tER sites are specialized cup-shaped ER subdomains characterized by the focused budding of COPII vesicles. Sec16 has been proposed to be involved in the biogenesis of tER sites by binding to COPII coat components and clustering nascent-coated vesicles. Here, we show that Drosophila Sec16 (dSec16) acts instead as a tER scaffold upstream of the COPII machinery, including Sar1. We show that dSec16 is required for Sar1-GTP concentration to the tER sites where it recruits in turn the components of the COPII machinery to initiate coat assembly. Last, we show that the dSec16 domain required for its localization maps to an arginine-rich motif located in a nonconserved region. We propose a model in which dSec16 binds ER cups via its arginine-rich domain, interacts with Sar1-GTP that is generated on ER membrane by Sec12 and concentrates it in the ER cups where it initiates the formation of COPII vesicles, thus acting as a tER scaffold.
The primary embryonic axes in flies, frogs and fish are formed through translational regulation of localized transcripts before fertilization 1 . In Drosophila, the axes are established through the transport and translational regulation of gurken (grk) and bicoid (bcd) messenger RNA (mRNA) in the oocyte and embryo 1 . bcd and grk mRNA are both translationally silent while being localized within the oocyte along microtubules by cytoplasmic Dynein 1-4 . Once localized, grk is translated at the dorsoanterior of the oocyte to send a TGF-alpha signal to the overlying somatic cells 5 . In contrast, bcd is translationally repressed in the oocyte until its activation in early embryos to form an anteroposterior morphogenetic gradient 6 . How this differential translational regulation is achieved is not fully understood. Here, we address this question using ultrastructural analysis, super-resolution microscopy and live cell imaging. We show that grk and bcd ribonucleoprotein (RNP) complexes associate with electron dense bodies that lack ribosomes and contain translational repressors, characteristic of Processing bodies (P bodies), which are regions of cytoplasm where translational decisions are made. Endogenous grk mRNA forms dynamic RNP particles that become docked and translated at the periphery of P bodies, where we show that the translational activator Orb/CEPB and the anchoring factor Squid (Sqd) are also enriched. In contrast, an excess of grk mRNA becomes localized inside the P bodies, where endogenous bcd mRNA is localized and translationally repressed. Interestingly, bcd mRNA dissociates from P bodies in embryos following egg activation, when it is known to become translationally active. We propose a general principle of translational regulation during axis specification involving remodeling of transport RNPs and dynamic partitioning of different transcripts between the translationally active edge of P bodies and their silent core.
RNAi screening for kinases regulating the functional organization of the early secretory pathway in Drosophila S2 cells has identified the atypical Mitotic-Associated Protein Kinase (MAPK) Extracellularly regulated kinase 7 (ERK7) as a new modulator. We found that ERK7 negatively regulates secretion in response to serum and amino-acid starvation, in both Drosophila and human cells. Under these conditions, ERK7 turnover through the proteasome is inhibited, and the resulting higher levels of this kinase lead to a modification in a site within the C-terminus of Sec16, a key ER exit site component. This post-translational modification elicits the cytoplasmic dispersion of Sec16 and the consequent disassembly of the ER exit sites, which in turn results in protein secretion inhibition. We found that ER exit site disassembly upon starvation is TOR complex 1 (TORC1) independent, showing that under nutrient stress conditions, cell growth is not only inhibited at the transcriptional and translational levels, but also independently at the level of secretion by inhibiting the membrane flow through the early secretory pathway. These results reveal the existence of new signalling circuits participating in the complex regulation of cell growth.
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