Analysis of the expression patterns, subcellular localisations and interaction partners of Drosophila proteins using a pigP protein trap library

Abstract

Although we now have a wealth of information on the transcription patterns of all the genes in the Drosophila genome, much less is known about the properties of the encoded proteins. To provide information on the expression patterns and subcellular localisations of many proteins in parallel, we have performed a large-scale protein trap screen using a hybrid piggyBac vector carrying an artificial exon encoding yellow fluorescent protein (YFP) and protein affinity tags. From screening 41 million embryos, we recovered 616 verified independent YFP-positive lines representing protein traps in 374 genes, two-thirds of which had not been tagged in previous P element protein trap screens. Over 20 different research groups then characterized the expression patterns of the tagged proteins in a variety of tissues and at several developmental stages. In parallel, we purified many of the tagged proteins from embryos using the affinity tags and identified co-purifying proteins by mass spectrometry. The fly stocks are publicly available through the Kyoto Drosophila Genetics Resource Center. All our data are available via an open access database (Flannotator), which provides comprehensive information on the expression patterns, subcellular localisations and in vivo interaction partners of the trapped proteins. Our resource substantially increases the number of available protein traps in Drosophila and identifies new markers for cellular organelles and structures.

Keywords: Affinity purification; Cytoophidia; Live imaging; Protein trap; piggyBac.

Fig. 1.

Design of the pigP protein trap screen. (A) Schematic of the pigP vector. (B) Schematic showing the inclusion of the affinity tags and Venus-YFP into the middle of a trapped protein after insertion of the pigP vector in the correct reading frame into an intron between protein-coding exons. (C) The crossing scheme used to generate pigP protein trap insertions. (D) Scheme for the recovery of YFP-positive pigP insertions. (E) Summary of the results of all pigP protein trap screens.

Fig. 2.

Examples of protein trap lines with tissue-specific expression patterns. (A) Zasp52 (CPTI-000408) is expressed in two rows of ventral mesectoderm cells in the germ band extending embryo. (B,C) The putative glutamate transporter MSF3 (CPTI-002305) is expressed in the first instar larva in protrusive cells that envelop the central nervous system, which are likely to be the surface glia that form the blood-brain barrier. (D) The CPTI-001473 insertion in Complexin labels the neuromuscular junctions in the first instar larva. (E) An insert in Nervana 2 (CPTI-001459), the β subunit of the Na⁺/K⁺ ATPase, is strongly expressed in the central nervous system and labels the axons of the motor neurons extending to their target muscles. (F) A Babos protein trap insertion (CPTI-0001423) labels the peripheral nervous system, including the sensory axons projecting towards the CNS. (G) A maximum intensity projection of a z-stack through the adult brain showing the expression pattern of Gad1 (CPTI-000977; green) and stained for Bruchpilot (magenta). (H) A western blot probed with mouse monoclonal anti-GFP, showing the circadian expression of the CPTI-100059 insert in Trailer hitch (Tral) in extracts from adult heads. Flies were grown at 18°C under a 12 h light/12 h dark regime with samples taken at the times indicated.

Fig. 3.

Examples of protein trap insertions with specific subcellular localisations. (A) CPTI-002342 (α-catenin) labels the adherens junctions that outline the apical margins of the cells of the larval epidermis. (B) Gliotactin (CPTI-003903) strongly accumulates at the tricellular junctions at the apical vortices of the larval epidermal cells. (C) The N-acetylgalactosaminyltransferase CG30463 (CPTI-003680) labels the dispersed Golgi ministacks in the larval epidermis. (D) CG30463 (CPTI-002151) localisation in the cells of the accessory gland of the adult male testis. (E) The CPTI-100048 insertion in the chitin-binding protein Obst-E marks whorls of chitin fibres in the first instar larval cuticle. (F) The CPTI-004445 insertion in CG14207 labels the z bands of the muscles in the ovarian sheath. (G,H) Paxillin (CPTI-000546) and the leucine-rich repeat protein CG1399 (CPTI-001765) are highly enriched at muscle-attachment sites in the first instar larva.

Fig. 4.

Protein trap lines that label intranuclear structures in primary spermatocytes that are likely to correspond to the giant loops of the Y chromosome. (A) Pasilla (CPTI-000668) marks the C-loop of the Y chromosome in primary spermatocytes. (B-F) Smooth (CPTI-002828) (B), NonA (CPTI-003091) (C), Hrb98DE (CPTI-000205) (D), Squid (CPTI-000239) (E), ZAP3 (CPTI-004292) (F) and Muscleblind (CPTI-003555) (G) label similar structures that are likely to be giant loops of the Y chromosome. (H-J) NonA (H), ZAP3 (I) and Hrb98DE (J) also mark intranuclear speckles in the nurse cells and follicle cells of the ovary. (K) Hrb98DE localisation on the polytene chromosomes of the larval salivary gland.

Fig. 5.

Protein trap lines that label large cytoplasmic aggregates in the female germ line. (A) A protein trap insertion in Cytidine synthase (CPTI-001881) labels the rod-like cytoophidia that form in the cytoplasm of the germ cells of the developing egg chamber. (B) Ade3 (CPTI-003733) forms similar rod-like structures in the female germ line. (C) Ade5 (CPTI-002207) forms more spherical aggregates in the nurse cell and oocyte cytoplasm. (D) Fax (CPTI-002774) also localises to large spherical cytoplasmic structures in these cells.

Fig. 6.

Annotation of the protein interaction data from the affinity purifications of protein trap lines. (A) A Venn diagram showing the number of proteins that co-purified with PKA-R2 (CPTI-001580) in the affinity purifications using the 3×FLAG, StrepTagII and YFP tags. (B) A section of a table generated in Flannotator listing the three proteins that were detected in all three purifications of PKA-R2. The bait protein is highlighted in yellow and likely contaminants are shown in grey. (C) The details of PKA-C1 peptides identified by the mass spectrometer in some of the 3×FLAG, StrepTagII and YFP affinity purifications of PKA-R2 (taken from Flannotator). The ‘Protein Matches’ section at the bottom shows the amino acid sequence of PKA-C1 with the positions of identified peptides highlighted in red. (D) A section of a table generated in Flannotator listing the proteins that co-purified with SmD3 in the 3×FLAG affinity purifications. These include the other six Sm proteins that form a heptameric ring with SmD3 (CPTI-002164) and components of the U1, U2, U4, U5 and U6 snRNPs, with which the Sm proteins associate. The ‘Validation’ column indicates the probability score that the observed interaction is real calculated using a Generalized Iterative Scaling-Maximum Entropy supervised machine-learning approach. The ‘Pubmed’ column lists the PubMed identification numbers of any publications that have also described an interaction between SmD3 and the identified protein.