Northern Kentucky University
Sub-cellular Localization of Two P-type ATPases, Atp13a1 and Atp13a2
Institution
Northern Kentucky University
Faculty Advisor/ Mentor
Patrick Schultheis; Kristi Martines; Diane McGill
Abstract
P-type ATPases are membrane proteins that transport their substrates against their concentration gradients. The focus of this study is two novel P-type ATPases termed Atp13a1 and Atp13a2. To date, nothing is known about their substrate specificity, subcellular localization or physiological function. The aim of this study was to determine the membrane location of Atp13a1 and Atp13a2 via western blotting and immunofluorescence. To facilitate their localization, HeLa cell lines expressing GFP and V5-His tagged versions of Atp13a1 and Atp13a2 were produced. Cell lysates from these cell lines were layered onto a sucrose density gradient (40%-10% sucrose) and were centrifuged at 100,000 x g for 16 hours to separate cell compartments. One milliliter fractions were harvested from the gradient after centrifugation and tested via western blot. Antibodies to organelle markers in ER, Golgi, and plasma membrane were used with antibodies to the GFP and V5-His tags to co-localize the ATPases with the organelles. To verify the sub-cellular fractionation data, immunofluorescence was also used to independently test the localization of each of the ATPases.
Sub-cellular Localization of Two P-type ATPases, Atp13a1 and Atp13a2
P-type ATPases are membrane proteins that transport their substrates against their concentration gradients. The focus of this study is two novel P-type ATPases termed Atp13a1 and Atp13a2. To date, nothing is known about their substrate specificity, subcellular localization or physiological function. The aim of this study was to determine the membrane location of Atp13a1 and Atp13a2 via western blotting and immunofluorescence. To facilitate their localization, HeLa cell lines expressing GFP and V5-His tagged versions of Atp13a1 and Atp13a2 were produced. Cell lysates from these cell lines were layered onto a sucrose density gradient (40%-10% sucrose) and were centrifuged at 100,000 x g for 16 hours to separate cell compartments. One milliliter fractions were harvested from the gradient after centrifugation and tested via western blot. Antibodies to organelle markers in ER, Golgi, and plasma membrane were used with antibodies to the GFP and V5-His tags to co-localize the ATPases with the organelles. To verify the sub-cellular fractionation data, immunofluorescence was also used to independently test the localization of each of the ATPases.