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Photoreceptors are among the most metabolically active cells in the body, relying on both oxidative phosphorylation and glycolysis to satisfy their high energy needs. Local glycolysis is thought to be particularly crucial in supporting the function of the photoreceptor's light-sensitive outer segment compartment, which is devoid of mitochondria. Accordingly, it has been commonly accepted that the facilitative glucose transporter Glut1 responsible for glucose entry into photoreceptors is localized in part to the outer segment plasma membrane. However, we now demonstrate that Glut1 is entirely absent from the rod outer segment and is actively excluded from this compartment by targeting information present in its cytosolic C-terminal tail. Our data indicate that glucose metabolized in the outer segment must first enter through other parts of the photoreceptor cell. Consequently, the entire energy supply of the outer segment is dependent on diffusion of energy-rich substrates through the thin connecting cilium that links this compartment to the rest of the cell.
Fig.2. Glut1 immunolocalization in amphibian retina. (A) Western blot analysis of retinal extracts from mouse (lane 1) and Xenopus (lane 2), probed with anti-human Glut1 polyclonal antibody. The Xenopus isoform was not recognized. (B) The polyclonal antibody raised against Xenopus Glut1 specifically recognized this protein in an immunoblot of Xenopus retinal extract. (C) A section of Xenopus retina immunostained for Glut1 (green). Nuclei were counter-stained with Hoechst 33342 (blue). To the left is a schematic of a frog rod photoreceptor with an overlying RPE cell, illustrating the relative positions of individual subcellular compartments. Abbreviations are the same as in Fig. 1. Scale bar: 20 μm. (D–I) Xenopus retinal cryosections stained for Glut1 (green) and Na+/K+-ATPase (NKA, red; D–F) or phalloidin (red; G–I). Glut1 localized to the RPE and its microvilli, and to the rod photoreceptor plasma membrane in the synaptic, nuclear and inner segment layers. Labeling of the Na+/K+-ATPase (D–F) was used as a marker for the photoreceptor inner segment plasma membrane and calycal processes (arrowheads). Labeling of F-actin with phalloidin (G–I) was used as a marker for the RPE microvilli, in addition to the photoreceptor calycal processes. (J–L) Staining of two isolated rod photoreceptor fragments revealed Glut1 (green) on inner segment (IS) membranes and calycal processes (arrowheads) but not along the plasma membrane of outer segments (OS) marked by wheat germ agglutinin (WGA, red). Scale bars: 10 μm (D–L).
Fig.3. Immunogold analysis of Glut1 in Xenopus retina sections. Longitudinal sections (A,B) and cross-sections (C,D) through Xenopus retina were processed for immunogold analysis. Gold particles (representatives marked by red arrowheads) were abundant on the fingerlike RPE microvilli (A) and their cross-sections (C). Rare, nonspecific particles (green arrowheads) were occasionally found within the rod outer segments, with no preference for the lateral plasma membrane; they were found at similar frequencies in control sections incubated with rabbit IgG control (B,D). (E) Gold particle labeling densities (particles per μm2 of membrane) were calculated for the outer segments and microvilli in both Glut1-stained and control sections. The mean labeling density for each type of structure in ten separate images is plotted with error bars representing 95% confidence intervals. MV, microvilli; OS, outer segment; ROS, rod outer segment. Scale bars: 200 nm.
Fig.4. GFP-tagged Glut1 is actively excluded from rod outer segments of transgenic Xenopus tadpoles. (A,B) GFP-tagged Glut1 was expressed in transgenic tadpole rods and localized to the plasma membrane of the synaptic terminal, nuclear region and inner segment, including the calycal processes (arrowheads). The outer segments extend well beyond the GFP signal in the calycal processes (B). (C) GFP-tagged Glut1 mutant lacking its 38-residue C-terminal tail (GFP–Glut1δ38) displayed prominent outer segment localization in tadpole rods. OS, outer segment; IS, inner segment; N, nucleus; ST, synaptic terminal. Scale bars: 10 μm.
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