Significance The connecting cilium (CC) of the photoreceptor provides the only route for the trafficking of the outer segment (OS) proteins. Failure of OS protein transport causes degenerative photoreceptor diseases, including retinitis pigmentosa. We demonstrate that Tmem138, a protein linked to ciliopathy, is localized to the photoreceptor CC. Germline deletion of Tmem138 abolished OS morphogenesis, followed by rapid photoreceptor degeneration. Tmem138 interacts with rhodopsin and two additional CC compartment proteins, Ahi1 and Tmem231, likely forming a membrane complex to facilitate trafficking of rhodopsin and other OS-bound proteins across the CC. The study thus implicates a new line of regulation on the delivery of OS proteins through interactions with CC membrane complex(es) and provides insights into photoreceptor ciliopathy diseases.

Photoreceptor connecting cilium (CC) is structurally analogous to the transition zone (TZ) of primary cilia and gates the molecular trafficking between the inner and the outer segment (OS). Retinal dystrophies with underlying CC defects are manifested in a broad array of syndromic conditions known as ciliopathies as well as non-syndromic retinal degenerations. Despite extensive studies, protein trafficking across the photoreceptor CC is largely unknown. Here we genetically inactivated mouse Tmem138, a gene encoding a ciliary membrane protein localized to the ciliary TZ and linked to Joubert syndrome (JBTS). Germline deletion of Tmem138 abolished OS morphogenesis followed by rapid photoreceptor degeneration. Tmem138 was found localized to the photoreceptor CC and, accordingly, the molecular compartments of the CC and axoneme of the mutant photoreceptors were altered despite ciliogenesis proceeding normally at the early stage of photoreceptor development. To gain further insights into Tmem138 function in OS biogenesis, we focused on trafficking of rhodopsin, the most abundant protein of the OS. Mislocalization of rhodopsin was readily observed as early as P5 in the mutant photoreceptors prior to growth of the OS. Ablation of Tmem138 in mature rods recapitulated the molecular changes in the germline mutants, causing well-formed outer segment discs to disintegrate accompanied by mislocalization of rhodopsin in the cell body. Furthermore, Tmem138 interacted with rhodopsin, and two additional CC compartment proteins Ahi1 and Tmem231, which were both altered in the mutant photoreceptors. Taken together, these results suggest that Tmem138 has a distinct role in gating the transport of rhodopsin and likely other OS bound proteins through formation of CC transport complex(es).