An Arabidopsis Clathrin Assembly Protein with a Predicted Role in Plant Defense Can Function as an Adenylate Cyclase

Abstract

Adenylate cyclases (ACs), much like guanylate cyclases (GCs), are increasingly recognized as essential parts of many plant processes including biotic and abiotic stress responses. In order to identify novel ACs, we have applied a search motif derived from experimentally tested GCs and identified a number of Arabidopsis thaliana candidates including a clathrin assembly protein (AT1G68110; AtClAP). AtClAP contains a catalytic centre that can complement the AC-deficient mutant cyaA in E. coli, and a recombinant AtClAP fragment (AtClAP^261-379) can produce cyclic adenosine 3',5' monophosphate (cAMP) from adenosine triphosphate (ATP) in vitro. Furthermore, an integrated analysis of gene expression and expression correlation implicate cAMP in pathogen defense and in actin cytoskeletal remodeling during endocytic internalization.

Keywords: Arabidopsis thaliana; adenylate cyclase; cAMP; clathrin assembly; endocytosis; pathogen responses.

Figure 1

(a) The 14 amino acid adenylate cyclase (AC) search motif derived from annotated and experimentally tested guanylate cyclase (GC) and AC catalytic centres. The residue forming hydrogen bonding with purine at position 1 is highlighted in red, the residue conferring substrate specificity in position 3 is highlighted in blue, while the aa in position 14, stabilizing the transition state from ATP to cAMP, is highlighted in red. The amino acid [DE] at 1–3 residue downstream from position 14, participates in Mg²⁺/Mn²⁺-binding and is coloured green. Arabidopsis thaliana candidates including AtClAP were retrieved with the motif devoid of the sequences upstream of position 1; (b) The complete amino acid sequence of AtCIAP with the AC catalytic centre highlighted in bold and the 119 amino acid fragments tested for AC activity indicated within the inverted red triangles. The underlined amino acids mark an N-terminal phosphatidylinositol 4,5-bisphosphate (also referred to as PtdIns(4,5)P₂, PIP₂ or PI(4,5)P₂) binding site; (c) Alignment of the AC centres of NbAC (ACR77530), ZmPSiP (AJ307886), AtKUP7 (AT5G09400), AtPPR-AC (AT1G62590) and AtClAP (AT1G68110); (d) Full-length models of AtClAP with the AC centre (gold) and the PIP₂ binding site (brown) represented in the left and right panels. The Epsin N-terminal homology (ENTH) and clathrin adaptor domains are shown as green and blue, respectively, and they both make up the membrane-binding region of the protein; (e) Docking of ATP at the AC centre and the interaction of ATP with the key residues at the catalytic centre of AtClAP shown as surface (left) and ribbon (right) models. The residues implicated in interactions with ATP are coloured Ca according to their charges and shown as individual atoms in the ribbon model. AtCIAP was modeled using the iterative threading assembly refinement (I-TASSER) method [22] and ATP docking simulation was performed using AutoDock Vina (ver. 1.1.2) [23].

Figure 2

(a) The recombinant AC domain of AtCIAP^261–379 complemented the cyaA mutant E. coli (SP850). The wild-type E. coli shows a strong reddish colour while both the cyaA mutant and the cyaA mutant with an un-induced recombinant AtCIAP^261−379 yielded yellowish colonies; (b) Cyclic AMP generated by the recombinant AtCIAP^261–379 at different time points in reaction mixes containing at final concentrations, 5 µg protein, 1 mM ATP and 5 mM Mn²⁺ or Mg²⁺. Inset: A Coomassie brilliant blue-stained gel after resolution of the affinity purified His-tagged recombinant AtCIAP^261−379 (arrow) by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (left plane) and a Western blot analysis of the resolved AtCIAP^261−379 with an anti-HIS antibody (right plane); (c) Cyclic AMP generated by 5 µg AtCIAP^261−379 in the presence (at final concentrations) of 1 mM ATP or GTP, or 1 mM ATP and 250 µM Ca²⁺ when 5 mM Mn²⁺ ion is the cofactor (control reaction contained all other components except the protein and Ca²⁺). Data are mean values (n = 3) and error bars show standard error (SE) of the mean. Asterisks denote values significantly different from those of control (p < 0.05) determined by analysis of variance (ANOVA) and post hoc Student–Newman–Keuls multiple range tests.

Figure 3

(a) An extracted mass chromatogram of the m/z 328 [M-1]⁻¹ ion of cAMP generated by 5 µg of the AtCIAP^261−379 in a reaction system containing at final concentrations, 50 mM Tris-Cl; pH 8.0, 1 mM ATP and 5 mM Mn²⁺ after 20 min. Inset: Incubation time course; (b) Mass of the resultant peak in the chromatogram. Inset: Detection calibration curve.

Figure 4

The heatmap constructed to illustrate the fold change (log2) in expression of AtClAP and 25 selected expression-correlated genes (ECG25) in response to selected microarray experiments. The experiments presented include; P. syringae (12 h after treatment (hat), GSE17464 and E-MEXP-1094), G. cichoracearum (18 and 36 hat, GSE26679), S. sclerotiorum (48 hat, E-MEXP-3122), syringolin (12 hat, AT-00258/FGCZ and E-MEXP-739), flg22 (12 hat, GSE17464), salicylic acid npr1-1 sni1 double and npr1-1 sni1 ssn2-1 triple mutants (16 hat, GSE23617) and methyl jasmonate (22 hat, GSE17464). Details of the microarray experimental conditions are presented in Appendix D.

Figure 5

A model illustrating the dual-role of AtClAP in clathrin-mediated endocytosis. In the primary function, AtClAP assembles clathrin at protein adaptors attached to the membrane of a newly forming vesicle and recruits components essential for endocytosis such as Epsin and other accessory proteins to internalize membrane proteins, effectors or receptor–ligand complexes. ATPases generate ATP, which combines with heat shock proteins (e.g., Hsc70) to release the bound clathrin and adaptors during invagination. In the secondary role, ATP is converted to cAMP by the solvent-exposed AC centre of AtClAP and cAMP, in turn, assembles actin to aid endocytosis and to assist vesicle translocation in the cell. Internalized cargoes are transported into early endosomes, where they may be recycled to the membrane or tagged by ubiquitin for endosomal sorting and degradation in vacuoles. The ubiquitin tagged effectors may also be targeted for degradation through the ubiquitin-proteasome system. Boxed terms represent processes or genes that are found in the list of top-200 most expression correlated genes with AtClAP.