Leishmania parasitophorous vacuoles interact continuously with the host cell's endoplasmic reticulum; parasitophorous vacuoles are hybrid compartments

Macrophages that express representative endoplasmic reticulum (ER) molecules tagged with green fluorescence protein were generated to assess the recruitment of ER molecules to Leishmania parasitophorous vacuoles (PVs). More than 90% of PVs harbouring Leishmania pifanoi or Leishmania donovani parasites recruited calnexin, to their PV membrane. An equivalent proportion of PVs also recruited the membrane‐associated soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs), Sec22b. Both ER molecules appeared to be recruited very early in the formation of nascent PVs. Electron microscopy analysis of infected Sec22b/YFP expressing cells confirmed that Sec22b was recruited to Leishmania PVs. In contrast to PVs, it was found that no more than 20% of phagosomes that harboured Zymosan particles recruited calnexin or Sec22b to their limiting phagosomal membrane. The retrograde pathway that ricin employs to access the cell cytosol was exploited to gain further insight into ER–PV interactions. Ricin was delivered to PVs in infected cells incubated with ricin. Incubation of cells with brefeldin A blocked the transfer of ricin to PVs. This implied that molecules that traffic to the ER are transferred to PVs. Moreover the results show that PVs are hybrid compartments that are composed of both host ER and endocytic pathway components.     

The Q-soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (Q-SNARE) SNAP-47 Regulates Trafficking of Selected Vesicle-associated Membrane Proteins (VAMPs)

SNAREs constitute the core machinery of intracellular membrane fusion, but vesicular SNAREs localize to specific compartments via largely unknown mechanisms. Here, we identified an interaction between VAMP7 and SNAP-47 using a proteomics approach. We found that SNAP-47 mainly localized to cytoplasm, the endoplasmic reticulum (ER), and ERGIC and could also shuttle between the cytoplasm and the nucleus. SNAP-47 preferentially interacted with the trans-Golgi network VAMP4 and post-Golgi VAMP7 and -8. SNAP-47 also interacted with ER and Golgi syntaxin 5 and with syntaxin 1 in the absence of Munc18a, when syntaxin 1 is retained in the ER. A C-terminally truncated SNAP-47 was impaired in interaction with VAMPs and affected their subcellular distribution. SNAP-47 silencing further shifted the subcellular localization of VAMP4 from the Golgi apparatus to the ER. WT and mutant SNAP-47 overexpression impaired VAMP7 exocytic activity. We conclude that SNAP-47 plays a role in the proper localization and function of a subset of VAMPs likely via regulation of their transport through the early secretory pathway.     

Sec22 and Memb11 are v-SNAREs of the anterograde endoplasmic reticulum-Golgi pathway in tobacco leaf epidermal cells

Distinct sets of soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) are distributed to specific intracellular compartments and catalyze membrane fusion events. Although the central role of these proteins in membrane fusion is established in nonplant systems, little is known about their role in the early secretory pathway of plant cells. Analysis of the Arabidopsis (Arabidopsis thaliana) genome reveals 54 genes encoding SNARE proteins, some of which are expected to be key regulators of membrane trafficking between the endoplasmic reticulum (ER) and the Golgi. To gain insights on the role of SNAREs of the early secretory pathway in plant cells, we have cloned the Arabidopsis v-SNAREs Sec22, Memb11, Bet11, and the t-SNARE Sed5, and analyzed their distribution in plant cells in vivo. By means of live cell imaging, we have determined that these SNAREs localize at the Golgi apparatus. In addition, Sec22 was also distributed at the ER. We have then focused on understanding the function of Sec22 and Memb11 in comparison to the other SNAREs. Overexpression of the v-SNAREs Sec22 and Memb11 but not of the other SNAREs induced collapse of Golgi membrane proteins into the ER, and the secretion of a soluble secretory marker was abrogated by all SNAREs. Our studies suggest that Sec22 and Memb11 are involved in anterograde protein trafficking at the ER-Golgi interface.     

