RESUMEN
N-myristoylation and S-acylation promote protein membrane association, allowing regulation of membrane proteins. However, how widespread this targeting mechanism is in plant signaling processes remains unknown. Through bioinformatics analyses, we determined that among plant protein kinase families, the occurrence of motifs indicative for dual lipidation by N-myristoylation and S-acylation is restricted to only five kinase families, including the Ca2+ -regulated CDPK-SnRK and CBL protein families. We demonstrated N-myristoylation of CDPK-SnRKs and CBLs by incorporation of radiolabeled myristic acid. We focused on CPK6 and CBL5 as model cases and examined the impact of dual lipidation on their function by fluorescence microscopy, electrophysiology and functional complementation of Arabidopsis mutants. We found that both lipid modifications were required for proper targeting of CBL5 and CPK6 to the plasma membrane. Moreover, we identified CBL5-CIPK11 complexes as phosphorylating and activating the guard cell anion channel SLAC1. SLAC1 activation by CPK6 or CBL5-CIPK11 was strictly dependent on dual lipid modification, and loss of CPK6 lipid modification prevented functional complementation of cpk3 cpk6 guard cell mutant phenotypes. Our findings establish the general importance of dual lipid modification for Ca2+ signaling processes, and demonstrate their requirement for guard cell anion channel regulation.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Proteínas de Unión al Calcio/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Calcio/metabolismo , Canales Iónicos/metabolismo , Proteínas de la Membrana/metabolismo , Ácido Mirístico/metabolismo , Procesamiento Proteico-Postraduccional , Ácido Abscísico/farmacología , Acilación , Secuencias de Aminoácidos , Animales , Aniones , Arabidopsis/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Lípidos/química , Modelos Biológicos , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/enzimología , Estomas de Plantas/citología , Estomas de Plantas/efectos de los fármacos , Estomas de Plantas/fisiología , Unión Proteica/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Nicotiana/enzimología , XenopusRESUMEN
A large series of conjugated carbazole dimers, namely bicarbazoles 1-12, were synthesized by Suzuki-Miyaura, Sonogashira, Hay, and McMurry coupling reactions. In 1-12, the two carbazole moieties are linked at the 1-, 2-, or 3-position directly or via an acetylenic or olefinic spacer. The structure-property relationships, particularly the effects of the conjugation connectivity and the π-conjugated spacers on the electronic, photophysical, and electrochemical properties of 1-12, were studied by extensive UV-vis and fluorescence spectroscopic measurements, cyclic voltammetry (CV), and theoretical calculations as well as X-ray crystallographic analyses. The connection at the 1-position of carbazole ensures high extent of π-conjugation, while that at the 3-position enhances the electron-donating ability. Both acetylenic and olefinic spacers allow the extension of π-conjugation, and the latter also causes the increase of the donor ability. Moreover, the structural variations were found to affect the fluorescence quantum yields significantly, which are up to 0.84.