Background

In Drosophila, the early secretory apparatus comprises discrete paired Golgi stacks in close proximity to exit sites from the endoplasmic reticulum (tER sites), thus forming tER-Golgi units. Although many components involved in secretion have been identified, the structural components sustaining its organisation are less known. Here we set out to identify novel ER resident proteins involved in the of tER-Golgi unit organisation.

Results

To do so, we designed a novel screening strategy combining a bioinformatics pre-selection with an RNAi screen. We first selected 156 proteins exhibiting known or related ER retention/retrieval signals from a list of proteins predicted to have a signal sequence. We then performed a microscopy-based primary and confirmation RNAi screen in Drosophila S2 cells directly scoring the organisation of the tER-Golgi units. We identified 49 hits, most of which leading to an increased number of smaller tER-Golgi units (MG for “more and smaller Golgi”) upon depletion. 16 of them were validated and characterised, showing that this phenotype was not due to an inhibition in secretion, a block in G2, or ER stress. Interestingly, the MG phenotype was often accompanied by an increase in the cell volume. Out of 6 proteins, 4 were localised to the ER.

Conclusions

This work has identified novel proteins involved in the organisation of the Drosophila early secretory pathway. It contributes to the effort of assigning protein functions to gene annotation in the secretory pathway, and analysis of the MG hits revealed an enrichment of ER proteins. These results suggest a link between ER localisation, aspects of cell metabolism and tER-Golgi structural organisation.

The Golgi apparatus is essential for protein sorting and transport. Many researchers have long been fascinated with the form and function of this organelle. Yet, despite decades of scrutiny, the mechanisms by which proteins are transported across the Golgi remain controversial. At a recent meeting, many prominent Golgi researchers assembled to critically evaluate the core issues in the field. This report presents the outcome of their discussions and highlights the key open questions that will help guide the field into a new era.

Ceramides are central intermediates of sphingolipid metabolism with critical functions in cell organization and survival. They are synthesized on the cytosolic surface of the endoplasmic reticulum (ER) and transported by ceramide transfer protein to the Golgi for conversion to sphingomyelin (SM) by SM synthase SMS1. In this study, we report the identification of an SMS1-related (SMSr) enzyme, which catalyses the synthesis of the SM analogue ceramide phosphoethanolamine (CPE) in the ER lumen. Strikingly, SMSr produces only trace amounts of CPE, i.e., 300-fold less than SMS1-derived SM. Nevertheless, blocking its catalytic activity causes a substantial rise in ER ceramide levels and a structural collapse of the early secretory pathway. We find that the latter phenotype is not caused by depletion of CPE but rather a consequence of ceramide accumulation in the ER. Our results establish SMSr as a key regulator of ceramide homeostasis that seems to operate as a sensor rather than a converter of ceramides in the ER.

Background

Muscle fibres are formed by elongation and fusion of myoblasts into myotubes. During this differentiation process, the cytoskeleton is reorganized, and proteins of the centrosome re-localize to the surface of the nucleus. The exact timing of this event, and the underlying molecular mechanisms are still poorly understood.

Results

We performed studies on mouse myoblast cell lines that were induced to differentiate in culture, to characterize the early events of centrosome protein re-localization. We demonstrate that this re-localization occurs already at the single cell stage, prior to fusion into myotubes. Centrosome proteins that accumulate at the nuclear surface form an insoluble matrix that can be reversibly disassembled if isolated nuclei are exposed to mitotic cytoplasm from Xenopus egg extract. Our microscopy data suggest that this perinuclear matrix of centrosome proteins consists of a system of interconnected fibrils.

Conclusion

Our data provide new insights into the reorganization of centrosome proteins during muscular differentiation, at the structural and biochemical level. Because we observe that centrosome protein re-localization occurs early during differentiation, we believe that it is of functional importance for the reorganization of the cytoskeleton in the differentiation process.

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.

An RNAi screen in Drosophila cells has identified about 100 TANGO proteins, which may regulate protein exocytosis or secretion.

Although the organization and functions of the constitutive secretory pathway have been intensively studied for decades, a recent genome-wide RNAi screen in Drosophila cells has identified about 100 genes encoding novel so-called TANGO proteins (for transport and Golgi organization) that may be direct regulators of various aspects of protein exocytosis or secretion.

The exocyst is an octameric complex required for polarized secretion. Some components of the exocyst are found on the plasma membrane, whereas others are recruited to Golgi membranes, suggesting that exocyst assembly tethers vesicles to their site of fusion. We have found that in Drosophila melanogaster oocytes the majority of the exocyst component Sec5 is unexpectedly present in clathrin-coated pits and vesicles at the plasma membrane. In oocytes, the major substrate for clathrin-dependent endocytosis is the vitellogenin receptor Yolkless. A truncation mutant of Sec5 (sec5E13) allows the formation of normally sized oocytes but with greatly reduced yolk uptake. We find that in sec5E13 oocytes Yolkless accumulates aberrantly in late endocytic compartments, indicating a defect in the endocytic cycling of the receptor. An analogous truncation of the yeast SEC5 gene results in normal secretion but a temperature-sensitive defect in endocytic recycling. Thus, the exocyst may act in both Golgi to plasma membrane traffic and endocytic cycling, and hence in oocytes is recruited to clathrin-coated pits to facilitate the rapid recycling of Yolkless.

