Functional characterization of an unusual phytochelatin synthase, LjPCS3, of Lotus japonicus.
In plants and many other organisms, phytochelatin synthase (PCS) catalyzes the synthesis of phytochelatins from glutathione in the presence of certain metals and metalloids. We have used budding yeast (Saccharomyces cerevisiae) as a heterologous system to characterize two PCS proteins, LjPCS1 and LjPCS3, of the model legume Lotus japonicus. Initial experiments revealed that the metal tolerance of yeast cells in vivo depends on the concentrations of divalent cations in the growth medium. Detailed in vivo (intact cells) and in vitro (broken cells) assays of PCS activity were performed with yeast expressing the plant enzymes, and values of phytochelatin production for each metal tested were normalized with respect to those of cadmium to correct for the lower expression level of LjPCS3. Our results showed that lead was the best activator of LjPCS1 in the in vitro assay, whereas, for both assays, arsenic, iron, and aluminum were better activators of LjPCS3 and mercury was similarly active with the two enzymes. Most interestingly, zinc was a powerful activator, especially of LjPCS3, when assayed in vivo, whereas copper and silver were the strongest activators in the in vitro assay. We conclude that the in vivo and in vitro assays are useful and complementary to assess the response of LjPCS1 and LjPCS3 to a wide range of metals and that the differences in the C-terminal domains of the two proteins are responsible for their distinct expression levels or stabilities in heterologous systems and patterns of metal activation.
Ramos
Javier
J
Departamento de Nutrición Vegetal, Estación Experimental de Aula Dei, Consejo Superior de Investigaciones Científicas, 50080 Zaragoza, Spain.
Naya
Loreto
L
Gay
Marina
M
Abián
Joaquín
J
Becana
Manuel
M
D019881:Aminoacyltransferases / Q000235:genetics / Q000378:metabolism*; D003470:Culture Media; D004789:Enzyme Activation; D000070116:Lotus / Q000201:enzymology* / Q000235:genetics; D019216:Metals, Heavy / Q000378:metabolism*; D054811:Phytochelatins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
D003470:Culture Media; D019216:Metals, Heavy; D054811:Phytochelatins; D019881:Aminoacyltransferases; C093784:glutathione gamma-glutamylcysteinyltransferase