Identification of a VxP Targeting Signal in the Flagellar Na+ /K+ -ATPase

Abstract

Na(+) /K(+) -ATPase (NKA) participates in setting electrochemical gradients, cardiotonic steroid signaling and cellular adhesion. Distinct isoforms of NKA are found in different tissues and subcellular localization patterns. For example, NKA α1 is widely expressed, NKA α3 is enriched in neurons and NKA α4 is a testes-specific isoform found in sperm flagella. In some tissues, ankyrin, a key component of the membrane cytoskeleton, can regulate the trafficking of NKA. In the retina, NKA and ankyrin-B are expressed in multiple cell types and immunostaining for each is striking in the synaptic layers. Labeling for NKA is also prominent along the inner segment plasma membrane (ISPM) of photoreceptors. NKA co-immunoprecipitates with ankyrin-B, but on a subcellular level colocalization of these two proteins varies dependent on the cell type. We used transgenic Xenopus laevis tadpoles to evaluate the subcellular trafficking of NKA in photoreceptors. GFP-NKA α3 and α1 are localized to the ISPM, but α4 is localized to outer segments (OSs). We identified a VxP motif responsible for the OS targeting by using a series of chimeric and mutant NKA constructs. This motif is similar to previously identified ciliary targeting motifs. Given the structural similarities between OSs and flagella, our findings shed light on the subcellular targeting of this testes-specific NKA isoform.

Keywords: ATP1a3; ATP1a4; ATPase; Muller glia; ankyrin; cilia; flagella; photoreceptors; retina; sperm; synapse; trafficking.

Figure 1. NKA and AnkB in bovine retina

A) Co-immunoprecipitations from bovine retinal lysates, blotted for NKA and AnkB. Either anti-NKA antibodies (upper panel) or anti-AnkB antibodies (lower panel) alongside IgG as a negative control were used as the precipitating antibodies. B) Bovine retina, after depletion of photoreceptor outer segments and nuclei, separated into soluble versus membrane fractions. Membrane fraction verified by blotting for NKA and HCN1, soluble fraction verified by blotting for actin and GAPDH. AnkB was enriched in the soluble fraction. C) Double labeling of bovine retina with antibodies against NKA (green) and AnkB (red). Scale bar is 20 μm and nuclei labeled with Hoechst in merged images in C and D. Abbreviations: OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GC, ganglion cell layer; NFL, nerve fiber layer.

Figure 2. NKA and AnkB localization in mouse retina

A) Serial tangential sections through a dark-adapted mouse retina blotted for AnkB, NKA, arrestin (marker for photoreceptor IS, soma, and synapse), transducin alpha (Tα) (marker for OS) and Rom-1 (marker for OS). AnkB and NKA are present in the same sections throughout the retina. B, C) Double labeling of mouse retina with antibodies against NKA (red) and AnkB (green) or D) CRALBP (red) and AnkB (green). Scale bar is 20 μm (B–D), abbreviations as in Fig 1; OLM is outer limiting membrane, the band of adherens junctions between Muller glia and photoreceptor inner segments.

Figure 3. NKA and AnkB localization in dissociated mouse retina cell fragments

Immunostaining for AnkB (red) and NKA (green) in cellular fragments identified by morphology. The schematic in (K) places the images of the cell fragments shown in (A–J) in context of their original location in intact retina. A^I-III–C^I-III) Three examples of isolated photoreceptor inner segments (myoid) attached to the soma containing the nucleus. AnkB labeling is interior to the NKA labeling, as seen in the merged images (C^I-III) and line scan plots (D^I-III) where the normalized signal intensity of AnkB (red, dashed line) and NKA (green, solid line) along the length of lines arbitrarily drawn perpendicular to the long axis of the photoreceptor myoid (yellow lines in C^I-III) is plotted. E–G) Fragment of a Muller glial cell; identified by the thick diameter, length, and numerous small processes. AnkB labeled the entirety of this fragment, but NKA was not detected. Asterisk indicates isolated nuclei from broken rod photoreceptors identified by the unique central condensation of heterochromatin (C,G,J). H–J) Bipolar neuron as identified by tufts of dendrites adjacent to the nucleus, long axon and large synapse. AnkB labeling is interior to the NKA labeling in the dendrites, soma, and axon; AnkB but not NKA was enriched in the synaptic terminal. Scale bar is 5 μm for each set of panels.

