DOI: 10.5061/dryad.wwpzgmssx
title: Comprehensive study for SNARE involved in the post-Golgi transport in Drosophila photoreceptors

description: Polarized transport is essential for the construction of multiple plasma
 membrane domains within cells. Drosophilaphotoreceptors serve as excellent
 model systems for studying the mechanisms of polarized transport. We
 conducted a comprehensive SNARE (soluble N-ethylmaleimide-sensitive factor
 attachment protein receptor) screening of the fly genome using RNAi
 knockdown and CRISPR/Cas9 somatic knockout combined with the CoinFLP
 system to identify SNAREs involved in post-Golgi trafficking. The results
 suggest that, in post-Golgi transport, no SNARE is exclusively responsible
 for transport to a single specific plasma membrane domain. However, each
 SNARE shows some preference for certain membrane domains: the loss of
 nSyb, Ykt6, and Snap24/25 results in severe defects in rhabdomere
 transport, while the loss of Syx1A and Snap29 leads to significant
 impairments in basolateral transport. Together with the function of Syx1A,
 Snap25, and nSyb in the fusion of synaptic vesicles with the synaptic
 plasma membrane, these results suggest that SNAREs are not the sole
 determinants for vesicles to specify their target subdomains in the plasma
 membrane. Furthermore, rhodopsin transport to the rhabdomere requires two
 kinds of R-SNAREs, Ykt6 and nSyb, suggesting that multiple sets of
 post-Golgi SNAREs are contributing in tandem or in cooperation, rather
 than in parallel.
description: Plasmids were constructed as follows. 
 <strong>pU62-wsh1</strong><br>pU62-wsh1 is a
 plasmid carrying a cassette that works as a "dominant white-eye"
 marker in Drosophila, consist of Drosophila U6-2 promoter and shRNA
 against white gene. This vector was constructed based on a vector,
 pAc-sgRNA-Cas9 (Bassett et al., 2014) by inserting a synthetic shRNA unit
 against white gene, HMS00017, predesigned in TRiP RNAi & CRISPR
 fly project (Zirin et al., 2020). Four oligonucleotides TTC-wsh1-F1,
 wsh1-R1p, wsh1-F2p and wsh1-Ap-GTT were ligated into pAc-sgRNA-Cas9
 digested with BbsI, to obtain pU62-wsh1.
 <strong>pBIDwsh-UASC</strong><br>pBIDwsh-UASC
 is a phi31 transgenic vector based on pBID-UASC (Wang et al., 2012) but
 carrying "dominant white-eye" marker instead of miniature-white
 “red-eye” marker. From pU62-wsh1, U6-wshRNA cassette was amplified with
 primers Nh-U62 and gCas9puro-F, then digested with NheI and ApaI, and then
 cloned between NheI and ApaI sites of pBID-UASC to obtain
 pBID-wsh-UASC.
 <strong>pBIDwsh-Act-coinFLP-Gal4</strong><br>pBIDwsh-Act-coinFLP-Gal4 is a phi31 transgenic vector carrying "dominant white-eye" marker and coinFLP-Gal4 cassette under control of Act5C promoter. From original coinFLP vector, pAct-FRT-stop-FRT3-FRT-FRT3-Gal4 attB (Bosch et al., 2015), the DNA fragment including Act5C promoter and FRT-stop-FRT3-FRT-FRT3-Gal4 cassette was amplified with primers Sbf-BID-Act-F and Gal4-BID-Xh-R. Using Gibson assembly, the fragment was cloned between SbfI and BglII sites of pBIDwsh-UASC, replacing UASC promoter and multiple cloning sites, to obtain pBIDwsh-Act-coinFLP-Gal4. <strong>5x longGMR promoter</strong><br>The longGMR promoter (Wernet et al., 2003) consists of five copies of enhancer sequence in the upstream of Rh1 promoter and a basal promoter core of hsp70. Since the detail of the construct was not described, the region containing longGMR promoter was amplified using PCR with primers wdelR_Nhe and Gal4DBD-R2 from a transgenic fly caring P{longGMR-Gal4} (Bloomington Drosophila Stock Center #8605)..