GPI biosynthesis is essential for rhodopsin sorting at the trans-Golgi network in Drosophila photoreceptors

Abstract

SUMMARYSorting of integral membrane proteins plays crucial roles in establishing and maintaining the polarized structures of epithelial cells and neurons. However, little is known about the sorting mechanisms of newly synthesized membrane proteins at the trans-Golgi network (TGN). To identify which genes are essential for these sorting mechanisms, we screened mutants in which the transport of Rhodopsin 1 (Rh1), an apical integral membrane protein in Drosophila photoreceptors, was affected. We found that deficiencies in glycosylphosphatidylinositol (GPI) synthesis and attachment processes cause loss of the apical transport of Rh1 from the TGN and mis-sorting to the endolysosomal system. Moreover, Na + K + -ATPase, a basolateral membrane protein, and Crumbs (Crb), a stalk membrane protein, were mistransported to the apical rhabdomeric microvilli in GPI-deficient photoreceptors. These results indicate that polarized sorting of integral membrane proteins at the TGN requires the synthesis and anchoring of GPI-anchored proteins. Little is known about the cellular biological consequences of GPI deficiency in animals in vivo. Our results provide new insights into the importance of GPI synthesis and aid the understanding of pathologies involving GPI deficiency.

The roles of Syx5 in Golgi morphology and Rhodopsin transport inDrosophilaphotoreceptors

“…In a previous study, we identified several mutants that failed to accumulate Rh1 in the rhabdomeres, using retinal mosaic screening of P-element or piggy-Bac inserted lines maintained at stock centers (Satoh et al, 2013). Among them, two lines, KY114446 and KY114472 with P-elements EP2313 and EY07901, respectively, were inserted into the 5′-UTR of Syx5 gene (Fig.…”

“…5F), suggesting that most of the Rh1 is degraded 3 h after BLICS in Syx5-deficient photoreceptors. In other mutants associated with Rh1 transport defects, such as Rab6 and PIG mutants, 3 h after BLICS there is a large number of late endosomal compartments and multi-vesicular bodies (MVBs) containing Rh1, indicating that Rh1 is degraded within MVBs, however, it takes more than 3 h to degrade Rh1 in the MVBs (Iwanami et al, 2016; Satoh et al, 2013). Therefore, we conclude that in Syx5-deficient photoreceptors, Rh1 is first transported to Golgi units and is then quickly degraded by MVBs-independent processes.…”

“…Membrane proteins synthesized on the ER membrane are transported to cis- Golgi cisternae by Rab1 (Satoh et al, 1997), Syx5, and possibly by Sec22 (Zhao et al, 2015). Probably after Gos28 dependent processes (Rosenbaum et al, 2014), basolateral membrane proteins are sorted by AP1 at trans- Golgi/TGN (Satoh et al, 2013) and the two apical membrane proteins are transported together to the recycling endosome (RE) by Rab6 (Iwanami et al, 2016). There is another round of sorting for the two apical pathways at the RE.…”

“…For live-imaging and immunostaining in the Bruin flies, the following tester lines were used: y w; P3RFP FRT40A/SM1; Rh1Arr2GFP eye-FLP/TM6B were used for live-imaging, while y w ey-FLP; P3RFP FRT40A / SM1 were used for immunostaining (Satoh et al, 2013). For P-element excision, wg SP-1 /CyO; ry, Sb, Delta2-3/TM6B (Kyoto stock number, 107139) lines was crossed to Syx5 EP2313 , FRT40A/CyO , or Syx5 EY07901 , FRT40A/CyO line.…”

“…Therefore, an assay for rhodopsin accumulation in late pupal rhabdomeres offers a sensitive method to identify the genes involved in biosynthetic membrane transport. In a previous study, we screened P-element/piggyBac insertion lines by visualizing Rh1, using Arrestin2::GFP and a water immersion technique, this led to the identification of several lines which failed to accumulate Rh1 at the rhabdomeres (Satoh et al, 2013). In two of these lines, P-elements were inserted in the 5′-UTR of Syx5 , which encodes a SNARE protein.…”