The de novo model for Golgi stack biogenesis predicts that membrane exiting the ER at transitional ER (tER) sites contains and recruits all the necessary molecules to form a Golgi stack, including the Golgi matrix proteins, p115, GM130, and GRASP65/55. These proteins leave the tER sites faster than Golgi transmembrane resident enzymes, suggesting that they act as a template nucleating the formation of the Golgi apparatus. However, the localization of the Golgi matrix proteins at tER sites is only shown under conditions where exit from the ER is blocked. Here, we show in Drosophila S2 cells, that dGRASP, the single Drosophila homologue of GRASP65/55, localizes both to the Golgi membranes and the tER sites at steady state and that the myristoylation of glycine 2 is essential for the localization to both compartments. Its depletion for 96 h by RNAi gave an effect on the architecture of the Golgi stacks in 30% of the cells, but a double depletion of dGRASP and dGM130 led to the quantitative conversion of Golgi stacks into clusters of vesicles and tubules, often featuring single cisternae. This disruption of Golgi architecture was not accompanied by the disorganization of tER sites or the inhibition of anterograde transport. This shows that, at least in Drosophila, the structural integrity of the Golgi stacks is not required for efficient transport. Overall, dGRASP exhibits a dynamic association to the membrane of the early exocytic pathway and is involved in Golgi stack architecture.

Oculocerebrorenal syndrome of Lowe is caused by mutation of OCRL1, a phosphatidylinositol 4,5-bisphosphate 5-phosphatase localized at the Golgi apparatus. The cellular role of OCRL1 is unknown, and consequently the mechanism by which loss of OCRL1 function leads to disease is ill defined. Here, we show that OCRL1 is associated with clathrin-coated transport intermediates operating between the trans-Golgi network (TGN) and endosomes. OCRL1 interacts directly with clathrin heavy chain and promotes clathrin assembly in vitro. Interaction with clathrin is not, however, required for membrane association of OCRL1. Overexpression of OCRL1 results in redistribution of clathrin and the cation-independent mannose 6-phosphate receptor (CI-MPR) to enlarged endosomal structures that are defective in retrograde trafficking to the TGN. Depletion of cellular OCRL1 also causes partial redistribution of a CI-MPR reporter to early endosomes. These findings suggest a role for OCRL1 in clathrin-mediated trafficking of proteins from endosomes to the TGN and that defects in this pathway might contribute to the Lowe syndrome phenotype.

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-α 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.

Here, we describe that depletion of the Drosophila homologue of p115 (dp115) by RNA interference in Drosophila S2 cells led to important morphological changes in the Golgi stack morphology and the transitional ER (tER) organization. Using conventional and immunoelectron microscopy and confocal immunofluorescence microscopy, we show that Golgi stacks were converted into clusters of vesicles and tubules, and that the tERs (marked by Sec23p) lost their focused organization and were now dispersed throughout the cytoplasm. However, we found that this morphologically altered exocytic pathway was nevertheless largely competent in anterograde protein transport using two different assays. The effects were specific for dp115. Depletion of the Drosophila homologues of GM130 and syntaxin 5 (dSed5p) did not lead to an effect on the tER organization, though the Golgi stacks were greatly vesiculated in the cells depleted of dSed5p. Taken together, these studies suggest that dp115 could be implicated in the architecture of both the Golgi stacks and the tER sites.

NSF and p97 are ATPases required for the heterotypic fusion of transport vesicles with their target membranes and the homotypic fusion of organelles. NSF uses ATP hydrolysis to dissociate NSF/SNAPs/SNAREs complexes, separating the v- and t-SNAREs, which are then primed for subsequent rounds of fusion. In contrast, p97 does not dissociate the p97/p47/SNARE complex even in the presence of ATP. Now we have identified a novel essential factor for p97/p47-mediated membrane fusion, named VCIP135 (valosin-containing protein [VCP][p97]/p47 complex-interacting protein, p135), and show that it binds to the p97/p47/syntaxin5 complex and dissociates it via p97 catalyzed ATP hydrolysis. In living cells, VCIP135 and p47 are shown to function in Golgi and ER assembly.

We provide a detailed description of Golgi stack biogenesis that takes place in vivo during one of the morphogenetic events in the lifespan of Drosophila melanogaster. In early third-instar larvae, small clusters consisting mostly of vesicles and tubules were present in epithelial imaginal disk cells. As larvae progressed through mid- and late-third instar, these larval clusters became larger but also increasingly formed cisternae, some of which were stacked. In white pupae, the typical Golgi stack was observed. We show that larval clusters are Golgi stack precursors by 1) localizing various Golgi-specific markers to the larval clusters by electron and immunofluorescence confocal microscopy, 2) driving this conversion in wild-type larvae incubated at 37°C for 2 h, and 3) showing that this conversion does not take place in an NSF1 mutant (comt 17). The biological significance of this conversion became clear when we found that the steroid hormone 20-hydroxyecdysone (ecdysone) is critically involved in this conversion. In its absence, Golgi stack biogenesis did not occur and the larval clusters remained unaltered. We showed that dGM130 and sec23p expression increases approximately three- and fivefold, respectively, when discs are exposed to ecdysone in vivo and in vitro. Taken together, these results suggest that we have developed an in vivo system to study the ecdysone-triggered Golgi stack biogenesis.