Role of Asparagine-linked Oligosaccharides in Rhodopsin Maturation and Association with Its Molecular Chaperone, NinaA

Abstract

Many proteins require N-linked glycosylation for conformational maturation and interaction with their molecular chaperones. In Drosophila, rhodopsin (Rh1), the most abundant rhodopsin, is glycosylated in the endoplasmic reticulum (ER) and requires its molecular chaperone, NinaA, for exit from the ER and transport through the secretory pathway. Studies of vertebrate rhodopsins have generated several conflicting proposals regarding the role of glycosylation in rhodopsin maturation. We investigated the role of Rh1 glycosylation and Rh1/NinaA interactions under in vivo conditions by analyzing transgenic flies expressing Rh1 with isoleucine substitutions at each of the two consensus sites for N-linked glycosylation (N20I and N196I). We show that Asn 20 is the sole site for glycosylation. The Rh1 N20I protein is retained within the secretory pathway, causing an accumulation of ER cisternae and dilation of the Golgi complex. NinaA associates with nonglycosylated Rh1 N20I ; therefore, retention of nonglycosylated rhodopsin within the ER is not due to the lack of Rh1 N20I /NinaA interaction. We further show that Rh1 N20I interferes with wild type Rh1 maturation and triggers a dominant form of retinal degeneration. We conclude that during maturation Rh1 is present in protein complexes containing NinaA and that Rh1 glycosylation is required for transport of the complexes through the secretory pathway. Failure of this transport process leads to retinal degeneration.The correct folding, assembly, and subcellular distribution of many glycoproteins is dependent on the proteins undergoing Nlinked glycosylation within the endoplasmic reticulum (ER) (1, 2).

Phototransduction and retinal degeneration in Drosophila

“…Mutation of asparagine 20 decreases the level of mature Rh1 and results in retention of this protein in the secretory pathway. In contrast to many proteins that require N-linked glycosylation for interactions with chaperons, N-linked glycosylation of Rh1 does not affect binding to its chaperon, NINAA [69]. Another ER transmembrane molecular chaperon, calnexin, is required in photoreceptor cells specifically for rhodopsin maturation [70].…”

“…The glycosylation takes place at single site (asparagine 20) and is absent from the mature form of Rh1 [67][68][69]. Mutation of asparagine 20 decreases the level of mature Rh1 and results in retention of this protein in the secretory pathway.…”

“…In Drosophila photoreceptor cells, rhodopsin is synthesized and core-glycosylated in the endoplasmic reticulum (ER), transported through the Golgi, and delivered to the rhabdomeres where it functions in phototransduction. Among the mutations in rhodopsin that affect maturation is one (N20I) that disrupts the N-linked glycosylations site of Rh1 [69]. The Rh1 N20I protein is retained in the secretory pathway, resulting in accumulation of ER cisternae and retinal degeneration.…”

Cyclophilin A Mediates Vid22p Function in the Import of Fructose-1,6-bisphosphatase into Vid Vesicles

“…ninaA forms a stable complex with rhodopsin and co-localizes with Rh1 in the secretary vesicles, suggesting that Rh1 may require ninaA as it travels through the distal compartments of the secretory pathway (38). Furthermore, ninaA mutations inhibit Rh1 transport from the ER, leading to accumulation of ER cisternae (40). Another example of protein-protein interaction comes from studies showing that human cyclophilin A and the human immunodeficiency virus 1 GAG protein form a complex (32,41,42).…”

Molecular genetics of retinal degeneration: A Drosophila perspective

“…8,9 Opsin, like other integral membrane proteins, is translated in the ribosome of the rough endoplasmic reticulum (ER). Following translation, Rh1 undergoes N-linked glycosylation at Asp (20), 59,60 rendering it sensitive to endoglycosidase H. However, the carbohydrate moiety is subsequently removed when Rh1 exits the ER, resulting in a mature Rh1 without glycosylation. 61 In the ER, the carbohydrate moiety is required for folding and maturation of Rh1, 61 which is aided by two chaperones, NINAA 51 and CNX.…”