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

Abstract

The anteroposterior and dorsoventral axes of the future embryo are specified within Drosophila oocytes by localizing gurken mRNA, which targets the secreted Gurken transforming growth factor-␣ synthesis and transport to the same site. A key question is whether gurken mRNA is targeted to a specialized exocytic pathway to achieve the polar deposition of the protein. Here, we show, by (immuno)electron microscopy that the exocytic pathway in stage 9 -10 Drosophila oocytes comprises a thousand evenly distributed transitional endoplasmic reticulum (tER)-Golgi units. Using Drosophila mutants, we show that it is the localization of gurken mRNA coupled to efficient sorting of Gurken out of the ER that determines which of the numerous equivalent tER-Golgi units are used for the protein transport and processing. The choice of tER-Golgi units by mRNA localization makes them independent of each other and represents a nonconventional way, by which the oocyte implements polarized deposition of transmembrane/secreted proteins. We propose that this pretranslational mechanism could be a general way for targeted secretion in polarized cells, such as neurons. INTRODUCTIONPolarized localization of proteins is achieved by at least two different mechanisms. The first one relies on restricted localization of mRNAs that encode cytosolic proteins, allowing local protein translation, thus creating differential protein activity and generating cell asymmetry and polarity (Bashirullah et al., 1998). This is, for instance, the case of actin mRNA localization in the nerve terminal of neurons (Lee and Hollenbeck, 2003), oskar localization at the posterior pole of Drosophila oocytes (Ephrussi et al., 1991), and ash1 localization in dividing yeast cells (Long et al., 1997). On the other hand, the mechanism ensuring the polarized deposition of transmembrane or secreted proteins is thought, in mammalian cells, to be achieved by posttranslational sorting events in the trans-Golgi network (TGN). There, signals in their cytoplasmic tail or lumenal domain are deciphered, resulting in the directed movement of specialized transport vesicles to allow specific deposition at the apical or basolateral plasma membrane of epithelial cells (Ikonen and Simons, 1998;Nelson and Yeaman, 2001).However, there is a growing number of transmembrane and secreted proteins for which their transcript also exhibits a restricted localization. The transcript for the yeast plasma membrane protein Ist2 is actively transported along the acto-myosin network to the bud tip (Takizawa et al., 2000), and the protein is deposited locally (Juschke et al., 2004). wingless mRNA is localized apically in epithelial cells (Simmonds et al., 2001). In a stage 9 -10 Drosophila oocytes, gurken mRNA is transported and deposited exclusively in the dorsal/anterior corner (D/A; Neuman-Silberberg and Schupbach, 1993; Figure 1B). gurken encodes a protein that is synthesized in the endoplasmic reticulum (ER) as a 285-amino acid type I transmembrane protein precursor and transported to the Golgi apparat...

Distinct functional units of the Golgi complex in Drosophila cells

“…For example, we found that FRC was localized to all Golgi units in salivary gland cells at the larval stage (data not shown). It was also recently shown that all of the Golgi complexes dispersed in oocytes may have the ability to process the GURKEN precursor protein, which is usually cleaved in a subset of the Golgi complexes residing in the dorso-anterior region (29). The Golgi units may thus be altered in a manner dependent on development, cell type, and signaling processes.…”

A Dual Role for Actin and Microtubule Cytoskeleton in the Transport of Golgi Units from the Nurse Cells to the Oocyte Across Ring Canals

“…Unlike mammalian cells in which Golgi units are found close to the nucleus, Drosophila egg chambers present Golgi dispersed both throughout the NC and the oocyte cytoplasm (Herpers and Rabouille, 2004). Its correct distribution is essential for the proper development of the oocyte (Januschke et al, 2007;Coutelis and Ephrussi, 2007), but the process controlling the transport of Golgi to the oocyte has not been addressed.…”

“…We interpreted the accumulation of the Golgi in the oocyte as an indication of active transport of Golgi into the oocyte against a concentration gradient. In Drosophila egg chamber, the Golgi is observed in close vicinity of ER-exit sites and forms functional units called "tER-Golgi units" (Kondylis and Rabouille, 2003;Herpers and Rabouille, 2004). To discriminate the transport that we were following, either Golgi vesicles or tER-Golgi units, we performed a double labeling with a dCOG5-GFP transgene, which labels ER-exit sites (Herpers and Rabouille, 2004), and a lectin wheat germ agglutinin (WGA), which distribution is similar to GalT (Januschke et al, 2007).…”

“…In Drosophila egg chamber, the Golgi is observed in close vicinity of ER-exit sites and forms functional units called "tER-Golgi units" (Kondylis and Rabouille, 2003;Herpers and Rabouille, 2004). To discriminate the transport that we were following, either Golgi vesicles or tER-Golgi units, we performed a double labeling with a dCOG5-GFP transgene, which labels ER-exit sites (Herpers and Rabouille, 2004), and a lectin wheat germ agglutinin (WGA), which distribution is similar to GalT (Januschke et al, 2007). We observed that 45% of the WGA signal is associated with the dCOG5 labeling, either at the vicinity or inside RCs (Supplemental Figure S1), indicating that whole tER-Golgi units were transported into the oocyte.…”

“…But, when stacks are present, they do not form a single copy organelle. Instead, they remain scattered throughout the cytoplasm (Kondylis and Rabouille, 2003;Herpers and Rabouille, 2004), an organization that is similar to that in yeast (Rossanese et al, 1999). Whatever the morphology of the Golgi apparatus, they are in proximity to tER sites (trans-endoplasmic reticulum).…”