Legionella pneumophila Promotes Functional Interactions between Plasma Membrane Syntaxins and Sec22b

Biogenesis of a specialized organelle that supports intracellular replication of Legionella pneumophila involves the fusion of secretory vesicles exiting the endoplasmic reticulum (ER) with phagosomes containing this bacterial pathogen. Here, we investigated host plasma membrane SNARE proteins to determine whether they play a role in trafficking of vacuoles containing L. pneumophila. Depletion of plasma membrane syntaxins by RNA interference resulted in delayed acquisition of the resident ER protein calnexin and enhanced retention of Rab1 on phagosomes containing virulent L. pneumophila, suggesting that these SNARE proteins are involved in vacuole biogenesis. Plasma membrane‐localized SNARE proteins syntaxin 2, syntaxin 3, syntaxin 4 and SNAP23 localized to vacuoles containing L. pneumophila. The ER‐localized SNARE protein Sec22b was found to interact with plasma membrane SNAREs on vacuoles containing virulent L. pneumophila, but not on vacuoles containing avirulent mutants of L. pneumophila. The addition of α‐SNAP and N‐ethylmaleimide‐sensitive factor (NSF) to the plasma membrane SNARE complexes formed by virulent L. pneumophila resulted in the dissociation of Sec22b, indicating functional pairing between these SNAREs. Thus, L. pneumophila stimulates the non‐canonical pairing of plasma membrane t‐SNAREs with the v‐SNARE Sec22b to promote fusion of the phagosome with ER‐derived vesicles. The mechanism by which L. pneumophila promotes pairing of plasma membrane syntaxins and Sec22b could provide unique insight into how the secretory vesicles could provide an additional membrane reserve subverted during phagosome maturation.     

SNARE membrane trafficking dynamics in vivo

The ER/Golgi soluble NSF attachment protein receptor (SNARE) membrin, rsec22b, and rbet1 are enriched in approximately 1-micrometer cytoplasmic structures that lie very close to the ER. These appear to be ER exit sites since secretory cargo concentrates in and exits from these structures. rsec22b and rbet1 fused to fluorescent proteins are enriched at approximately 1-micrometer ER exit sites that remained more or less stationary, but periodically emitted streaks of fluorescence that traveled generally in the direction of the Golgi complex. These exit sites were reused and subsequent tubules or streams of vesicles followed similar trajectories. Fluorescent membrin- enriched approximately 1-micrometer peripheral structures were more mobile and appeared to translocate through the cytoplasm back and forth, between the periphery and the Golgi area. These mobile structures could serve to collect secretory cargo by fusing with ER-derived vesicles and ferrying the cargo to the Golgi. The post-Golgi SNAREs, syntaxin 6 and syntaxin 13, when fused to fluorescent proteins each displayed characteristic patterns of movement. However, syntaxin 13 was the only SNARE whose life cycle appeared to involve interactions with the plasma membrane. These studies reveal the in vivo spatiotemporal dynamics of SNARE proteins and provide new insight into their roles in membrane trafficking.     

Toxoplasma gondii Vps11, a subunit of HOPS and CORVET tethering complexes,is essential for the biogenesis of secretory organelles

Apicomplexan parasites harbour unique secretory organelles (dense granules, rhoptries and micronemes) that play essential functions in host infection. Toxoplasma gondii parasites seem to possess an atypical endosome‐like compartment, which contains an assortment of proteins that appear to be involved in vesicular sorting and trafficking towards secretory organelles. Recent studies highlighted the essential roles of many regulators such as Rab5A, Rab5C, sortilin‐like receptor and syntaxin‐6 in secretory organelle biogenesis. However, little is known about the protein complexes that recruit Rab‐GTPases and SNAREs for membrane tethering in Apicomplexa. In mammals and yeast, transport, tethering and fusion of vesicles from early endosomes to lysosomes and the vacuole, respectively, are mediated by CORVET and HOPS complexes, both built on the same Vps‐C core that includes Vps11 protein. Here, we show that a T. gondii Vps11 orthologue is essential for the biogenesis or proper subcellular localization of secretory organelle proteins. TgVps11 is a dynamic protein that associates with Golgi endosomal‐related compartments, the vacuole and immature apical secretory organelles. Conditional knock‐down of TgVps11 disrupts biogenesis of dense granules, rhoptries and micronemes. As a consequence, parasite motility, invasion, egress and intracellular growth are affected. This phenotype was confirmed with additional knock‐down mutants of the HOPS complex. In conclusion, we show that apicomplexan parasites use canonical regulators of the endolysosome system to accomplish essential parasite‐specific functions in the biogenesis of their unique secretory organelles.     

