Inhibition of copper uptake in yeast reveals the copper transporter Ctr1p as a potential molecular target of saxitoxin.
Saxitoxin is a secondary metabolite produced by several species of dinoflagellates and cyanobacteria which targets voltage-gated sodium and potassium channels in higher vertebrates. However, its molecular target in planktonic aquatic community members that co-occur with the toxin producers remains unknown. Previous microarray analysis with yeast identified copper and iron-homeostasis genes as being differentially regulated in response to saxitoxin. This study sought to identify the molecular target in microbial cells by comparing the transcriptional profiles of key copper and iron homeostasis genes (CTR1, FRE1, FET3, CUP1, CRS5) in cells exposed to saxitoxin, excess copper, excess iron, an extracellular Cu(I) chelator, or an intracellular Cu(I) chelator. Protein expression and localization of Ctr1p (copper transporter), Fet3p (multicopper oxidase involved in high-affinity iron uptake), and Aft1p (iron regulator) were also compared among treatments. Combined transcript and protein profiles suggested saxitoxin inhibited copper uptake. This hypothesis was confirmed by intracellular Cu(I) imaging with a selective fluorescent probe for labile copper. On the basis of the combined molecular and physiological results, a model is presented in which the copper transporter Ctr1p serves as a molecular target of saxitoxin and these observations are couched in the context of the eco-evolutionary role this toxin may serve for species that produce it.
Center for Environmental Biotechnology, The University of Tennessee, 676 Dabney Hall, Knoxville, Tennessee 37966, United States.
Kathleen D
KD
Cusick
Steven C
SC
Minkin
Sheel C
SC
Dodani
Christopher J
CJ
Chang
Steven W
SW
Wilhelm
Gary S
GS
Sayler
D001692:Biological Transport / Q000187:drug effects; D027682:Cation Transport Proteins / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D000079923:Copper Transporter 1; D020869:Gene Expression Profiling; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D005800:Genes, Fungal / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006706:Homeostasis / Q000187:drug effects / Q000235:genetics; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D008954:Models, Biological; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D012530:Saxitoxin / Q000494:pharmacology*
C490078:CTR1 protein, S cerevisiae; D027682:Cation Transport Proteins; D000079923:Copper Transporter 1; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D012530:Saxitoxin; D003300:Copper; D007501:Iron