding abilities (Batistic and Kudla, 2004Go).

Palmitoylation has been shown to affect protein trafficking, protein stability, and protein aggregation (Linder and Deschenes, 2007Go). Our investigation of the protein levels of the CBL1 forms when expressed in N. benthamiana leaves (Figure 5; see Supplemental Figure 1E online) suggests that an influence of myristoylation or S-acylation on CBL1 stability and accumulation is rather unlikely. Instead, our biochemical investigation of CBL1 distribution in soluble and insoluble protein extracts pointed to a requirement of dual lipid modification of CBL1 for membrane association. Again, both myristoylation and S-acylation appear to be required for efficient membrane association. These results are in agreement with a required but not sufficient role of myristoylation in membrane tethering and a function of palmitoylation as an essential secondary signal for membrane association (Greaves and Chamberlain, 2007Go). The lipid modification–dependent membrane association of CBL1 distinguishes this protein from CBL4/SOS3. A previous study of this calcium sensor (Ishitani et al., 2000Go) did not detect an influence of myristoylation on the membrane association of CBL4/SOS3 in either biochemical separation analyses or localization studies using GFP fusion proteins. In both instances, a G2A mutation preventing myristoylation did not alter the subcellular distribution of the protein, and why myristoylation was important for CBL4 function remained a mystery (Ishitani et al., 2000Go). A potential explanation for this difference may be provided by our observation that CBL4 harbors a polybasic stretch of five successive amino acids within the first 12 residues of its N terminus. This structural feature of CBL4 may mediate a certain extent of membrane association even in the absence of lipid modification of the N terminus.

Our investigation of the cellular localization of CBL1:GFP fusion proteins established distinct functions of myristoylation and S-acylation for CBL1 sorting. While CBL1G2A was exclusively localized in the nucleus and cytoplasm, CBL1C3S accumulated in the ER and did not reach the plasma membrane, like the dual lipid–modified CBL1 protein (Figure 7). Importantly, colocalization analysis of CBL1C3S:GFP with the ER marker protein OFP:HDEL corroborated the ER localization of the myristoylated but not the acylated CBL1 protein. These results imply that myristoylation of CBL1 is required for cytoplasm-to-ER sorting of the protein, where it subsequently undergoes palmitoylation by an ER-localized palmitoyl transferase (PAT) as an essential step for effective trafficking to the plasma membrane. Remarkably, treatment with BFA, or coexpression with the dominant negative Sar1 mutant, did not impede CBL1 trafficking or palmitoylation (Figure 8; see Supplemental Figure 5 online). This distinguishes CBL1 from the well-characterized cytosolic proteins, like Lck, N-Ras, SNAP-25, and G{alpha}z, for which palmitoylation as a signal for protein sorting has been established (Greaves and Chamberlain, 2007Go). Like CBL1, Lck as well as N-Ras and H-Ras are palmitoylated at intracellular compartments. However, the transport of these proteins to the plasma membrane is blocked by BFA (Bijlmakers and Marsh, 2003Go). In the case of SNAP-25 and GAP-43, palmitoylation at intracellular compartments is prevented by BFA. Similar to CBL1, sorting of G{alpha}z is not affected by BFA. However, this protein undergoes palmitoylation at the plasma membrane. As BFA inhibits vesicle transport between the ER and the Golgi, negative effects of BFA treatment on protein sorting or palmitoylation likely reflect an involvement of the Golgi compartment in these processes. The findings that nonacylatable CBL1 resides at the ER and that the coexpression of the dominant negative Sar1 mutant or treatment with BFA does not impede ER-to-plasma membrane sorting of CBL1 point to direct trafficking of CBL1 from the ER to the plasma membrane, without any involvement of the Golgi organelle. Similar to our observations, Hasdemir et al. (2005)Go reported that KChIP-dependent trafficking of the Kv4.2 channel was Sar1(H79G)-independent, indicating that ER-to-Golgi transport was independent of COPII vesicles. However, transport of CBL1/CIPK1 complexes is distinguished from the trafficking of KChIP/Kv4.2 complexes by their dependence on COPI vesicles. While blocking of ARF GTPase function interferes with KChIP/Kv4.2 exocytic transport, such treatment does not affect CBL1/CIPK1 trafficking.