A Targeted siRNA Screen to Identify SNAREs Required for Constitutive Secretion in Mammalian Cells

The role of SNAREs in mammalian constitutive secretion remains poorly defined. To address this, we have developed a novel flow cytometry‐based assay for measuring constitutive secretion and have performed a targeted SNARE and Sec1/Munc18 (SM) protein‐specific siRNA screen (38 SNAREs, 4 SNARE‐like proteins and 7 SM proteins). We have identified the endoplasmic reticulum (ER)/Golgi SNAREs syntaxin 5, syntaxin 17, syntaxin 18, GS27, SLT1, Sec20, Sec22b, Ykt6 and the SM protein Sly1, along with the post‐Golgi SNAREs SNAP‐29 and syntaxin 19, as being required for constitutive secretion. Depletion of SNAP‐29 or syntaxin 19 causes a decrease in the number of fusion events at the cell surface and in SNAP‐29‐depleted cells causes an increase in the number of docked vesicles at the plasma membrane as determined by total internal reflection fluorescence (TIRF) microscopy. Analysis of syntaxin 19‐interacting partners by mass spectrometry indicates that syntaxin 19 can form SNARE complexes with SNAP‐23, SNAP‐25, SNAP‐29, VAMP3 and VAMP8, supporting its role in Golgi to plasma membrane transport or fusion. Surprisingly, we have failed to detect any requirement for a post‐Golgi‐specific R‐SNARE in this process.     

Ultrastructural studies of a vesicle system associated with endoplasmic reticulum in exo-erythrocytic forms of Plasmodium berghei

Fine structural studies of a specialized vesicle system associated with the endoplasmic reticulum (ER) of exo-erythrocytic Plasmodium berghei suggest that this system may be the equivalent of a Golgi apparatus. Patches of ER, randomly distributed in the cytoplasm of developing parasites, are formed of smooth and ribosome-studded cisternae intermingled with each other. The vesicle systems are located between as well as at the edges of ER aggregates and appear to be in different stages of budding from the cisternae. Prolonged osmication reveals distinct staining of the nuclear envelope and ER of the parasites as well as part of the Golgi apparatus of the hepatocytes. However, the small vesicles associated with the parasite's ER are unstained, as are the coated vesicles in the Golgi region of the liver cell. These sites in the parasite cytoplasm seem comparable to the concave surface of the Golgi apparatus in liver cells. The pinched-off vesicles fuse with others to form the prominent peripheral vacuolization characteristic of the nearly mature exoerythrocytic form. The formation of these peripheral vacuoles and their subsequent fusion with the parasite membrane may be an exocytosis mechanism supplying the rapidly expanding parasite with new plasma membrane material.     

Members of a mammalian SNARE complex interact in the endoplasmic reticulum in vivo and are found in COPI vesicles

Retrograde traffic between the Golgi apparatus and the endoplasmic reticulum (ER) is largely mediated by COPI-coated transport vesicles. In mammalian cells, retrograde traffic can pass through an intermediate compartment. Here, we report that the mammalian soluble N-ethylmaleimide-sensitive factor (NSF) attachment receptor (SNARE) proteins mSec22b, mUse1/D12, mSec20/BNIP1, and syntaxin 18 form a quaternary SNARE complex. Fluorescence resonance energy transfer (FRET) experiments prove that these interactions occur in the ER of living cells. In addition, mUse1/D12 and mSec20/BNIP1 form homo-oligomers in vivo. Furthermore, we show that mSec22b, mUse1/D12, mSec20/BNIP1, and syntaxin 18 are recruited into COPI-coated vesicles formed in vitro. Immunogold electron microscopy confirmed that these SNARE proteins colocalize with the KDEL receptor ERD2 in COPI-coated vesicles. Moreover, both FRET and immunoprecipitation experiments reveal interactions of these SNAREs with both ERD2 and COPI subunits. We conclude that the SNAREs described here are sorted via interaction with components of the COPI-dependent budding complex into Golgi-to-ER retrograde COPI vesicles and function in retrograde transport from the Golgi to the ER Golgi intermediate compartment (ERGIC) or the ER.     

