Tian G , Ropelewski P , Nemet I , Lee R , Lodowski KH , Imanishi Y .

R01 EY020826 NEI NIH HHS , P30 EY011373 NEI NIH HHS , T32 DK007319 NIDDK NIH HHS

	Figure 1. Cilia targeting of P/rds in hTERT-RPE1 cells. A–D, bP/rds with HA and FLAG tags (A), SSTR3-GFP (B), bP/rds-Dend2 (C), or bP/rds (D; green) colocalized with acetylated tubulin (ATub, red) or α tubulin (αTub, red) in the primary cilia of hTERT-RPE1 cells. E, When expressed in hTERT-RPE1 cells, the majority bP/rds was Endo H (En] sensitive, whereas SSTR3 and rhodopsin were Endo H resistant. F, G, Endogenous bovine P/rds (F; detected by mAb anti-bovine P/rds) and endogenous X. laevis P/rds (G; detected by mAb anti-X. laevis P/rds) were sensitive to Endo H (En]. P/rds, SSTR3, and rhodopsin were all sensitive to PNGase F (P), confirming that they are glycoproteins. Arrow indicates the position of the Endo H-resistant form of P/rds; arrowhead indicates the position of EndoH-processed P/rds. The images are confocal images of a single x–y plane. Scale bar, 10 μm.
	Figure 2. Brefeldin A causes resorption of cis-Golgi structure to ER, but does not abolish the cilia targeting of P/rds. A, Western blot analysis of BFA (0.5 μg/ml for 4 h)-treated hTERT-RPE1 cells stably expressing bP/rds-HA-FLAG. Total cell lysate (T), and cell surface fraction (PM) isolated by biotinylation method were treated with Endo H (En). BFA treatment led to abolishment of the small amount of the Endo H-resistant form of P/rds on the cell surface. The arrowhead indicates the position of Endo H-processed P/rds. B, bP/rds (green) was colabeled with anti-GM130 (cis-Golgi, red) in the absence (Con) and presence (BFA) of BFA treatment. BFA treatment caused GM130 redistribution, which indicates the disruption of cis-Golgi structures. C, bP/rds (green) was colabeled with anti-acetylated tubulin (red) in the absence (Con) and presence (BFA) of BFA treatment. Cilia localization of bP/rds was maintained after BFA treatment. D, E, Trafficking of bP/rds-Dend2 or SSTR3, stably expressed in hTERT-RPE1 cells, was tested by FRAP in the absence (Con) and presence of (BFA) treatment. D, One hour after photobleaching, P/rds recovered in the cilia of control cells. P/rds recovered in 50% of cilia treated with BFA. Under the same conditions, SSTR3 recovery was blocked by BFA treatment. E, The amounts of cilia localized P/rds and SSTR3 immediately following and 1 h after photobleaching. The amount was normalized to the prebleach levels. Significant recovery of bP/rds-Dend2, but no significant recovery of SSTR3, was observed in BFA-treated cells. ***p < 0.001 by MWRST. The data are represented as mean ± SD. The numbers of cilia analyzed from at least three sample preparations are indicated in parentheses. F, Cells stably expressing bP/rds-Dend2 were treated with 150 μM cycloheximide for up to 24 h and then analyzed for the bP/rds-Dend2 protein level by Western blots. The half-life of bP/rds-Dend2 is estimated to be ∼1.8 h. The images are confocal images of a single x–y plane. Scale bars: B and C, 10 μm; D, 5 μm.
	Figure 3. Trans-Golgi exit is not essential for cilia targeting of P/rds. A, B, hTERT-RPE1 cells stably expressing bP/rds-HA-FLAG were treated with 0.5 μM 30N12 or 1 μM monensin, which block the function of trans-Golgi structures, for 4 h. A, The samples treated with 30N12 were analyzed by Western blots. Total lysate (T) and cell surface fraction (PM) were also treated with Endo H (En). After 30N12 treatment, the small amount of the Endo H-resistant form of P/rds on PM became nearly undetectable. B, bP/rds (green) was colabeled with anti-acetylated tubulin (red) with (30N12, Mon) or without (Con) treatment. Cilia localization of bP/rds was maintained after 30N12 or monensin treatment. C, D, The trafficking of bP/rds-Dend2 or SSTR3-GFP, stably expressed in hTERT-RPE1 cells, was tested by FRAP in the absence of (Con) and presence of 30N12 (30N12) or monensin (Mon). The effect of cycloheximide (CYH) treatment was also tested on the trafficking of bP/rds-Dend2. C, One hour after photobleaching, P/rds recovered in the cilia of 30N12- or monensin-treated cells. Under the same conditions, SSTR3 recovery was blocked by 30N12 or monensin treatment. P/rds recovery was blocked by CYH treatment, suggesting that protein synthesis is required for P/rds recovery in cilia. D, The amounts of cilia-localized P/rds and SSTR3 immediately following and 1 h after photobleaching. The amount was normalized to the prebleach levels. Significant recovery of bP/rds-Dend2 was observed in 30N12- or monensin-treated cells, but not in CYH-treated cells. SSTR3 recovered in untreated control cells; however, the recovery was blocked by 30N12 or monensin. Therefore, 30N12 and monensin blocks the conventional trafficking of SSTR3 effectively, but does not block the unconventional trafficking of P/rds. The data are represented as mean ± SD. ***p < 0.001 by MWRST. The numbers of cilia analyzed from at least three sample preparations are indicated in parentheses. The images are confocal images of a single x–y plane. Scale bars, 5 μm.
