Rab6 Is Required for Multiple Apical Transport Pathways but Not the Basolateral Transport Pathway in Drosophila Photoreceptors

Abstract

Polarized membrane trafficking is essential for the construction and maintenance of multiple plasma membrane domains of cells. Highly polarized Drosophila photoreceptors are an excellent model for studying polarized transport. A single cross-section of Drosophila retina contains many photoreceptors with 3 clearly differentiated plasma membrane domains: a rhabdomere, stalk, and basolateral membrane. Genome-wide high-throughput ethyl methanesulfonate screening followed by precise immunohistochemical analysis identified a mutant with a rare phenotype characterized by a loss of 2 apical transport pathways with normal basolateral transport. Rapid gene identification using whole-genome resequencing and single nucleotide polymorphism mapping identified a nonsense mutation of Rab6 responsible for the apical-specific transport deficiency. Detailed analysis of the trafficking of a major rhabdomere protein Rh1 using blue light-induced chromophore supply identified Rab6 as essential for Rh1 to exit the Golgi units. Rab6 is mostly distributed from the trans-Golgi network to a Golgi-associated Rab11-positive compartment that likely recycles endosomes or transport vesicles going to recycling endosomes. Furthermore, the Rab6 effector, Rich, is required for Rab6 recruitment in the trans-Golgi network. Moreover, a Rich null mutation phenocopies the Rab6 null mutant, indicating that Rich functions as a guanine nucleotide exchange factor for Rab6. The results collectively indicate that Rab6 and Rich are essential for the trans-Golgi network–recycling endosome transport of cargoes destined for 2 apical domains. However, basolateral cargos are sorted and exported from the trans-Golgi network in a Rab6-independent manner.

The roles of Syx5 in Golgi morphology and Rhodopsin transport inDrosophilaphotoreceptors

“…Additionally, there is a reduction in the number of medial- and trans- Golgi cisternae owing to a lack of cis- Golgi cisternae that can progressively maturate into medial - and trans -Golgi cisternae. These phenotypes are distinct from that observed in Rab6 mutants, where cytoplasmic condensations of medial - and trans- Golgi resident enzymes occurs, but the amount and localization of cis -Golgi proteins remains unaffected (Iwanami et al, 2016; Satoh et al, 2016). …”

“…We confirmed Golgi association of Syx5 in late pupal photoreceptors by overexpressing Syx5::Myc and found that it predominantly localizes to the puncta closely associated with the trans-side of Golgi units marked by CFP::GalT (Satoh et al, 2005) (Fig. 3A), unlike Rab6 (another Golgi protein) which localizes to Golgi units and post-Golgi vesicles at the base of the rhabdomeres (Iwanami et al, 2016). Golgi units in photoreceptors are well-developed at the early pupal stages with their width reaching above 1 µm (Satoh et al, 2005).…”

“…2A). Interestingly, unlike the mutants of genes involved in Rh1 transport, such as Rab11, dRip11, MyoV and Rab6 (Iwanami et al, 2016; Li et al, 2007; Satoh et al, 2005), Rh1 was not detected in the cytoplasm of Syx5 EP2313 photoreceptors. First, we observed that in Syx5 EP2313 photoreceptors, levels of other rhabdomeric proteins like TRP, Chp (Montell and Rubin, 1989; Reinke et al, 1988) and basolateral membrane protein Na + K + ATPase (Yasuhara et al, 2000), were severely reduced (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.…”

Hedgehog signaling regulates ciliary localization of mouse odorant receptors

“…4F and Fig. S4), or Rab6B and Rab8A required for multiple apical transport pathways, including rhodopsin transport to the cilium in Drosophila photoreceptors (25,26) (Fig. 4 G and H).…”