Endosomal Fusion upon SNARE Knockdown is Maintained by Residual SNARE Activity and Enhanced Docking

SNARE proteins mediate membrane fusion in the secretory pathway of eukaryotic cells. Genetic deletion and siRNA-based knockdown have been instrumental in assigning given SNAREs to defined intracellular transport steps. However, SNARE depletion occasionally results in barely detectable phenotypes. To understand how cells cope with SNARE loss, we have knocked down several SNAREs functioning in early endosome fusion. Surprisingly, knockdown of syntaxin 13, syntaxin 6 and vti1a, alone or in combinations, did not result in measurable changes of endosomal trafficking or fusion. We found that the residual SNARE levels (typically ∼10%) were sufficient for a substantial amount of SNARE–SNARE interactions. Conversely, in wild-type cells, most SNARE molecules were concentrated in clusters, constituting a spare pool not readily available for interactions. Additionally, the knockdown organelles exhibited enhanced docking. We conclude that SNAREs are expressed at much higher levels than needed for maintenance of organelle fusion, and that loss of SNAREs is compensated for by the co-regulation of the docking machinery.     

Toxoplasma gondii salvages sphingolipids from the host Golgi through the rerouting of selected Rab vesicles to the parasitophorous vacuole

The obligate intracellular protozoan Toxoplasma gondii actively invades mammalian cells and, upon entry, forms its own membrane-bound compartment, named the parasitophorous vacuole (PV). Within the PV, the parasite replicates and scavenges nutrients, including lipids, from host organelles. Although T. gondii can synthesize sphingolipids de novo, it also scavenges these lipids from the host Golgi. How the parasite obtains sphingolipids from the Golgi remains unclear, as the PV avoids fusion with host organelles. In this study, we explore the host Golgi–PV interaction and evaluate the importance of host-derived sphingolipids for parasite growth. We demonstrate that the PV preferentially localizes near the host Golgi early during infection and remains closely associated with this organelle throughout infection. The parasite subverts the structure of the host Golgi, resulting in its fragmentation into numerous ministacks, which surround the PV, and hijacks host Golgi–derived vesicles within the PV. These vesicles, marked with Rab14, Rab30, or Rab43, colocalize with host-derived sphingolipids in the vacuolar space. Scavenged sphingolipids contribute to parasite replication since alterations in host sphingolipid metabolism are detrimental for the parasite''s growth. Thus our results reveal that T. gondii relies on host-derived sphingolipids for its development and scavenges these lipids via Golgi-derived vesicles.     

Coordination of golgin tethering and SNARE assembly: GM130 binds syntaxin 5 in a p115-regulated manner

During membrane traffic, transport carriers are first tethered to the target membrane prior to undergoing fusion. Mechanisms exist to connect tethering with fusion, but in most cases, the details remain poorly understood. GM130 is a member of the golgin family of coiled-coil proteins tat is involved in membrane tethering at the endoplasmic reticulum (ER) to Golgi intermediate compartment and cis-Golgi. Here, we demonstrate that GM130 interacts with syntaxin 5, a t-SNARE also localized to the early secretory pathway. Binding to syntaxin 5 is specific, direct, and mediated by the membrane-proximal region of GM130. Interestingly, interaction with syntaxin 5 is inhibited by the binding of the vesicle docking protein p115 to a distal binding site in GM130. The interaction between GM130 and the small GTPase Rab1 is also inhibited by p115 binding. Our findings suggest a mechanism for coupling membrane tethering and fusion at the ER to Golgi intermediate compartment and cis-Golgi, with GM130 playing a central role in linking these processes. Consistent with this hypothesis, we find that depletion of GM130 by RNA interference slows the rate of ER to Golgi trafficking in vivo. The interactions of GM130 with syntaxin 5 and Rab1 are also regulated by mitotic phosphorylation, which is likely to contribute to the inhibition of ER to Golgi trafficking that occurs when mammalian cells enter mitosis.     

