Rab6-mediated retrograde trafficking from the Golgi: the trouble with tubules

A characteristic feature of every eukaryotic cell is its division into different compartments. This subdivision into different intracellular organelles like the endoplasmic reticulum (ER), the Golgi apparatus or the endosomal/lysosomal system enables cells to provide the appropriate environment for a great variety of biochemical processes. However, it also necessitates an elaborate machinery for the communication between these compartments or organelles. On one hand, material has to be exchanged between organelles, but on the other hand, their integrity with respect to their protein and lipid content, has to be maintained to fulfil their function. Transport processes between different organelles are mediate by intracellular traffic pathways. Proteins enter the secretory pathway at the ER, where they acquire first posttranslational modifications. From the ER, they are delivered to the Golgi, where they are further modified and sorted to their target compartments. In the secretory pathway, transport carriers, so-called vesicles, bud from one organelle (donor) and fuse with the next organelle (acceptor) along their trafficking route. Understanding the molecular mechanisms and regulations underlying vesicular transport is crucial and therefore has been a main topic of research over the last decades. The machinery required for budding and fusion of vesicles along their trafficking pathways is conserved from yeast to human. Therefore, the yeast Saccharomyces cerevisiae represents a suitable organism to study the secretory pathway. In this thesis, we used S. cerevisiae to examine the regulation of vesicular traffic at the ER-Golgi interface, more specifically the fusion of vesicles with ER membranes. The consumption of a vesicle at its target membrane is mediated by the orchestrated action of various members of conserved protein families that act in a regulated manner. Main players involved in vesicular fusion are Rab GTPases, tethering factors and SNAREs. The tethering factors and the Rab GTPases mediate the first contact of an incoming vesicle with its acceptor organelle, whereas the SNARE proteins are responsible for the final fusion event between vesicles and target membranes. Here, we identified the Rab GTPase Ypt1p as mediator of vesicle fusion with the ER. Moreover, Ypt1p was not only required for vesicle fusion at the ER, but also for the maintenance of the morphology and protein composition of the Golgi, and for vesicle formation at the Golgi. In addition, the tethering complex responsible for the docking of Golgi-derived vesicles with the ER, the Dsl1 tethering complex was analyzed. We found that this complex, apart from mediating the first contact of the incoming vesicles with the ER membrane, seems to play an additional role in proofreading and stabilization of SNARE complexes that are responsible for vesicle fusion at the ER

The eukaryotic cell is divided into a number of structurally and functionally distinct membrane-bounded compartments. Each compartment contains its distinct set of molecules, and an elaborate molecular machinery ensures that proteins, lipids, and sugars can be transported between compartments while maintaining organelle identity. Figure 1 shows schematically the most important stations of the intracellular membrane trafficking pathways in mammalian cells. The first station of the exocytic pathway is the endoplasmic reticulum (ER), the membrane of which is continuous with the nuclear envelope. Membrane traffic from the ER to the Golgi apparatus occurs via a vesiculotubular intermediate compartment (IC) (1). From the Golgi apparatus, molecules can be further transported to the trans-Golgi network (TGN). Here, cargo molecules are sorted to distinct domains of the plasma membrane as well as to the endocytic pathway (2). The endocytic pathway begins with the formation of vesicles from the plasma membrane. The endocytic vesicles fuse with early endosomes (EE). Like the TGN, the EE is an important sorting station, which shunts molecules to alternative destinations, including the plasma membrane and later endocytic compartments (3). Late endosomes (LEs) frequently contain internal vesicles and are sometimes referred to as multivesicular bodies (MVBs) (4, 5). These organelles appear to have a two-way communication with the TGN: they receive lysosomal enzymes, complexed to mannose 6-phosphate receptors, via TGN vesicles, and they recycle ligand-free mannose 6-phosphate receptors back to the TGN (6). LEs may fuse with primary (or dense-core) lysosomes, to form secondary lysosomes. Here, the degradation of luminal material occurs very efficiently. There is evidence that primary lysosomes may be reformed from secondary lysosomes through a selective membrane recovery (7).

Kinesin-1 drives the movement of diverse cargoes, and it has been proposed that specific kinesin light chain (KLC) isoforms target kinesin-1 to these different structures. Here, we test this hypothesis using two in vitro motility assays, which reconstitute the movement of rough endoplasmic reticulum (RER) and vesicles present in a Golgi membrane fraction. We generated GST-tagged fusion proteins of KLC1B and KLC1D that included the tetratricopeptide repeat domain and the variable C-terminus. We find that preincubation of RER with KLC1B inhibits RER motility, whereas KLC1D does not. In contrast, Golgi fraction vesicle movement is inhibited by KLC1D but not KLC1B reagents. Both RER and vesicle movement is inhibited by preincubation with the GST-tagged C-terminal domain of ubiquitous kinesin heavy chain (uKHC), which binds to the N-terminal domain of uKHC and alters its interaction with microtubules. We propose that although the TRR domains are required for cargo binding, it is the variable C-terminal region of KLCs that are vital for targeting kinesin-1 to different cellular structures.

