Mutations in Four Glycosyl Hydrolases Reveal a Highly Coordinated Pathway for Rhodopsin Biosynthesis and N-Glycan Trimming in Drosophila melanogaster

Abstract

As newly synthesized glycoproteins move through the secretory pathway, the asparagine-linked glycan (N-glycan) undergoes extensive modifications involving the sequential removal and addition of sugar residues. These modifications are critical for the proper assembly, quality control and transport of glycoproteins during biosynthesis. The importance of N-glycosylation is illustrated by a growing list of diseases that result from defects in the biosynthesis and processing of N-linked glycans. The major rhodopsin in Drosophila melanogaster photoreceptors, Rh1, is highly unique among glycoproteins, as the N-glycan appears to be completely removed during Rh1 biosynthesis and maturation. However, much of the deglycosylation pathway for Rh1 remains unknown. To elucidate the key steps in Rh1 deglycosylation in vivo, we characterized mutant alleles of four Drosophila glycosyl hydrolases, namely α-mannosidase-II (α-Man-II), α-mannosidase-IIb (α-Man-IIb), a β-N-acetylglucosaminidase called fused lobes (Fdl), and hexosaminidase 1 (Hexo1). We have demonstrated that these four enzymes play essential and unique roles in a highly coordinated pathway for oligosaccharide trimming during Rh1 biosynthesis. Our results reveal that α-Man-II and α-Man-IIb are not isozymes like their mammalian counterparts, but rather function at distinct stages in Rh1 maturation. Also of significance, our results indicate that Hexo1 has a biosynthetic role in N-glycan processing during Rh1 maturation. This is unexpected given that in humans, the hexosaminidases are typically lysosomal enzymes involved in N-glycan catabolism with no known roles in protein biosynthesis. Here, we present a genetic dissection of glycoprotein processing in Drosophila and unveil key steps in N-glycan trimming during Rh1 biosynthesis. Taken together, our results provide fundamental advances towards understanding the complex and highly regulated pathway of N-glycosylation in vivo and reveal novel insights into the functions of glycosyl hydrolases in the secretory pathway.

The Gos28 SNARE Protein Mediates Intra-Golgi Transport of Rhodopsin and Is Required for Photoreceptor Survival

“…This was accomplished by monitoring the molecular weight of Rh1 via Western blot analysis. We have previously shown that Rh1 is glycosylated in the ER and then completely deglycosylated as it moves through the Golgi, such that defects in Rh1 transport lead to the accumulation of Rh1 in hyperglycosylated high molecular weight forms (39,47). The gos28 1 EMS allele displayed a defect in Rh1, with a substantial amount of the protein accumulating in a higher molecular weight form, compared with wild type (Fig.…”

“…mutant, we screened ϳ12,000 ethyl methylsulfonate (EMS) mutagenized lines from the Zuker collection (37) for the presence or absence of the deep pseudopupil (38). We identified ϳ900 deep pseudopupil-defective mutants, which were further screened for defects in Rh1 by Western blot analysis (39). The wild-type stocks used in this study were bw;st and cn,bw (parental stocks from the EMS mutagenesis), as well as Canton-S. We also used the eyes absent mutant allele, eya 1 .…”

“…In wild-type flies, Rh1 was initially detected as glycosylated high molecular weight forms that were processed down to the mature form, beginning ϳ10 h post heat shock ( Fig. 4A) (39,43). At later time points, Rh1 was detected exclusively in the mature low molecular weight form in wild-type flies.…”

“…Two transgenic stocks, each containing an inducible UAS-RNAi construct directed against the gos28 transcript, P[GD3051]v12152 and P[KK107479]v100289, were obtained from the Vienna Drosophila RNAi Center (41). Eyespecific knockdown of the Gos28 target mRNA transcripts was achieved by performing standard genetic crosses between the UAS-RNAi strains and an eye-specific Gal4 driver line provided by Claude Desplan as follows: P[UAS-Dcr2];P[ey-Gal4], P[IGMR-Gal4];ϩ, as described previously (39,42).…”

“…In wild-type flies, the N-glycosylation group is completely removed from Rh1 during its transport to the rhabdomere (39,47,(51)(52)(53). Accordingly, the mature 34-kDa form of Rh1 is insensitive to both Endo H and PNGase F (Fig.…”