Figure 4. Expression of NKA in X. laevis photoreceptors

A, B) Immunohistochemical labeling for NKA (green) in adult Xenopus laevis retina B) higher magnification of labeled photoreceptors shown adjacent to transmitted light image. Transgenic X. laevis tadpoles expressing C) GFP-NKA α3, D) GFP-NKA α1, or E) GFP-NKA α4 in rods. Nuclei labeled with Hoechst. Abbreviations: OS, outer segment; IS, inner segment; CP, calycal processes; ONL, outer nuclear layer; N, nuclei; OPL, outer plexiform layer; ST, synaptic terminal; INL, inner nuclear layer; IPL, inner plexiform layer; GC, ganglion cell layer.

Figure 5. The N-terminus of NKA alpha subunits contains targeting information

A) A GFP-tagged chimera consisting of amino acids 1-82 from α3 and 89-1029 from α4 localizes to the ISPM. B) The complimentary chimera consisting amino acids 1-88 from α4 and 83-1025 from α3 localizes to OS. Nuclei labeled with Hoechst. Abbreviations: OS, outer segment; IS, inner segment; N, nuclei; ST, synaptic terminal. Scale bars are 5 μm.

Figure 6. Targeting information is within the first 14 amino acids of NKA

GFP-tagged chimeras containing the N-terminus of NKA α3 localize to the ISPM, A) amino acids 1-38 from α3 and 45-1029 from α4, C) 1-24 from α3 and 23-1029 from α4, E) 1-14 from α3 and 13-1029 from α4. GFP-tagged chimeras containing the N-terminus of NKA α4 localize to the OS, B) amino acids 1-44 from α4 and 39-1025 from α3, D) 1-24 from α4 and 25-1025 from α3, F) 1-14 from α4 and 15-1025 from α3. Nuclei labeled with Hoechst, abbreviations and scale bars as in Figure 4.

Figure 7. The first 14 amino acids of NKA α4 are required for outer segment localization

A) Deleting the first 14 amino acids from NKA α3 does not prevent ISPM localization. B) Deleting the first 14 amino acids from NKA α4 results in ISPM localization. Nuclei labeled with Hoechst, abbreviations and scale bars as in Figure 4.

Figure 8. Identification of individual residues required for outer segment localization of NKA α4

A) Alignment of the N-terminal amino acids from Xenopus NKA α3 and human NKA α4; dashed line indicates sequence required for OS localization, triangles indicate a VxP motif, red asterisks indicate other residues mutated in the following panels. Color coding of amino acids: green, small polar; purple, polar; white, hydrophobic; blue, positively charged; red, negatively charged; brown, proline. B) Mutations, V10A and P12A results in ISPM localization of NKA α4. The individual mutations, V10A (C), or P12A (D) have the same affect. E) The T13P mutation in NKA α3 introducing a VxP motif was insufficient to change ISPM localization. F) Mutation of prolines 17 and 21 downstream of the VxP motif in NKA α4 did not prevent OS targeting. Deletions of amino acids G) 15-24, or H) 1-44 in NKA α3 did not prevent ISPM targeting. Nuclei labeled with Hoechst, abbreviations and scale bars as in Figure 4.

Figure 9. A summary of the design and targeting behavior of all constructs expressed in transgenic Xenopus photoreceptors

The linear arrangement of sequence motifs in NKA are shown on top. Xenopus laevis NKA α3 is shown in orange, human NKA α4 is shown in black, red asterisks indicate individual amino acid substitutions. Abbreviations: TMD, transmembrane domain; ISPM, inner segment plasma membrane; OS, outer segment.