The secretory apparatus of an ancient eukaryote: protein sorting to separate export pathways occurs before formation of transient Golgi-like compartments

Transmission of the protozoan parasite Giardia intestinalis to vertebrate hosts presupposes the encapsulation of trophozoites into an environmentally resistant and infectious cyst form. We have previously shown that cyst wall proteins were faithfully sorted to large encystation-specific vesicles (ESVs), despite the absence of a recognizable Golgi apparatus. Here, we demonstrate that sorting to a second constitutively active pathway transporting variant-specific surface proteins (VSPs) to the surface depended on the cytoplasmic VSP tail. Moreover, pulsed endoplasmic reticulum (ER) export of chimeric reporters containing functional signals for both pathways showed that protein sorting was done at or very soon after export from the ER. Correspondingly, we found that a limited number of novel transitional ER-like structures together with small transport intermediates were generated during encystation. Colocalization of transitional ER regions and early ESVs with coat protein (COP) II and of maturing ESVs with COPI and clathrin strongly suggested that ESVs form by fusion of ER-derived vesicles and subsequently undergo maturation by retrograde transport. Together, the data supported the hypothesis that in Giardia, a primordial secretory apparatus is in operation by which proteins are sorted in the early secretory pathway, and the developmentally induced ESVs carry out at least some Golgi functions.     

The Yip1p.Yif1p complex is required for the fusion competence of endoplasmic reticulum-derived vesicles

Here we report that Yip1p and Yif1p, two members of an integral membrane protein complex that bind to the Rab Ypt1p, are required for membrane fusion with the Golgi in vitro. To block fusion, anti-Yip1p or anti-Yif1p antibodies must be added before vesicles bud from the endoplasmic reticulum (ER). These antibodies do not block the packaging of Yip1p, Yif1p, or the soluble NSF attachment protein receptor (SNAREs) into vesicles. We propose that Yip1p and Yif1p perform a critical role in establishing the fusion competence of ER to Golgi vesicles at the time of budding. Consistent with this proposal, we observe that the Yip1p.Yif1p complex binds to the ER to Golgi SNAREs Bos1p and Sec22p, two components of the membrane fusion machinery.     

All three components of the neuronal SNARE complex contribute to secretory vesicle docking

Before exocytosis, vesicles must first become docked to the plasma membrane. The SNARE complex was originally hypothesized to mediate both the docking and fusion steps in the secretory pathway, but previous electron microscopy (EM) studies indicated that the vesicular SNARE protein synaptobrevin (syb) was dispensable for docking. In this paper, we studied the function of syb in the docking of large dense-core vesicles (LDCVs) in live PC12 cells using total internal reflection fluorescence microscopy. Cleavage of syb by a clostridial neurotoxin resulted in significant defects in vesicle docking in unfixed cells; these results were confirmed via EM using cells that were prepared using high-pressure freezing. The membrane-distal portion of its SNARE motif was critical for docking, whereas deletion of a membrane-proximal segment had little effect on docking but diminished fusion. Because docking was also inhibited by toxin-mediated cleavage of the target membrane SNAREs syntaxin and SNAP-25, syb might attach LDCVs to the plasma membrane through N-terminal assembly of trans-SNARE pairs.     

Ultrastructural Studies of a Vesicle System Associated with Endoplasmic Reticulum in Exo-Erythrocytic Forms of Plasmodium berghei1

ABSTRACT. Fine structural studies of a specialized vesicle system associated with the endoplasmic reticulum (ER) of exo-erythrocytic Plasmodium berghei suggest that this system may be the equivalent of a Golgi apparatus. Patches of ER, randomly distributed in the cytoplasm of developing parasites, are formed of smooth and ribosome-studded cisternae intermingled with each other. The vesicle systems are located between as well as at the edges of ER aggregates and appear to be in different stages of budding from the cisternae. Prolonged osmication reveals distinct staining of the nuclear envelope and ER of the parasites as well as part of the Golgi apparatus of the hepatocytes. However, the small vesicles associated with the parasite's ER are unstained, as are the coated vesicles in the Golgi region of the liver cell. These sites in the parasite cytoplasm seem comparable to the concave surface of the Golgi apparatus in liver cells. The pinched-off vesicles fuse with others to form the prominent peripheral vacuolization characteristic of the nearly mature exo-erythrocytic form. The formation of these peripheral vacuoles and their subsequent fusion with the parasite membrane may be an exocytosis mechanism supplying the rapidly expanding parasite with new plasma membrane material.     