In addition, cellular transport of CBL1 resembles the trafficking of Ras2p in Saccharomyces cerevisiae (Linder and Deschenes, 2007Go). In yeast, Ras2p proteins are localized to the plasma membrane even when the secretory pathway is blocked by treatment with BFA (Dong et al., 2003Go). Ras2p proteins from S. cerevisiae are palmitoylated at the ER by the ERF2/ERF4 PAT (Linder and Deschenes, 2007Go). It is tempting, therefore, to speculate that in Arabidopsis a similar pathway for CBL calcium sensor palmitoylation exists in that ER-localized ERF2/ERF4-like PATs modify these proteins at the ER and thereby enable BFA-independent trafficking to the plasma membrane by a yet unknown targeting pathway. Although the analysis of S-acylation processes in plants is still in its infancy, a survey of the Arabidopsis genome identified 23 PATs of the DHHC-CRD type, of which at least 2 proteins display significant similarity with ERF2/ERF4 (Hemsley et al., 2005Go). Although this situation does not exclude the possibility that CBL1 represents a substrate for PATs that are unrelated to ERF2/ERF4, a potential modification of CBL1 by ERF-like PATs needs to be seriously considered.

Our results demonstrating that the first 12 amino acids of CBL1 in either their lipid-modifiable form or as mutated versions absolutely mimic the localization, trafficking, and BFA resistance of the native CBL1 protein make the occurrence of a calcium-myristoyl-palmitoyl switch for the membrane association of CBL1 rather unlikely. Instead, since the GFP moiety of these fusion proteins does not bind calcium, and since the N terminus of CBL1 does not contain EF hands and is sufficient for fusion protein targeting, these data point to a calcium-independent plasma membrane association of CBL1. In agreement with this conclusion are our observations that treatment with Ca2+ or chelators of divalent cations did not affect the plasma membrane association of CBL1 (see Supplemental Figure 2B online). In addition, the recently solved crystal structure of CBL4/CIPK24 (Sanchez-Barrena et al., 2007Go) does not support lipid-dependent rearrangements of the N terminus, and our inspection of the CBL1 amino acid sequence revealed nonconserved substitutions in residues critical for a calcium-myristoyl switch (e.g., residues corresponding to Gly-96 in recoverin). Therefore, we conclude that CBL1 as well as the other plasma membrane–localized CBL proteins are not regulated by a calcium-myristoyl switch.

CBL proteins exert their function by interacting with and activating CIPK-type Ser/Thr protein kinases. We show here that CBL1 interacts with its target kinase CIPK1 in vivo independently of the calcium sensor lipid modification status (Figure 9). Most importantly, our results provide evidence that the lipid modification status of the CBL calcium sensor moiety determines the localization of the respective CBL/CIPK complex. These findings support a model in which CBL1/CIPK1 complexes assemble in the cytoplasm and the myristoylation of CBL1 allows for sorting to the ER. Here, additional acylation of CBL1 enables trafficking of the calcium sensor/protein kinase complex to the plasma membrane. Our results demonstrating that preassembled CBL/CIPK complexes are subject to sorting to their final cellular destination are important for understanding the formation and function of alternative CBL/CIPK complexes. While CBL1/CIPK1 complexes are localized at the plasma membrane, CBL2/CIPK1 complexes accumulate at the tonoplast (Figure 10; see Supplemental Figure 5 online). Our data suggest that the molecular events contributing to decoding calcium signals and leading to alternative complex formation of CIPK1 with either CBL1 or CBL2 already occur in the cytoplasm and that subsequent trafficking of the complex to its respective target localization relies on cellular sorting machinery. Although this model does not exclude regulated complex sorting as a mechanism of calcium signaling in the CBL/CIPK system, it emphasizes the regulation of CBL/CIPK complex formation as a crucial step in decoding calcium signals.

Altogether, our results establish the importance of myristoylation and S-acylation of CBL1 for its proper function. The crucial importance of both lipid modifications is due to their function as signals in cytoplasm-to-ER-to-plasma membrane trafficking of this calcium sensor protein. Modification of CBL1 by S-acylation is identified as the key in targeting CBL1/CIPK1 complexes by a novel, BFA- and Sar1-independent sorting pathway to the plasma membrane. This acylation-directed, CBL-dependent sorting of calcium sensor/protein kinase complexes represents an important mechanism in calcium signaling that likely contributes to generating signaling specificity and efficient signal processing.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 METHODS
REFERENCES
 
General Methods, Plant Material and Cultivation, and Ras Recruitment Assays
Molecular biology methods were performed according to standard procedures (Sambrook and Russell, 2001Go). A list of primers used in this work is presented in Supplemental Table 1 online. The identity of all plasmid constructs generated in this study was verified by sequencing. Arabidopsis thaliana cv Wassilewskija plants were used in this work and, except for the stress treatments indicated, cultivated in a growth chamber under a 16-h-light/8-h-dark cycle with 70% atmospheric humidity and a 22°C (day)/18°C (night) temperature regimen. Nicotiana benthamiana plants were cultivated in a greenhouse under a 12-h-light/12-h-dark cycle with 60% atmospheric humidity at 20°C.