	Figure 4. Cilia targeting of P/rds in cilia is dependent on COPII, but not on the GRASP55-mediated mechanism. A, Cells stably expressing bP/rds that are also transiently expressing either wild-type or the H79G mutant of Sar1 (red) were colabeled with anti-GRASP55 (green). Expression of Sar1(H79G), but not wild-type Sar1, caused the disruption of cis-Golgi structure. B, P/rds (green) was colabeled with wild-type or the H79G mutant of Sar1 (red). The primary cilia is also labeled by anti-acetylated tubulin (red, the same color as Sar1). Cilia targeting of P/rds was inhibited by Sar1(H79G). C, P/rds (green) was colabeled for Sar1 (blue) and calreticulin (red, an ER marker). P/rds localized in ER in the cell expressing Sar1(H79G). D, E, Cells stably expressing bP/rds were transfected with GRASP55 siRNA and then analyzed for GRASP55 protein level (D) and the localization of P/rds (E; green) in primary cilia (acetylated tubulin, red). The siRNA effectively reduced the protein expression of GRASP55 by more than 10-fold, but did not affect the cilia targeting of P/rds. The images are confocal images of a single x–y plane. Scale bars, 10 μm.
	Figure 5. The C-terminal tail region of P/rds is essential for the cilia targeting. A, hTERT-RPE1 cells transiently expressing full-length or CT-truncated bP/rds were analyzed for the percentage of the primary cilia positive to P/rds. Strong (black bar), bP/rds is enriched in cilia; weak (red bar), bP/rds is in cilia but not enriched; NA (green bar), P/rds is not observed in cilia. ***p < 0.001 compared with bP/rds by t test. Error bars represent SD. bP/rds (1060 cilia from n = 6 preparations), bP/rds1-342 (635 cilia from n = 3 preparations), bP/rds1-336 (416 cilia from n = 3 preparations), bP/rds1-330 (245 cilia from n = 3 preparations), bP/rdsΔ289-312 (501 cilia from n = 3 preparations). A gradual reduction in the cilia-targeting efficacy was observed by CT truncations. B, The alignment of the P/rds CT tail region indicates that this region is highly conserved among vertebrate species: identical (*) or homologous (:) residues are shown. C, bP/rds1-342 (green) was concentrated in cilia like full-length protein. bP/rds1-336 (green) and bP/rdsΔ289-312 (green) localized in cilia but were not enriched and bP/rds1-330 (green) did not localize in cilia. Acetylated tubulin (red) was used as a cilia marker. The images are confocal images of a single x–y plane. Scale bar, 10 μm.
	Figure 6. C-terminal tail region of P/rds is essential for Golgi retention. A–C, hTERT-RPE1 cells transiently expressing full-length or CT-truncated bP/rds (green) were colabeled for GM130 (A; cis-Golgi), for GMII (B; medial and trans-Golgi), or for P230 (C; TGN). The images are confocal images of a single x–y plane. D, Maximum projection images of bP/rds and CT truncations. bP/rds is concentrated in cilia when the expression level is low (arrow) and localized to Golgi and other parts of cells except cilia when the expression level is high (arrowhead). CT truncation, down to the position 312, led to the accumulation of P/rds in the Golgi apparatus. Further truncation to position 288 led to exit of P/rds from the Golgi. Scale bars, 10 μm. E, CT-truncated bP/rds transiently expressed in hTERT-RPE1cells were treated with PNGase F (P) or Endo H (En). Truncation of the CT tail led to acquisition of Endo H resistance. Therefore, P/rds is capable of serving as a substrate for the GMII.
