In order for HCN1 to reach the plasma membrane, the di-arginine ER retention motif should be balanced by the action of an as yet unknown forward trafficking signal. HCN1 has four intracellular domains all of which are spatially available to trafficking regulators; the NT loop 1 connecting transmembrane domains 2-3, loop 2 connecting transmembrane domains 4-5, and the C-terminus (
Fig. 1A). We fused each of these HCN1 domains to a palmitoylated GFP reporter and examined their subcellular localization when expressed in rods of transgenic
X. laevis. For reference, the design and results from all HCN1 constructs generated in this study are summarized in the
Table. The membrane reporter consists of EGFP followed by a palmitoylated peptide consisting of amino acids 311 to 349 from
X. laevis rhodopsin (
Fig. 1B) and has been used in multiple previous studies of protein trafficking in this model system. When expressed alone, the reporter localizes mainly to the outer segment (OS) with some variable but relatively minor levels of signal in the ISPM or ER/Golgi compartments (
Fig. 1C).
22,23 Consistent with our previous finding using a similar integral membrane reporter, fusing the C-terminus (CT) of HCN1 to the reporter redirects the protein to the ER (
Fig. 1D).
15 Fusion of loop 1 (L1) did not affect the default localization of the reporter (
Fig. 1E). Fusion of loop 2 (L2) redirected the reporter to the ellipsoid (
Fig. 1F). We did not pursue this observation further because full-length HCN1 is not associated with mitochondria. Fusion of the NT of HCN1 to the reporter prevented it from localizing to OS. It was found in the IS with variable localization to both internal membranes with morphology consistent with the ER and Golgi, and to the ISPM (
Fig. 1G; note: our use of the term ISPM encompasses the plasma membrane surrounding all components of the IS [calycal processes, ellipsoid, myoid and soma, axon, and syntaptic terminal]). This indicates that the NT of HCN1 contains trafficking information.
Because the NT only displays partial ISPM targeting when fused to the reporter, we brought back other elements in HCN1 to test if the NT is required for the targeting of full-length HCN1 to the ISPM. When the full-length GFP-tagged
Xenopus tropicalis HCN1 (aa 1-839) was expressed in transgenic
X. laevis photoreceptors, the majority of the protein was found at the ISPM (
Fig. 2A). HCN1 lacking the NT (GFP-HCN1; aa122-839) was retained in the ER, evidenced by colocalization with calnexin, an ER marker (
Figs. 2B–D). Several examples of cells expressing wild-type or NT-truncated HCN1 are shown in
Supplementary Figure S1, with line scans of the GFP fluorescence intensity across the cell, which demonstrates that the lack of the NT resulted in greater retention within the internal membranes of the cell. In total, these data show that the NT of HCN1 contains an ISPM targeting signal.