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.

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).

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).

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.

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.

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.

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 (αGFP).
(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).

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.

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).

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.