Membrane targeting of CIPK1 by CBL1, CBL1 mutants, and CBL2 was investigated using the Ras recruitment system as described by D'Angelo et al. (2006)Go.

In Vitro Myristoylation Assays
To generate plasmids suitable for coupled in vitro transcription/translation, cDNAs of CBL1, and respective mutants of CBL1 as well as CBL2 and CBL2nCBL1, were amplified by PCR and cloned in the pET24b(+) vector (Novagen-EMD Chemicals). For coupled in vitro transcription/translation, the TNT T7-coupled reticulocyte lysate system (Promega) was used according to the manufacturer's instructions. For control reactions, 0.5 µg of plasmid was incubated in reticulocyte lysates containing 20 µCi of [35S]Met in a total reaction volume of 25 µL.

For myristoylation assays, [35S]Met was replaced by nonlabeled Met and 25 µCi of [3H]myristic acid was added and incubated as stated in the manufacturer's manual. After the synthesis, proteins were separated on a 14% SDS-PAGE gel, fixed on the gel by incubation in 10% acetic acid, and treated with Amplify solution (GE Healthcare). 35S-labeled proteins were detected overnight at –80°C using Kodak Biomax MR films (GE Healthcare). Detection of [3H]myristic acid–labeled proteins was performed after an exposure time of 4 weeks at –80°C with Hyperfilm MP films (GE Healthcare).

Protein Isolation, Protein Mobility Assays, and Membrane Fractionation
N. benthamiana leaves were infiltrated with Agrobacterium tumefaciens expressing the respective plasmid combination, and leaf samples were harvested at 3 to 5 d after infiltration and immediately frozen in liquid nitrogen. Denatured protein extracts were obtained by incubating homogenized plant material in sample buffer (62.5 mM Tris, pH 6.8, 4% SDS, 10% glycerol, and 10 mM DTT) for 10 min at 80°C. The material was then centrifuged (15 min, 20,000g, 4°C), and the supernatant was stored at –80°C. To remove thioester-linked lipid groups from proteins, the material was incubated in buffer for 10 min at 95°C supplemented with 200 mM DTT (Fukata et al., 2004Go). To analyze protein mobility, proteins were separated by long-range SDS-PAGE (15.5%) using the PerfectBlue Dual Gel System Twin L from Peqlab.

To obtain native protein extracts, material was resuspended in extraction buffer (50 mM HEPES, pH 7.5, 10 mM KCl, supplemented with protease inhibitor cocktail from Sigma-Aldrich) at 4°C for 30 min by slow rotation. After extraction, the suspension was centrifuged for 10 min at 3,000g, and the supernatant was additionally centrifuged for 15 min at 10,000g and for 5 min at 20,000g. Finally, to separate soluble and insoluble proteins, the supernatant was centrifuged for 1 h at 100,000g. The resulting pellet was resuspended in 50 mM HEPES, pH 7.5, 50 mM NaCl, 1.5% SDS, and 1% Triton. All steps were performed at 4°C or on ice.

To detect GFP, CBLnGFP, and CBL:GFP, proteins were blotted to a polyvinylidene difluoride membrane after SDS-PAGE and incubated with 1:4000 rabbit anti-GFP antibody (Invitrogen) and 1:10,000 (CBL:GFP) or 1:50,000 (GFP, CBLnGFP) goat anti-rabbit antibody (Bio-Rad) conjugated with horseradish peroxidase. Detection was performed by an enhanced chemiluminescence reaction.

Fluorescence Microscopy
The production of CBL1.GFP, CBL1.SPYCE, and CIPK1.SPYNE constructs has been described (D'Angelo et al., 2006Go). Mutated versions of CBL1 cDNA, CBL2, and mutated versions of CBL2nCBL1, as well as CBL1nGFP and its mutated versions, were introduced into either pGPTVII.GFP or pSPYCE (Walter et al., 2004Go). Plasmids were introduced into A. tumefaciens (GV3101 pMP90), and infiltration of N. benthamiana leaves was performed as described (Walter et al., 2004Go; Waadt and Kudla, 2008Go). Protoplast preparations were performed according to D'Angelo et al. (2006)Go. Fluorescence microscopy was performed with an inverted microscope (Leica DMIRE2) equipped with the Leica TCS SP2 laser-scanning device (Leica Microsystems). Detection of fluorescence was performed as follows: GFP, excitation at 488 nm (Ar/Kr laser), scanning at 500 to 535 nm; yellow fluorescent protein (bimolecular fluorescence complementation), excitation at 514 nm (Ar/Kr laser), scanning at 525 to 600 nm; OFP, excitation at 543 nm (He/Ne laser), scanning at 565 to 595 nm. Autofluorescence of plastids was detected at 650 to 720 nm. All images were acquired using a 63x/1.20 water-immersion objective (HCX PL Apo CS) from Leica. All laser-scanning confocal micrographs presented in this study are optical sections of the middle position in the cell.

