Isolation of secreted proteins from Drosophila ovaries and embryos through in vivo BirA-mediated biotinylation

Abstract

The extraordinarily strong non-covalent interaction between biotin and avidin (kD = 10−14–10−16) has permitted this interaction to be used in a wide variety of experimental contexts. The Biotin Acceptor Peptide (BAP), a 15 amino acid motif that can be biotinylated by the E. coli BirA protein, has been fused to proteins-of-interest, making them substrates for in vivo biotinylation. Here we report on the construction and characterization of a modified BirA bearing signals for secretion and endoplasmic reticulum (ER) retention, for use in experimental contexts requiring biotinylation of secreted proteins. When expressed in the Drosophila female germline or ovarian follicle cells under Gal4-mediated transcriptional control, the modified BirA protein could be detected and shown to be enzymatically active in ovaries and progeny embryos. Surprisingly, however, it was not efficiently retained in the ER, and instead appeared to be secreted. To determine whether this secreted protein, now designated secBirA, could biotinylate secreted proteins, we generated BAP-tagged versions of two secreted Drosophila proteins, Torsolike (Tsl) and Gastrulation Defective (GD), which are normally expressed maternally and participate in embryonic pattern formation. Both Tsl-BAP and GD-BAP were shown to exhibit normal patterning activity. Co-expression of Tsl-BAP together with secBirA in ovarian follicle cells resulted in its biotinylation, which permitted its isolation from both ovaries and progeny embryos using Avidin-coupled affinity matrix. In contrast, co-expression with secBirA in the female germline did not result in detectable biotinylation of GD-BAP, possibly because the C-terminal location of the BAP tag made it inaccessible to BirA in vivo. Our results indicate that secBirA directs biotinylation of proteins bound for secretion in vivo, providing access to powerful experimental approaches for secreted proteins-of-interest. However, efficient biotinylation of target proteins may vary depending upon the location of the BAP tag or other structural features of the protein.

ERp29 Attenuates Nicotine-Induced Endoplasmic Reticulum Stress and Inhibits Choroidal Neovascularization

“…Second, ERp29 is an escort protein that is involved in COP vesicle trafficking [28][29][30][31]. ERp29 is involved in the process of protein transport between the ER and Golgi apparatus or the process of protein transport to secretory vesicles [32][33][34]. Both of these functions are important for regulating ER stress and helping cells cope with stress in their microenvironment.…”

Physiological and Pathological Regulation of Peripheral Metabolism by Gut-Peptide Hormones in Drosophila

“…In vivo trafficking of hormones is difficult to achieve due to limited genetic tools. Recent studies, which engineered a promiscuous biotin ligase, BirA, to specifically label secreted proteins including peptide prohormones (Stevens et al, 2019;Droujinine et al, 2020), are very promising to address the limitation (Figure 4B). They used a fused BirA to biotinylate all proteins in the muscle ER and detected biotin-labeled proteins in the blood to identify potential myokines.…”

The Probable, Possible, and Novel Functions of ERp29

“…As mentioned above, ERp29 contains a C-terminal KEEL motif that interacts with the KDEL-R rather than the more classical C-terminal KDEL motif. KEEL-harboring ER constituents are less fully retained in the ER than those with KDEL; in other words, KEEL binding has lower affinity than KDEL to KDEL-R in the acidic Golgi (Giannotta et al, 2012;Stevens et al, 2019). In fact, our group has found ERp29 in more distal compartments, including at the surface of epithelial cells and secreted into culture media in CFBE41o-cells (Suaud et al, 2011a), suggesting that ERp29 may have other functional roles beyond promoting anterograde ER → Golgi trafficking of client cargo via KDEL-R and COP II machinery.…”