RESUMEN
Photoreceptors are complex ciliated sensory neurons. The basal body and periciliary ridge of photoreceptors function in association with the Golgi complex to regulate the export of proteins from the inner segment to the outer segment sensory axoneme. Here, we show that the retinitis pigmentosa protein RP2, which is a GTPase activating protein (GAP) for Arl3, localizes to the ciliary apparatus, namely the basal body and the associated centriole at the base of the photoreceptor cilium. Targeting to the ciliary base was dependent on N-terminal myristoylation. RP2 also localized to the Golgi and periciliary ridge of photoreceptors, which suggested a role for RP2 in regulating vesicle traffic and docking. To explore this hypothesis, we investigated the effect of RP2 depletion and the expression of a constitutively active form of Arl3 (Q71L) on pericentriolar vesicle transport. Kif3a, a component of intraflagellar transport (IFT), is important in cilia maintenance and transport of proteins through the connecting cilium in photoreceptors. Similar to Kif3a and Arl3 depletion, loss of RP2 led to fragmentation of the Golgi network. Depletion of RP2 and dysregulation of Arl3 resulted in dispersal of vesicles cycling cargo from the Golgi complex to the cilium, including the IFT protein IFT20. We propose that RP2 regulation of Arl3 is important for maintaining Golgi cohesion, facilitating the transport and docking of vesicles and thereby carrying proteins to the base of the photoreceptor connecting cilium for transport to the outer segment.
Asunto(s)
Centriolos/metabolismo , Cuerpo Ciliar/metabolismo , Proteínas del Ojo/fisiología , Aparato de Golgi/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas de la Membrana/fisiología , Células Fotorreceptoras/metabolismo , Retinitis Pigmentosa/metabolismo , Factores de Ribosilacion-ADP/metabolismo , Animales , Transporte Biológico , Células Cultivadas , Cilios/metabolismo , Proteínas de Unión al GTP , Humanos , Ratones , Vesículas Transportadoras/metabolismoRESUMEN
PURPOSE: AIPL1 mutations cause the severe inherited blindness Leber congenital amaurosis (LCA). The similarity of AIPL1 to tetratricopeptide repeat (TPR) cochaperones that interact with the chaperone Hsp90 and the ability of AIPL1 to suppress the aggregation of NUB1 fragments in a chaperone-like manner suggest that AIPL1 might function as part of a chaperone heterocomplex facilitating retinal protein maturation. In this study the interaction of AIPL1 with molecular chaperones is revealed and functionally characterized. METHODS: AIPL1-interacting proteins were identified using a yeast two-hybrid system, and the effect of AIPL1 pathogenic mutations and sequence requirements mediating the identified interactions were investigated. The interactions were validated by a comprehensive set of biochemical assays, and the ability of the AIPL1-binding partners to cooperate with AIPL1 in the suppression of NUB1 fragment aggregation was assessed. RESULTS: AIPL1 interacts with the molecular chaperones Hsp90 and Hsp70. Mutations within the TPR domain of AIPL1 or removal of the chaperone TPR acceptor site abolished the interactions. Importantly, LCA-causing mutations in AIPL1 also compromised these interactions, suggesting that the essential function of AIPL1 in photoreceptors may involve the interaction with Hsp90 and Hsp70. Examination of the role of these chaperones in AIPL1 chaperone activity demonstrated that AIPL1 cooperated with Hsp70, but not with Hsp90, to suppress the formation of NUB1 inclusions. CONCLUSIONS: These findings suggest that AIPL1 may cooperate with both Hsp70 and Hsp90 within a retina-specific chaperone heterocomplex and that the specialized role of AIPL1 in photoreceptors may therefore be facilitated by these molecular chaperones.
Asunto(s)
Proteínas Portadoras/metabolismo , Chaperonas Moleculares/metabolismo , Atrofia Óptica Hereditaria de Leber/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Benzoquinonas/farmacología , Células COS , Proteínas Portadoras/genética , Línea Celular Tumoral , Chlorocebus aethiops , Proteínas del Ojo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Lactamas Macrocíclicas/farmacología , Mutación , Atrofia Óptica Hereditaria de Leber/genética , Fragmentos de Péptidos/antagonistas & inhibidores , Estructura Terciaria de Proteína/genética , Neoplasias de la Retina/metabolismo , Neoplasias de la Retina/patología , Retinoblastoma/metabolismo , Retinoblastoma/patología , Factores de Transcripción/antagonistas & inhibidores , Técnicas del Sistema de Dos HíbridosAsunto(s)
Proteínas del Ojo/genética , Ligamiento Genético/genética , Retinitis Pigmentosa/genética , Proteínas del Ojo/química , Proteínas del Ojo/metabolismo , Proteínas de Unión al GTP , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Modelos Estructurales , Mutación , Retinitis Pigmentosa/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMEN
Mutations in RP2 cause X-linked retinitis pigmentosa and also macular and peripapillary atrophy. RP2 is a functional homologue of the tubulin folding cofactor, cofactor C, as it can replace the beta tubulin GTPase stimulating activity of cofactor C in an in vitro assay. An important difference between RP2 and cofactor C is their subcellular localization. RP2 is targeted to the cytoplasmic face of the plasma membrane by dual N-terminal acylation, and this post-translational modification is important for protein function. The activity of tubulin folding cofactors is modulated by certain ADP ribosylation factor-like (Arl) proteins. It has been shown that RP2 can interact directly with Arl3. Here we describe the methodologies that we have developed to analyze the interaction of RP2 with Arl3 and to investigate the effect of RP2 post-translational modifications on its subcellular and tissue localization.