For BFA (Applichem) treatments, this substance was diluted to 100 µM in water from a 100 mM stock solution, dissolved in 100% DMSO, and directly infiltrated into N. benthamiana leaves (Walter et al., 2004Go; Waadt and Kudla, 2008Go). After infiltration, leaf discs were collected and further incubated in water supplemented with 100 µM BFA and 0.1% Tween for 16 h at room temperature under continuous light. As a control, BFA was omitted and DMSO (0.1% in water) was used. To inhibit the vesicular transport in protoplasts, protoplasts were directly prepared in enzyme solution containing 100 µM BFA or 0.1% DMSO for 3 h. Treatments with 2-bromopalmitate were performed according to Lavy et al. (2002)Go.

Complementation of the cbl1 Mutant and Salt Stress Analysis
For complementation analysis of cbl1 mutant plants, cDNAs of CBL1 and the respective mutated versions of CBL1, as well as of CBL2 and CBL2nCBL1, were amplified by PCR and cloned into the pGPTV-Bar vector (Walter et al., 2004Go). Plant transformation was performed by the floral dip method (Clough and Bent, 1998Go). Transformed T1 plants were selected by spraying with 0.1% glufosinate-ammonium (200 g/L) (Basta; Bayer CropScience) containing 0.1% Tween. Homozygous T3 lines were used for complementation analysis.

To verify that the plant lines carried the correct recombinant DNA, genomic DNA was isolated using TriFast (Peqlab). Recombinant DNA was amplified by PCR, cloned into Xcm vector, and sequenced. Expression of recombinant DNA was investigated by isolating RNA using the NucleoSpin RNA plant kit (Machery-Nagel). cDNAs were prepared and analyzed as described by Albrecht et al. (2003)Go. RT-PCR analysis of CBL1 transcripts was performed in triplicate, and the amplification products were detected by staining with ethidium bromide as described by Albrecht et al. (2003)Go. Salt stress tolerance analyses were performed as described by D'Angelo et al. (2006)Go.

CBL1n Purification
To prepare protein extracts containing CBL1n:GFP or CBL1nC3S:GFP, N. benthamiana leaves were infiltrated with A. tumefaciens as described above. Protein expression was verified by fluorescence microscopy at 48 h after infiltration, and leaves were harvested and batch-frozen in liquid nitrogen at 48 h after infiltration. Proteins were extracted from 10 g of frozen leaf tissue by grinding in a mortar with a pestle in 3x volume (30 mL) of plant extraction buffer (50 mM NaH2PO4, pH 7.6, 300 mM NaCl, 10% glycerol, 2 mM β-mercaptoethanol, and plant protease inhibitor mixture [Sigma-Aldrich]). To precipitate insoluble material, extracts were centrifuged at 75,000g for 30 min. The resulting supernatants were discarded, and the insoluble pellet was incubated on ice for 30 min in the same volume (30 mL) of plant extraction buffer containing 1% Triton X-100 and 0.1% SDS. Solubilized extracts were centrifuged again, and the resulting supernatant was collected for further analysis. Protein extracts containing CBL1nC3S:GFP were prepared in a similar manner, except for the following changes. After extraction in a plant extraction buffer (without detergents) and centrifugation, the supernatants were collected and used as the source for protein purification.

CBL1n:GFP and CBL1nC3S:GFP were purified from total protein extracts in two steps. The first step involved differential ammonium sulfate precipitations. CBL1n:GFP or CBL1nC3S:GFP was precipitated in the 30 to 45% ammonium sulfate fraction. The precipitated samples were resuspended and dialyzed against 50 mM Tris-HCl, pH 8.0, 2 mM β-mercaptoethanol, 5% glycerol, and 0.2% Triton X-100. The dialyzed fractions were collected, and the CBL1n:GFP recombinant proteins were purified over a 5-mL bed volume of ready-to-use DEAE–cellulose ion-exchange columns. Purification was performed according to the manufacturer's instructions (Bio-Rad) with column buffer containing Tris-HCl, pH 8.0, 0.1% Triton X-100, and 2 mM β-mercaptoethanol. Fractions containing the recombinant proteins were eluted with 100 and 200 mM NaCl. CBL1n:GFP- or CBL1nC3S:GFP-containing fractions were pooled for further analysis.

