Understanding the Role of Accessory Proteins on ENaC Function
The epithelial sodium channel (ENaC) reabsorbs sodium in the distal convoluted tubules of the nephron in kidneys. Regulation and assembly of ENaC is not well characterized and the use of a yeast deletion library can shed light on the matter. The gene for Wasabi (a form of enhanced green fluorescent protein) was fused to the 5’ end of the gene for αENaC and then subcloned into the yeast expression vector pYES2/NTA. The vector with the new gene for Wasabi-αENaC was transformed into BY4742 yeast cells and into four single gene deletion mutants of yeast: scj1, jem1, ypk1 and lhs1. Survival assays, western blots, and co-localization studies were used to verify expression of the Wasabi-tagged αENaC and monitor localization of the fusion protein inside Saccharomyces cerevisae yeast cells. We report that deletion of SCJ1 did not affect ENaC function or localization, while deletions of JEM1 and LHS1 appear critical for degradation of misfolded ENaC in the ER and the deletions lead to localization of ENaC in the ER. We also report that YPK1 deletion prevented the yeast from expressing ENaC in the cell so localization and function could not be determined. Additional studies including cell surface biotinylation, co-immunoprecipitation, and additional confocal microscopy studies utilizing fluorophore-conjugated antibodies to illuminate cellular components (ER, Golgi, membrane, etc.) could be employed to further study localization.
ENaC, Wasabi, Hypertension, Localization
Horn, D. (2014). <i>Understanding the role of accessory proteins on ENaC function</i> (Unpublished thesis). Texas State University, San Marcos, Texas.