How the endoplasmic reticulum (ER) and the Golgi apparatus maintain their morphological and functional identity while working in concert to ensure the production of biomolecules necessary for the cell9s survival is a fundamental question in plant biology. Here, we isolated and characterized an Arabidopsis thaliana mutant that partially accumulates Golgi membrane markers and a soluble secretory marker in globular structures composed of a mass of convoluted ER tubules that maintain a connection with the bulk ER. We established that the aberrant phenotype was due to a missense recessive mutation in sec24A, one of the three Arabidopsis isoforms encoding the coat protomer complex II (COPII) protein Sec24, and that the mutation affects the distribution of this critical component at ER export sites. By contrast, total loss of sec24A function was lethal, suggesting that Arabidopsis sec24A is an essential gene. These results produce important insights into the functional diversification of plant COPII coat components and the role of these proteins in maintaining the dynamic identity of organelles of the early plant secretory pathway.

Giardia lamblia is an intestinal protozoan parasite required to survive in the environment in order to be transmitted to a new host. To ensure parasite survival, flagellated trophozoites colonizing the small intestine differentiate into non-motile environmentally-resistant cysts which are then shed in the environment. This cell differentiation process called encystation is characterized by significant morphological remodeling which includes secretion of large amounts of cyst wall material. Although much is known about the transcriptional regulation of encystation and the synthesis and trafficking of cyst wall material, the investigation of global changes in protein content and abundance during G. lamblia encystation is still unaddressed. In this study, we report on the quantitative analysis of the G. lamblia proteome during encystation using tandem mass spectrometry. Quantification of more than 1000 proteins revealed major changes in protein abundance in early, mid and late encystation, notably in constitutive secretory protein trafficking. Early stages of encystation were marked by a striking decrease of endoplasmic reticulum-targeted variant-specific surface proteins and significant increases in cytoskeleton regulatory components, NEK protein kinases and proteins involved in protein folding and glycolysis. This was in stark contrast to cells in the later stages of encystation which presented a surprisingly similar proteome composition to non-encysting trophozoites. Altogether these data constitute the first quantitative atlas of the Giardia proteome covering the whole process of encystation and point towards an important role for post-transcriptional control of gene expression in Giardia differentiation. Furthermore, our data provide a valuable resource for the community-based annotation effort of the G. lamblia genome, where almost 70% of all predicted gene models remains “hypothetical”.

Giardia lamblia is a parasitic protozoan that infects a wide range of vertebrate hosts including humans. Trophozoites are non-invasive but associate tightly with the enterocyte surface of the small intestine. This narrow ecological specialization entailed extensive morphological and functional adaptations during host-parasite co-evolution, including a distinctly polarized array of endocytic organelles termed peripheral vacuoles (PVs), which are confined to the dorsal cortical region exposed to the gut lumen and are in close proximity to the plasma membrane (PM). Here, we investigated the molecular consequences of these adaptations on the Giardia endocytic machinery and membrane coat complexes. Despite the absence of canonical clathrin coated vesicles in electron microscopy, Giardia possesses conserved PV-associated clathrin heavy chain (GlCHC), dynamin-related protein (GlDRP), and assembly polypeptide complex 2 (AP2) subunits, suggesting a novel function for GlCHC and its adaptors. We found that, in contrast to GFP-tagged AP2 subunits and DRP, CHC::GFP reporters have no detectable turnover in living cells, indicating fundamental differences in recruitment to the membrane and disassembly compared to previously characterized clathrin coats. Histochemical localization in electron tomography showed that these long-lived GlCHC assemblies localized at distinctive approximations between the plasma and PV membrane. A detailed protein interactome of GlCHC revealed all of the conserved factors in addition to novel or highly diverged proteins, including a putative clathrin light chain and lipid-binding proteins. Taken together, our data provide strong evidence for giardial CHC as a component of highly stable assemblies at PV-PM junctions that likely have a central role in organizing continuities between the PM and PV membranes for controlled sampling of the fluid environment. This suggests a novel function for CHC in Giardia and the extent of molecular remodeling of endocytosis in this species. In canonical clathrin mediated endocytosis (CME) models, the concerted action of ca. 50 proteins mediates the uptake of extracellular components. The key player in this process is clathrin which coats transport intermediates called clathrin coated vesicles (CCV). The intestinal parasite Giardia lamblia has undergone extensive remodeling during colonization of the mammalian duodenum. Here, we report on unique features of this parasite’s endocytic system, consisting of fixed peripheral vacuoles (PV) in close proximity to the exposed plasma membrane (PM), with no discernible CCVs. Using state-of-the-art imaging strategies, we show that the surface of Giardia trophozoites is pock-marked with PM invaginations reaching to the underlying PV membrane. Co-immunoprecipitation and analysis of protein dynamics reveal that, in line with the absence of CCVs, giardial clathrin assemblies have no dynamic behavior. CHC still remains associated to AP2 and dynamin, both conserved dynamic CME components, and to a newly identified putative clathrin light chain. The emerging model calls for giardial clathrin organized into static cores surrounded by dynamic interaction partners, and most likely involved in the regulation of fusion between the PM and the PVs in a “kiss-and-flush”-like mechanism. This suggests that Giardia harbors a conceptually novel function for clathrin in endocytosis, which might be a consequence of host-parasite co-evolution.

Protein secretion in eukaryotic cells is a well-studied process, which has been known for decades and is dealt with by any standard cell biology textbook. However, over the past 20 years, several studies led to the realization that protein secretion as a process might not be as uniform among different cargos as once thought. While in classic canonical secretion proteins carry a signal sequence, the secretory or surface proteome of several organisms demonstrated a lack of such signals in several secreted proteins. Other proteins were found to indeed carry a leader sequence, but simply circumvent the Golgi apparatus, which in canonical secretion is generally responsible for the modification and sorting of secretory proteins after their passage through the endoplasmic reticulum (ER). These alternative mechanisms of protein translocation to, or across, the plasma membrane were collectively termed “unconventional protein secretion” (UPS). To date, many research groups have studied UPS in their respective model organism of choice, with surprising reports on the proportion of unconventionally secreted proteins and their crucial roles for the cell and survival of the organism. Involved in processes such as immune responses and cell proliferation, and including far more different cargo proteins in different organisms than anyone had expected, unconventional secretion does not seem so unconventional after all. Alongside mammalian cells, much work on this topic has been done on protist parasites, including genera Leishmania, Trypanosoma, Plasmodium, Trichomonas, Giardia, and Entamoeba. Studies on protein secretion have mainly focused on parasite-derived virulence factors as a main source of pathogenicity for hosts. Given their need to secrete a variety of substrates, which may not be compatible with canonical secretion pathways, the study of mechanisms for alternative secretion pathways is particularly interesting in protist parasites. In this review, we provide an overview on the current status of knowledge on UPS in parasitic protists preceded by a brief overview of UPS in the mammalian cell model with a focus on IL-1β and FGF-2 as paradigmatic UPS substrates.