An Unconventional Secretory Pathway Mediates the Cilia Targeting of Peripherin/rds

Abstract

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.

PRPH2/RDS and ROM-1: Historical context, current views and future considerations

“…Using a variety of PRPH2/RDS mutants, pharmacological agents which block protein transport at specific well characterized steps, and high quality fluorescence microscopy in a tissue culture system it was found that PRPH2/RDS could transport to the cilia when trans-Golgi exit was blocked, but does require COPII-mediated exit from the ER. Consistent with a key role for the PRPH2/RDS C-terminal in trafficking, PRPH2/RDS was retained in the cis-Golgi when the C-terminal targeting sequence was eliminated (Tian et al, 2014). Various unconventional secretory pathways have been described (Nickel, 2010; Nickel and Rabouille, 2009), and it is not yet clear which of these is utilized by PRPH2/RDS, however it has been shown that unconventional trafficking of PRPH2/RDS does not require GRASP55 (a known non-conventional secretory protein) (Gee et al, 2011; Tian et al, 2014), and our recent work has suggested that Syntaxin 3 may be involved (Zulliger et al, 2015).…”

“…Further support for the idea that PRPH2/RDS traffics to the OS using a specialized transportation mechanism different from other OS components such as rhodopsin came from recent work showing that a large portion of PRPH2/RDS traffics to the OS through a non-conventional secretory pathway which bypasses the trans-Golgi (Tian et al, 2014). Hints that this might be the case had been observed many years earlier when it was discovered that the PRPH2/RDS N-glycan was sensitive to digestion by EndoH, a phenomenon usually associated with failure of a protein to be exposed to the enzyme Golgi mannosidase II in the medial to trans-Golgi (Connell and Molday, 1990; Velasco et al, 1993).…”

Three-dimensional organization of nascent rod outer segment disk membranes

“…In addition, proteins that function in the plasma membrane (e.g., the cGMP channel subunit proteins, such as CNGA1) must be separated from discrete disk membranes (12). ImmunoEM and trafficking studies of PRPH2 have shown that this protein is delivered to the cilium separately from RHO (38), and that it is not included in the most basal disks of rod OS (36,39), consistent with an open lamellar structure for the basal disks. The presence of PRPH2 in the OS coincides only with rim formation (39,40).…”

Gene Therapy for PRPH2-Associated Ocular Disease: Challenges and Prospects

“…Although the complete role of peripherin-2 in this process is not yet fully elucidated, the carboxy-terminal domain of peripherin-2 has been shown to play a role in membrane fusion (Boesze-Battagliaa andStefano 2002, 2003), initiation of rim membrane curvature (Khattree et al 2013), and OS targeting (Tam et al 2004;Tian et al 2014). The D2 loop and covalently linked peripherin-2 complexes are required for the maintenance of the flattened rim morphology (Wrigley et al 2000;Chakraborty et al 2009Chakraborty et al , 2010.…”