The transmembrane region of Gurken is not required for biological activity, but is necessary for transport to the oocyte membrane in Drosophila

Abstract

During Drosophila oogenesis, localization of the transforming growth factor alpha (TGFalpha)-like signaling molecule Gurken to the oocyte membrane is required for polarity establishment of the egg and embryo. To test Gurken domain functions, full-length and truncated forms of Gurken were expressed ectopically using the UAS/Gal4 expression system, or in the germline using the endogenous promoter. GrkDeltaC, a deletion of the cytoplasmic domain, localizes to the oocyte membrane and can signal. GrkDeltaTC, which lacks the transmembrane and cytoplasmic domains, retains signaling ability when ectopically expressed in somatic cells. However, in the germline, the GrkDeltaTC protein accumulates throughout the oocyte cytoplasm and cannot signal. In addition, we found that several strong gurken alleles contain point mutations in the transmembrane region. We conclude that secretion of Gurken requires its transmembrane region, and propose a model in which the gene cornichon mediates this process.

mRNA Localization and ER-based Protein Sorting Mechanisms Dictate the Use of Transitional Endoplasmic Reticulum-Golgi Units Involved in Gurken Transport inDrosophilaOocytes

“…This result suggests that g⌬TC is locally synthesized and diffuses throughout the oocyte ER, suggesting that the truncated domains are crucial for its efficient sorting at the D/A corner. A truncated version of Gurken lacking only its cytoplasmic domain (g⌬C) does not show such a strong diffusion and fully rescues the grk 2E12 /grk 2B6 phenotype (Queenan et al, 1999). Its diffusion in the ER was nevertheless slightly broader than in the WT (our unpublished data).…”

“…Gurken transmembrane domain has been suggested to be necessary for the efficient transport of the protein. The truncated Gurken protein lacking its transmembrane and cytoplasmic domain (g⌬TC) has been shown to be retained intracellularly (Queenan et al, 1999). When g⌬TC is expressed in a Gurken protein null background (grk 2E12 /grk 2B6 ), the truncated protein is present throughout the entire ER as seen by immunofluorescence (Figure 8, A and B) and by IEM (our unpublished data).…”

“…The antibodies used are the monoclonal mouse anti Gurken antibody 1D12 (Developmental Studies Hybridoma Bank, Iowa City, IA; Queenan et al, 1999); the anti-mammalian Sec23p rabbit polyclonal antibody (Affinity BioReagents, Golden, CO; Kondylis and Rabouille, 2003); the crude anti Yolkless rabbit polyclonal antibody 155 (gift from A. Mahowald, Chicago, IL); and the polyclonal anti-green fluorescent protein (GFP) antibody A6455 (Molecular Probes, Leiden, The Netherlands).…”

“…Mutant fly stocks used are dCOG5-GFP (w;GFP-Fws/Cyo) (Farkas et al, 2003); PDI-GFP (PDI::GFP/GFP-TM3,Sb) (Bobinnec et al, 2003) (Norvell et al, 1999); cni AR55 (b cni AR55 pr cn/Cyo), cni CF5 (b cni CF5 pr cn wx bw/Cyo) and cni AA12 (b cni AA12 /Cyo) (Roth et al, 1995); grk 2B6 (grk[2B6]b cn s L bo/Cyo) and grk 2E12 (w; grk[2E12]b/Cyo) (Neuman-Silberberg and Schupbach, 1993); and fly lines expressing Gurken lacking the transmembrane and the cytoplasmic domain g⌬TC4.1 and the cytoplasmic domain g⌬C100 (Queenan et al, 1999). The stocks carrying the constructs encoding the truncated versions of Gurken proteins were crossed into the grk 2B6 background and females from this cross were mated with male grk 2E12 to yield orange-eyed black flies (g⌬TC4.1; grk 2B6 /grk 2E12 and g⌬C100; grk 2B6 /grk 2E12 ).…”

“…(Neuman-Silberberg and Schupbach, 1993); and fly lines expressing Gurken lacking the transmembrane and the cytoplasmic domain g⌬TC4.1 and the cytoplasmic domain g⌬C100 (Queenan et al, 1999). The stocks carrying the constructs encoding the truncated versions of Gurken proteins were crossed into the grk 2B6 background and females from this cross were mated with male grk 2E12 to yield orange-eyed black flies (g⌬TC4.1; grk 2B6 /grk 2E12 and g⌬C100; grk 2B6 /grk 2E12 ).…”