Molecular mechanisms of urea uptake in marine diatoms
- Author(s): Oakley, Katherine Elizabeth
- et al.
The genome sequence and established expressed sequence tags for the pennate marine diatom Phaeodactylum tricornutum reveal a full complement of genes encoding proteins for a complete metazoan-type urea cycle, a urease enzyme and two urea transporters that could function to take up urea as an organic nutrient. Amidst this set, two genes involved in the uptake of urea as a nutrient source have been identified: JGI_Pt_20424, homologous to high- affinity plant urea transport proteins, and JGI_Pt_39772, with homology to bacterial and metazoan urea transporter proteins, including the well characterized vertebrate renal and erythrocyte transporters. Both genes were cloned into expression vectors containing a YFP fluorophore tag by Gateway technology (Invitrogen), and the subcellular localizations of the two urea transporter proteins were determined following overexpression studies and in vivo epifluorescence and confocal microscopy in transfected diatoms. By observing yellow fluorescence of the transporter-C-terminal YFP fusion protein in vivo, the urea transporter JGI_Pt_20424 localized to P. tricornutum's plasma membrane. The JGI_Pt_39772 urea transporter was localized to the mitochondrion using the same method. Urea uptake rates of ammonium grown cultures of both wild-type and transgenic P. tricornutum, containing the JGI_Pt_20424 urea transporter overexpression construct, were determined using ¹⁵N urea. The overexpression of JGI_Pt_20424 increased urea uptake approximately ten-fold compared to the wild-type; therefore, the protein efficiently transports urea. Work has begun on the molecular mechanisms and kinetics of urea transport by JGI_20424 and JGI_39772 using a heterologous expression system in Xenopus laevis oocytes