<br>pP-longGMR, pBID-longGMR and pBIDwsh-longGMR<br>The fragment containing longGMR promoter was digested with SphI and EcoRI and then ligated between SphI and EcoRI sites of pPdM-UAST (Yamashita et al., 2022), pBID-UASC, and pBIDwsh-UASC, to obtain pP-longGMR, pBID-longGMR, and pBIDwsh-longGMR, respectively.<br>pP-3xlongGMR, pBID-3xlongGMR and pBIDwsh-3xlongGMR<br>DNA fragments with various number of LGMR repeats were amplified from pP-longGMR, with primers Sph-longGMR and UAST-R4. The fragments were digested with SphI and EcoRI, then separated by size in agarose gel electrophoresis, and then ligated into pPdM-UAST, pBID-UASC, and pBIDwsh-UASC, to obtain pP-3xlongGMR, pBID-3xlongGMR, and pBIDwsh-3xlongGMR, respectively.<br>pP-longGMR-coinFLP-Gal4 and pBIDwsh-longGMR-coinFLP-Gal4<br>From pBIDwsh-Act-coinFLP-Gal4, DNA fragments including FRT-stop-FRT3-FRT-FRT3-Gal4 cassette were excised with BamHI and KpnI, and then ligated between BglII and KpnI sites in pP-longGMR, pP-3xlongGMR and pBIDwsh-longGMR to obtain pP-LongGMR-coinFLP-Gal4, pP-3xlongGMR-coinFLP-Gal4 and pBIDwsh-longGMR-coinFLP-Gal4. <strong>pX-HFCas9</strong><br>A mammalian CRISPR-Cas9 KO vector, pX-HFCas9, coding HiFiCas9R691A (Vakulskas et al., 2018) was constructed based on pSpCas9(BB)-2A-Puro PX459 V2.0 (Ran et al., 2013), by introducing R691A mutation in the Cas9 gene using Gibson assembly with primers HF-Cas9-F and HF-Cas9-R.<br>pBIDwsh-Act-coinFLP-HFCas9-H2B-2xNG and pP-3xlongGMR-coinFLP-HFCas9-H2B-2xNG<br>DNA fragments including HFCas9-T2A was amplified from pX-HF-Cas9 with Bg-Coin-Cas9 and ddT2A-R. DNA fragments including H2B-GFP was amplified from pcDNA3.1 miniSOG2 T2A H2B-EGFP (Makhijani et al., 2017) with ddT2A-F2 and H2B-EGFP-BID-K. Using Gibson assembly, fragments HFCas9-T2A and H2B-2xmNeonGreen were cloned between BglII and KpnI sites of pBIDwsh-Act-coin-Gal4 and pP-3xlongGMR-coinFLP-Gal4 to obtain pBIDwsh-Act-coinFLP-HFCas9-H2B-2xNG and pP-3xlongGMR-coinFLP-HFCas9-H2B-2xNG. <strong>pCFD4w-Syx6</strong><br>A phi31 vector pCFD4w was constructed by replacing promoter-gRNA scaffold of pCFD5w, a phi31 vector with white+ marker (gift from Michael Boutros, Addgene plasmid #112645), by those of pCFD4. DNA fragments including dU6:2-BbsI-gRNA scaffold were amplified with primers Sgf-U61F and dU6-wsh-fix and digested with EcoRI and NheI, then inserted between EcoRI and XbaI sites of pCFD5w. To construct pCFD4w-Syx6, oligonucleotides Syx6-gRNA3f and Syx6-gRNA3r were annealed and ligated with BbsI digested pCFD4w. <strong>pUAST-myc-Ykt6, pUAST-HA-Snap24 and pUAST-HA-Snap25 <br></strong>From third instar larvae cDNA, DNA fragment encoding Ykt6 were amplified using primers Xh-dYkt6 and dYkt6-Mlu, then digested with XhoI and MluI, and then ligated between XhoI and MluI sites in pMT-HA-NBgA-m, to generate pMT-HA-Ykt6. The fragment containing Ykt6 was excised with XhoI and XbaI and then inserted into pPdM-UAST-m2NBgAm to generate pUAST-myc-Ykt6. DNA fragment encoding snap24 and snap25 were amplified using primers Xh-Snap24, Snap24-Ap, Xh-Snap25 and Snap25-Ap, digested with XhoI and ApaI, and cloned into pMT-HA-NBgA-m to obtain pMT-HA-snap24 and pMT-HA-snap25. HA-snap24 and HA-snap25 fragments were excised with KpnI and MluI, and then inserted into pPdM-UAST-m2NBgAm to generate pUAST-HA-Snap24 and pUAST-HA-Snap25.
