XB-ART-48316J Neurosci January 15, 2014; 34 (3): 992-1006.
An unconventional secretory pathway mediates the cilia targeting of peripherin/rds.
It is unclear how unconventional secretion interplays with conventional secretion for the normal maintenance and renewal of membrane structures. The photoreceptor sensory cilium is recognized for fast membrane renewal, for which rhodopsin and peripherin/rds (P/rds) play critical roles. Here, we provide evidence that P/rds is targeted to the cilia by an unconventional secretion pathway. When expressed in ciliated hTERT-RPE1 human cell line, P/rd is localized to cilia. Cilium trafficking of P/rds was sustained even when the Golgi functions, including trans-Golgi-mediated conventional secretion, were inhibited by the small molecules brefeldin A, 30N12, and monensin. The unconventional cilia targeting of P/rds is dependent on COPII-mediated exit from the ER, but appears to be independent of GRASP55-mediated secretion. The regions in the C-terminal tail of P/rds are essential for this unconventional trafficking. In the absence of the region required for cilia targeting, P/rds was prohibited from entering the secretory pathways and was retained in the Golgi apparatus. A region essential for this Golgi retention was also found in the C-terminal tail of P/rds and supported the cilia targeting of P/rds mediated by unconventional secretion. In ciliated cells, including bovine and Xenopus laevis rod photoreceptors, P/rds was robustly sensitive to endoglycosidase H, which is consistent with its bypassing the medial Golgi and traversing the unconventional secretory pathway. Because rhodopsin is known to traffic through conventional secretion, this study of P/rds suggests that both conventional secretion and unconventional secretion need to cooperate for the renewal of the photoreceptor sensory cilium.
PubMed ID: 24431457
PMC ID: PMC3891973
Article link: J Neurosci
Grant support: R01 EY020826 NEI NIH HHS , P30 EY011373 NEI NIH HHS , T32 DK007319 NIDDK NIH HHS
Genes referenced: calr golga2 golga4 mtor prph prph2 rho sstr3
Article Images: [+] show captions
|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 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 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 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).|