Leishmania (L.) amazonensis: fusion between parasitophorous vacuoles in infected bone-marrow derived mouse macrophages

[Leishmania(L.)] amazonensis amastigotes reside in macrophages within spacious parasitophorous vacuoles (PVs) which may contain numerous parasites. After sporadic fusion events were detected by time-lapse cinemicrography, PV fusion was examined in two different models. In single infections, it was inferred from the reduction in PV numbers per cell. In a reinfection model, macrophages infected with unlabeled amastigotes were reinfected with GFP-transfected- or carboxyfluorescein diacetate succinimidyl ester-labeled parasites, and fusion was detected by the colocalization of labeled and unlabeled amastigotes in the same PVs. The main findings were: (1) as expected, fusion frequency increased with the multiplicity of infection; (2) most fusion events took place in the first 24h of infection or reinfection, prior to the multiplication of incoming parasites; (3) resident and incoming parasites multiplied at similar rates in fused PVs. The model should be useful in studies of parasite and host cell factors and mechanisms involved in PV fusogenicity.     

Retrograde vesicle transport in the Golgi

The Golgi apparatus is the central sorting and biosynthesis hub of the secretory pathway, and uses vesicle transport for the recycling of its resident enzymes. This system must operate with high fidelity and efficiency for the correct modification of secretory glycoconjugates. In this review, we discuss recent advances on how coats, tethers, Rabs and SNAREs cooperate at the Golgi to achieve vesicle transport. We cover the well understood vesicle formation process orchestrated by the COPI coat, and the comprehensively documented fusion process governed by a set of Golgi localised SNAREs. Much less clear are the steps in-between formation and fusion of vesicles, and we therefore provide a much needed update of the latest findings about vesicle tethering. The interplay between Rab GTPases, golgin family coiled-coil tethers and the conserved oligomeric Golgi (COG) complex at the Golgi are thoroughly evaluated.     

SNAREs are concentrated in cholesterol-dependent clusters that define docking and fusion sites for exocytosis

During exocytosis, SNARE proteins of secretory vesicles interact with the corresponding SNARE proteins in the plasmalemma to initiate the fusion reaction. However, it is unknown whether SNAREs are uniformly distributed in the membrane or whether specialized fusion sites exist. Here we report that in the plasmalemma, syntaxins are concentrated in 200 nm large, cholesterol-dependent clusters at which secretory vesicles preferentially dock and fuse. The syntaxin clusters are distinct from cholesterol-dependent membrane rafts since they are Triton X-100-soluble and do not co-patch with raft markers. Synaptosomal-associated protein (SNAP)-25 is also clustered in spots, which partially overlap with syntaxin. Cholesterol depletion causes dispersion of these clusters, which is associated with a strong reduction in the rate of secretion, whereas the characteristics of individual exocytic events are unchanged. This suggests that high local concentrations of SNAREs are required for efficient fusion.     

Cargo selection into COPII vesicles is driven by the Sec24p subunit

Transport of secretory proteins out of the endoplasmic reticulum (ER) is mediated by vesicles generated by the COPII coat complex. In order to understand how cargo molecules are selected by this cytoplasmic coat, we investigated the functional role of the Sec24p homolog, Lst1p. We show that Lst1p can function as a COPII subunit independently of Sec24p on native ER membranes and on synthetic liposomes. However, vesicles generated with Lst1p in the absence of Sec24p are deficient in a distinct subset of cargo molecules, including the SNAREs, Bet1p, Bos1p and Sec22p. Consistent with the absence of any SNAREs, these vesicles are unable to fuse with Golgi membranes. Furthermore, unlike Sec24p, Lst1p fails to bind to Bet1p in vitro, indicating a direct correlation between cargo binding and recruitment into vesicles. Our data suggest that the principle role of Sec24p is to discriminate cargo molecules for incorporation into COPII vesicles.