description: Nucleotide sequences of plasmids and DNA fragments used in the paper. All
 files are in standard Genbank format, which can be viewed using many DNA
 handling sofwares such as Ape
 ([https://jorgensen.biology.utah.edu/wayned/ape/](https://jorgensen.biology.utah.edu/wayned/ape/)).  ##### 5x\_longGMR\_promoter.gb  * Usage:  * The longGMR promoter (Wernet et al., 2003) consists of five copies of enhancer sequence in the upstream of Rh1 promoter and a basal promoter core of hsp70. Since the detail of the construct was not described in the original paper, the sequence was analysed. The region containing longGMR promoter was amplified using PCR from a transgenic fly caring P{longGMR-Gal4}  with primers wdelR_Nhe (GGGCTAGCTTCAATGATGTCCAGTGCAG) and Gal4DBD-R2 (GACCTTTTGGTTTTGGGAGAGTAGCG) and sequenced.  * Source:  * Drosophila strain P{longGMR-Gal4} * Bloomington Drosophila Stock Center #8605 * [http://flybase.org/reports/FBti0058798.htm](http://flybase.org/reports/FBti0058798.htm) * Wernet et al., 2003  * [https://doi.org/10.1016/S0092-8674(03)00848-1](https://doi.org/10.1016/S0092-8674\(03\)00848-1) ##### pAct\_FRT\_stop\_FRT3\_FRT\_FRT3\_Gal4\_attB(52889).gb * Usage:  * Used for plasmid construction as source of Coin-FLP casette * Source:  * Gift form Iswar Hariharan (Addgene plasmid # 52889) * [https://www.addgene.org/52889/](https://www.addgene.org/52889/) * CoinFLP: a system for efficient mosaic screening and for visualizing clonal boundaries in Drosophila * [https://doi.org/10.1242/dev.114603](https://doi.org/10.1242/dev.114603)  ##### pBID-3xlongGMR.gb * Usage:  * Used to construct phi31 Drosophila transformation vectors expressing insert under control of 3 copies of longGMR enhancers.  * Source: This work   ##### pBID-longGMR.gb * Usage:  * Used to construct phi31 Drosophila transformation vectors expressing insert under control of 5 copies of longGMR enhancers. A miniature-w marker can be used to screen transformant.   * Source: This work   ##### pBIDwsh-3xlongGMR-coinFLP-Gal4.gb * Usage:  * A phi31 Drosophila transformation vectors with coin_FLP casette under control of 3 copies of longGMR enhancers. A dominat-white-eye marker can be used to screen transformant.   * Source: This work   ##### pBIDwsh-3xlongGMR.gb * Usage:  * A phi31 Drosophila transformation vectors with coin_FLP casette under control of 3 copies of longGMR enhancers. A miniature-w marker can be used to screen transformant. Used to construct pBIDwsh-3xlongGMR-coinFLP-Gal4.  * Source: This work   ##### pBIDwsh-Act-coinFLP-Gal4.gb * Usage:  * A phi31 Drosophila transformation vectors with Gal4 activator and coin_FLP casette under control Act5C promoter. A dominat-white-eye marker can be used to screen transformant.   * Source: This work   ##### pBIDwsh-Act-coinFLP-HFCas9-H2B-NG.gb * Usage:  * A phi31 Drosophila transformation vectors with Cas9 gene and coin_FLP casette under control Act5C promoter. Histon 2B tagged with mNeonGreen is co-expressed with Cas9 when flipped-ON. A dominat-white-eye marker can be used to screen transformant.   * Source: This work   ##### pBIDwsh-longGMR.gb * Usage:  * A phi31 Drosophila transformation vectors with coin_FLP casette under control of 5 copies of longGMR enhancers. A miniature-w marker can be used to screen transformant. Used to construct pBIDwsh-3xlongGMR-coinFLP-Gal4.  * Source: This work   ##### pBIDwsh-UASC.gb * Usage:  * A phi31 Drosophila transformation vectors with coin_FLP casette under control of 10 copies of UAS enhancers. A dominat-white-eyemarker can be used to screen transformant. * Source: This work   ##### pCFD4w\.gb * Usage:  * A phi31 Drosophila transformation vectors with CRISPR gRNA under control of Drosophila U6 promoter. A miniature-w marker can be used to screen transformant.  * Source: This work   ##### pCFD5w(112645).gb * Usage:  * A phi31 Drosophila transformation vectors with CRISPR gRNA under control of Drosophila U6 promoter. A miniature-w marker can be used to screen transformant.  * Source:  * gift from Michael Boutros (Addgene plasmid # 112645) * [https://www.addgene.org/112645/](https://www.addgene.org/112645/)   ##### pMT-HA-NBgA-m.gb * Usage:  * A plasmid vector for Drosophila S2 cells. Used as intermediate vector to add linkers.  * Source: This work  **\ ** ##### pP-3xlongGMR-coinFLP-HFCas9-H2B-NG.gb * Usage:  * A P-element  Drosophila transformation vector with Cas9 gene and coin_FLP casette under control Act5C promoter. Histon 2B tagged with mNeonGreen is co-expressed with Cas9 when flipped-ON. A miniature-w marker can be used to screen transformant.   * Source: This work   ##### pP-3xlongGMR.gb * Usage:  * Used to construct P-element Drosophila transformation vectors expressing insert under control of 3 copies of longGMR enhancers. A miniature-w marker can be used to screen transformant.   * Source: This work   ##### pP-longGMR-coinFLP-Gal4.gb * Usage:  * A P-element  Drosophila transformation vector with GAL4 activator and coin_FLP casette under control of 3 copies of longGMR enhancers. A miniature-w marker can be used to screen transformant.   * Source: This work   ##### pP-longGMR.gb * Usage:  * Used to construct P-element Drosophila transformation vectors expressing insert under control of 5 copies of longGMR enhancers. A miniature-w marker can be used to screen transformant.   * Source: This work   ##### pPdM-UAST-vAX2m.gb * Usage:  * Used to construct P-element Drosophila transformation vectors expressing insert under control of 5 copies of UAS enhancers. A miniature-w marker can be used to screen transformant.   - Source: This work   ##### pU62-wsh1.gb * Usage:  * A phi31 Drosophila transformation vectors with CRISPR gRNA under control of Drosophila U6.2 promoter. A dominat-white-eye marker can be used to screen transformant.   * Source: This work   ##### pUAST-HA-Snap24.gb * Usage:  * A P-element Drosophila transformation vectors expressing Drosophila SNAP24 gene under control of 5 copies of UAS enhancers. A HA-tag was fused on the N-terminal. A miniature-w marker can be used to screen transformant.   - Source: This work   ##### pUAST-HA-Snap25.gb * Usage:  * A P-element Drosophila transformation vectors expressing Drosophila SNAP25 gene under control of 5 copies of UAS enhancers. A HA-tag was fused on the N-terminal. A miniature-w marker can be used to screen transformant.   - Source: This work   ##### pUAST-Myc-Ykt6.gb * Usage:  - A P-element Drosophila transformation vectors expressing Drosophila Ykt6 gene under control of 5 copies of UAS enhancers. A Myc-tag was fused on the N-terminal. A miniature-w marker can be used to screen transformant.   - Source: This work   ##### pX-HFCas9.gb * Usage:  * A mammalian CRISPR-Cas9 KO vector, carrying Cas9 gene with R691A high-fidelity mutation (Vakulskas et al., 2018, https://doi.org/10.1038/s41591-018-0137-0) . Used to construct Drosophila vectors with Cas9.  - Source: This work