	Figure 7. Unconventional cilia targeting of P/rds in IMCD3 cells. A, hTERT-RPE1and IMCD3 cells stably expressing bP/rds or bP/rds1-288 were separated into cytoplasm (Cyto) and plasma membrane (PM) fractions by the biotinylation method and analyzed by Western blots. bP/rds1-288 is barely observed in the plasma membrane. Na+/K+ ATPase (bottom) was used to demonstrate that the plasma membrane proteins were pulled down effectively. B, PNGase F (P) and Endo H (En) treatments of IMCD3 cells stably expressing bP/rds or its truncation mutants. C, Maximum projection images of full-length and CT-truncated bP/rds expressed in IMCD3 (green). The arrow indicates the cilium. D, Top, IMCD3 and hTERT-RPE1 cells stably expressing bP/rds1-312 (HA, green) were labeled for a cis-Golgi marker GM130 (red). Bottom, Intensity of bP/rds1-312 and GM130 (arbitrary intensity units, AI) along the white bars shown in the top. Significant overlap between bP/rds1-312 and cis-Golgi was observed in IMCD3 cells, whereas the overlap is less significant in hTERT-RPE1 cells. Scale bars, 10 μm.
	Figure 8. Mislocalization of CT-truncated xP/rds in rods. A, Localization of full-length or CT-truncated P/rds (green) in X. laevis rods. OS areas are labeled by wheat germ agglutinin (WGA, red). Nuclei are in blue. The bottom row of images are magnified views of the selected areas above. B, xP/rds-Dend2 (green) is localized to disk rims and incisures. C, xP/rds1-316-Dend2 (green) colocalizes with the turquoise Golgi marker (red). D–G, Immunoelectron microscopy localization of CT-truncated and full-length P/rds. D, xP/rds1-316-Dend2 mislocalizes in Golgi apparatus. E, xP/rds1-288-Dend2 mislocalizes in electron-dense vesicles of the IS. F, xP/rds-Dend2 does not localize in IS. G, xP/rds-Dend2 localizes in rim and incisures of OS. Asterisks indicate Golgi apparatus and arrows indicate electron dense vesicles. Animals were 14–15 d old.
	Figure 9. Quantitative analysis of P/rds mislocalization in X. laevis rods. A, Localization of P/rds and its truncation mutants in unfixed retinas (maximum projections). Scale bar, 10 μm. B, Concentrations (Conc) of Dend2 fusion proteins were measured for OS and IS structures of individual rods. The numbers of rods analyzed for each transgene construct are shown in parentheses. Those rods originated from three individual tadpoles for each transgene construct. C, The IS/OS concentration ratio was analyzed for each transgene. FL, xP/rds-Dend2; 1–316, xP/rds1-316-Dend2; 1–288, xP/rds1-288-Dend2. The data are represented as mean ± SD. p < 0.001 compared with xP/rds-Dend2 by MWRST. Animals were 9–10 d old.
	Figure 10. CT-truncated P/rds retained in IS are slow in renewal. A–C, Dend2 fluorescence of live retinal explants (maximum projections) 0 h (A), 4 h (B), or 48 h (C) after photoconversion. xP/rds1-316 in the IS does not exit the Golgi apparatus for >48 h. D, PNGase F (P) and Endo H (En) analysis of tadpole eyes expressing full-length and CT-truncated P/rds. CT-truncated P/rds are sensitive to Endo H similar to full-length P/rds. Animals were 10 d old in A–C and 14–15 d old in D.
	Figure 11. Model for unconventional cilia targeting of P/rds. A, The summarized localizations of P/rds and the CT truncations in photoreceptor cells and mammalian cells. ++, +, +/−, and − indicate that the transgenes' signals were strong, weak, very weak, and undetectable, respectively. Note: When overexpressed in mammalian cells, P/rds can also be detected in Golgi and other intracellular compartments. B, In both mammalian cells (left) and X. laevis rods (right), P/rds bypasses the Golgi apparatus, either the later portions or altogether, to reach the cilia, thus taking an unconventional secretory pathway (green arrow, P/rds). The Golgi retention signal prevents conventional secretion of P/rds1-312 or P/rds1-316 (red arrow, P/rds1-312 or P/rds1-316), but does not block unconventional cilia targeting in rods (red dash-dotted arrow), likely due to trafficking driven by oligomerization with endogenous P/rds. After release from Golgi (left, blue dotted arrow, P/rds1-288), P/rds1-288 is incapable of taking the secretory pathway in mammalian cells (left, blue arrow). Routes labeled 1 and 2 are the estimated routes explaining the cell-line-dependent variability observed between hTERT-RPE1 and IMCD3 cells. Neither 1 or 2 is a complete secretory pathway and cannot deliver P/rds1-288 to the cell surface. P/rds1-288 can reach the cilium through unconventional secretion in rods (right, blue dotted and dash-dotted arrows).

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