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Joseph G. Laird, Yuan Pan, David Yamaguchi, Sheila A Baker; Identifying targeting motifs necessary for the differential localization of Na+/K+-ATPase isoforms in photoreceptors. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):453.
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© ARVO (1962-2015); The Authors (2016-present)
Na+/K+-ATPase (NKA) is the sodium pump used by all cells to establish and maintain electrochemical gradients across cellular membranes. It can alternatively function in signaling as a receptor for cardiotonic steroids or participate in maintaining cell-cell adhesion. Ways to accommodate the unique requirements of different cell types include the utilization of different NKA isoforms or altering the subcellular localization of NKA. In photoreceptors, NKA composed of the α3 subunit is located in the plasma membrane of the inner segment. But in sperm, NKA is composed of the α4 subunit and localizes to the flagella. We observed that upon expression in photoreceptors NKA α4 localized to the analogous ciliary compartment, the outer segment. Our goal in this study was to take advantage of that observation to dissect the differential targeting information encoded within each of these NKA isoforms.
A series of GFP-tagged NKA constructs were cloned. These included a series of α3/α4 chimeras, deletion mutants and point mutants of each isoform. The proteins were expressed in Xenopus laevis rods by generating transgenic tadpoles. Retinas from stage 43-46 animals were immunostained with anti-GFP antibodies and imaged with confocal microscopy.
NKA α3 and α4 are most divergent in their cytoplasmic, unstructured N-termini. Chimeras with this region exchanged resulted in changing the localization of α3 from the inner segment plasma membrane to the outer segment and vice versa. Analysis of a series of paired chimeric constructs narrowed the region containing the motif responsible for this differential targeting to fourteen amino acids. Within this region we identified a valine and proline necessary for outer segment localization.
This study allowed us to uncover a targeting motif within a flagellar NKA isoform with similarity to the targeting motif of rhodopsin. We speculate that lack of this targeting motif in NKA α3 contributes to its inner segment localization where competition for energy resources between NKA, phototransduction in the outer segment, and the synaptic vesicle cycle in the synapse can be minimized.
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