Acyl Group Cleavage
Cleavage of acyl groups was performed as described previously (Sorek et al., 2007Go). Briefly, 25 µg of purified CBL1n:GFP or CBL1nC3S:GFP was dried in a vacuum concentrator inside sealable glass vials (2-mL reactive vial; Whatman catalog No. 986276) and resuspended in formic acid:ethanol (1:4, v/v). The samples were then washed three times with 0.5 mL of pentane:formic acid:ethanol (10:1:4, v/v) to wash away noncovalently bound lipids. A total of 70 mg of platinum (IV) oxide was added per 400-µL sample, and proteins were hydrogenated for 90 min. Following hydrogenation, extraction of released acyl groups was done by adding 0.5 mL of pentane. This extraction was repeated three times, and each time the pentane was transferred into a new tube. The pentane washes were pooled and concentrated under nitrogen to a final volume of 15 µL. Concentrated samples were analyzed by GC-MS.

GC-MS Analysis
Samples for analysis were manually injected in 1-µL aliquots into an Agilent Technologies GC/MSD system (6890N gas chromatography system and 5973N mass selective detector) equipped with an Rtx-5 SIL column (Restek). The column had the following dimensions: length, 30 m; inside diameter, 0.25 mm; film thickness, 0.25 µm. The column's stationary phase was 95% dimethyl and 5% diphenyl polysiloxane. Helium was used as the carrier gas at a flow rate of 0.8 mL/min. The injection temperature was set to 250°C (splitless mode), the interface was at 280°C, and the ion source was adjusted to 200°C. The analysis was performed under the following temperature program: 5 min of isothermal heating at 100°C, followed by a 5°C/min oven temperature ramp up to 280°C. The system was equilibrated for 1 min at 100°C before injection of the next sample. Mass spectra were recorded at 4.59 scans/s with a 41 to 350 m/z scanning range and an electron energy of 70 eV. Compounds were tentatively identified (>95% match) on the basis of the NIST98 Mass Spectral Library (data version NIST05, software version 2.0d) with the Chemstation V.D.038 program (Agilent Technologies). Further identification of major compounds was based on comparisons of mass spectra and retention times with those of authentic standards (farnesol, geranylgeraniol, palmitic acid, and stearic acid) analyzed under similar conditions.

Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome Initiative database under the following accession numbers: CBL1, At4g17615; CBL2, At5g55990; CBL4, At5g24270; CBL5, At4g01420; and CBL9, At5g47100.

Supplemental Data
The following materials are available in the online version of this article.

Supplemental Figure 1. Schematic Representation of CBL Constructs Used, in Vitro Myristoylation Assays, Characterization of Transgenic Lines, Function of CBL Acylation, and Protein Gel Blot Analysis of CBL:GFP Fusion Proteins.
Supplemental Figure 2. Colocalization Analysis of CBL1C3S:GFP and OFP:HDEL, and Membrane Association of CBL1, CBL2, and CBL1nCBL2.
Supplemental Figure 3. Magnifications of Microscopic Images of Selected Constructs.
Supplemental Figure 4. Inhibitor Studies with 2-Bromopalmitate, and Localization of CBL1n:GFP and Mutated Versions of CBL1n.
Supplemental Figure 5. Inhibitor Studies with BFA.
Supplemental Table 1. Primers Used in This Study.


   Acknowledgments
 
We gratefully acknowledge Sibylle Arendt for excellent technical assistance. We thank Efraim Lewinsohn and Einat Bar for access to GC-MC equipment, Thomas Merckle and Ami Aronheim for providing materials for the Ras recruitment system, Antje von Schaewen for OFP:HDEL, Chris Hawes for Nt Sar1H74L, and Nadine Paris for GFP:TM23. This work was supported by a grant from the Deutsche Forschungsgemeinshaft to J.K. and subsequently by Grant GIF 834/2005 from the German–Israeli Science Foundation to J.K. and S.Y.


   Footnotes
 
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Jörg Kudla (jkudla{at}uni-muenster.de).

[W] Online version contains Web-only data. Back

www.plantcell.org/cgi/doi/10.1105/tpc.108.058123

Received January 15, 2008; Revision received April 7, 2008. accepted May 3, 2008.


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