Parcas is the predominant Rab11-GEF for rhodopsin transport in Drosophila photoreceptors

Abstract

Rab11 is essential for polarized post-Golgi vesicle trafficking to photosensitive membrane rhabdomeres in Drosophila photoreceptors. Here, we found that Parcas (Pcs), recently shown to have guanine nucleotide exchange (GEF) activity toward Rab11, co-localizes with Rab11 on the trans-side of Golgi units and post-Golgi vesicles at the base of the rhabdomeres in pupal photoreceptors. Pcs fused with the electron micrography tag APEX2 localizes on 150-300 nm vesicles at the trans-side of Golgi units, which are presumably fly recycling endosomes. Loss of Pcs impairs Rab11 localization on the trans-side of Golgi units and induces the cytoplasmic accumulation of post-Golgi vesicles bearing rhabdomere proteins, as observed in Rab11 deficiency. In contrast, loss of Rab11-specific subunits of the TRAPPII complex, another known Rab11-GEF, does not cause any defects in eye development nor the transport of rhabdomere proteins; however, simultaneous loss of TRAPPII and Pcs results in severe defects in eye development. These results indicate that both TRAPPII and Pcs are required for eye development, but Pcs functions as the predominant Rab11-GEF for post-Golgi transport to photosensitive membrane rhabdomeres.

Recycling endosomes attach to the trans-side of Golgi stacks in Drosophila and mammalian cells

“…REs localized to the trans-side of Golgi stacks in Drosophila S2 cells By using electron microscopy, we also investigated the localization of RE markers in Schneider 2 (S2) cells derived from Drosophila embryos (Schneider, 1972). We have recently reported that Parcas (Pcs) is the predominant Rab11-GEF in fly photoreceptors and intensively colocalizes with Rab11 (Otsuka et al, 2019). In S2 cells, V5::Pcs localized to the trans-side of Golgi stacks (Fig.…”

“…3B-D). By using the genetic EM-tag ascorbate peroxidase 2 (APEX2; Martell et al, 2017), we also found Pcs localization to the cytoplasmic surface of 150-300 nm distorted vesicles or cisternae, which often associate with Golgi stacks (Otsuka et al, 2019). To clarify Pcs localization in regard to cistrans polarity of Golgi stacks, we performed double-APEX-labeling of GalT and Pcs.…”

“…1A, Fig. S1A arrows) (see Iwanami et al, 2016;Otsuka et al, 2019;. For a more-detailed analysis of Rab11 associated with Golgi stacks, we examined retinas at early pupal stage, which comprise larger Golgi stacks compared to those at late pupal stage.…”

“…To build pMT-HA-Rab11, N-terminal 3×HAtag (MYPYDVPDYAGGYPYDVPDYAGGYPYDVPDYAGGGGSGGG) followed by Drosophila Rab11 cDNA was cloned into pMT-puro. The myc::APEX2::myc fragment from pMT-myc-APEX2-myc (Otsuka et al, 2019) was inserted to pMT-hyg to create pMT-hyg-myc-APEX2-myc. The second Myc-tag in pMT-hyg-myc-APEX2-myc was replaced by EGFP to generate pMT-hyg-myc-APEX2-EGFP, then the first Myc-tag was replaced by aa 1-59 of the human B4GALT1 gene in pmTurquoise2-Golgi to create pMT-hyg-GalT-APEX2-EGFP.…”

“…Expression was induced by adding 1 mM CuSO 4 at 2 days after transfection. Cells were fixed in 4% paraformaldehyde in 1×PBS for 1 h on ice and immunostaining of S2 cells was performed as described previously (Otsuka et al, 2019). Primary antisera were as follows: rabbit anti-GM130 (1:300; Abcam #ab30637, Cambridge, UK) and guinea pig anti-Rab6 (1:300) (Iwanami et al, 2016).…”