In mammalian cells, membrane traffic pathways play a critical role in connecting the various compartments of the endomembrane system. Each of these pathways is highly regulated, requiring specific machinery to ensure their fidelity. In the early secretory pathway, transport between the endoplasmic reticulum (ER) and Golgi apparatus is largely regulated via cytoplasmic coat protein complexes that play a role in identifying cargo and forming the transport carriers. The secretory pathway is counterbalanced by the retrograde pathway, which is essential for the recycling of molecules from the Golgi back to the ER. It is believed that there are at least two mechanisms to achieve this - one using the cytoplasmic COPI coat complex, and another, poorly characterised pathway, regulated by the small GTPase Rab6. In this work, we describe a systematic RNA interference screen targeting proteins associated with membrane fusion, in order to identify the machinery responsible for the fusion of Golgi-derived Rab6 carriers at the ER. We not only assess the delivery of Rab6 to the ER, but also one of its cargo molecules, the Shiga-like toxin B-chain. These screens reveal that three proteins, VAMP4, STX5, and SCFD1/SLY1, are all important for the fusion of Rab6 carriers at the ER. Live cell imaging experiments also show that the depletion of SCFD1/SLY1 prevents the membrane fusion event, suggesting that this molecule is an essential regulator of this pathway.

COPII proteins are essential for exporting most cargo molecules from the endoplasmic reticulum. The membrane-facing surface of the COPII proteins (especially SEC23-SEC24) interacts directly or indirectly with the cargo molecules destined for exit. As we characterized the SEC23A mutations at the SEC31 binding site identified from patients with cranio-lenticulo-sutural dysplasia, we discovered that the SEC23-SEC31 interface can also influence cargo selection. Remarkably, M702V SEC23A does not compromise COPII assembly, vesicle size, and packaging of cargo molecules into COPII vesicles that we have tested but induces accumulation of procollagen in the endoplasmic reticulum when expressed in normal fibroblasts. We observed that M702V SEC23A activates SAR1B GTPase more than wild-type SEC23A when SEC13-SEC31 is present, indicating that M702V SEC23A causes premature dissociation of COPII from the membrane. Our results indicate that a longer stay of COPII proteins on the membrane is required to cargo procollagen than other molecules and suggest that the SEC23-SEC31 interface plays a critical role in capturing various cargo molecules.

Glycosylphosphatidylinositols (GPI) are essential components in the plasma membrane of the protozoan parasite Leishmania mexicana, both as membrane anchors for the major surface macromolecules and as the sole class of free glycolipids. We provide evidence that L.mexicana dolichol-phosphate-mannose synthase (DPMS), a key enzyme in GPI biosynthesis, is localized to a distinct tubular subdomain of the endoplasmic reticulum (ER), based on the localization of a green fluorescent protein (GFP)-DPMS chimera and subcellular fractionation experiments. This tubular membrane (termed the DPMS tubule) is also enriched in other enzymes involved in GPI biosynthesis, can be specifically stained with the fluorescent lipid, BODIPY-C5-ceramide, and appears to be connected to specific subpellicular microtubules that underlie the plasma membrane. Perturbation of microtubules and DPMS tubule structure in vivo results in the selective accumulation of GPI anchor precursors, but not free GPIs. The DPMS tubule is closely associated morphologically with the single Golgi apparatus in non-dividing and dividing cells, appears to exclude luminal ER resident proteins and is labeled, together with the Golgi apparatus, with another GFP chimera containing the heterologous human Golgi marker beta1,2-N-acetylglucosaminyltransferase-I. The possibility that the DPMS-tubule is a stable transitional ER is discussed.

Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer disease (AD) and likely contributes to neuropathology through various pathways. Here we report that the intracellular trafficking of apoE4 is impaired in Neuro-2a cells and primary neurons, as shown by measuring fluorescence recovery after photobleaching. In Neuro-2a cells, more apoE4 than apoE3 molecules remained immobilized in the endoplasmic reticulum (ER) and the Golgi apparatus, and the lateral motility of apoE4 was significantly lower in the Golgi apparatus (but not in the ER) than that of apoE3. Likewise, the immobile fraction was larger, and the lateral motility was lower for apoE4 than apoE3 in mouse primary hippocampal neurons. ApoE4 with the R61T mutation, which abolishes apoE4 domain interaction, was less immobilized, and its lateral motility was comparable with that of apoE3. The trafficking impairment of apoE4 was also rescued by disrupting domain interaction with the small-molecule structure correctors GIND25 and PH002. PH002 also rescued apoE4-induced impairments of neurite outgrowth in Neuro-2a cells and dendritic spine development in primary neurons. ApoE4 did not affect trafficking of amyloid precursor protein, another AD-related protein, through the secretory pathway. Thus, domain interaction renders more newly synthesized apoE4 molecules immobile and slows their trafficking along the secretory pathway. Correcting the pathological structure of apoE4 by disrupting domain interaction is a potential therapeutic approach to treat or prevent AD related to apoE4.

A key determinant for intracellular pathogenic bacteria to ensure their virulence within host cells is their ability to bypass the endocytic pathway and to reach a safe niche of replication. In the case of Brucella, the bacterium targets the ER (endoplasmic reticulum) to create a replicating niche called the BCV (Brucella-containing vacuole). The ER is a suitable strategic place for pathogenic Brucella. Indeed, bacteria can be hidden from host cell defences to persist within the host, and they can take advantage of the membrane reservoir delivered by the ER to replicate. Interaction with the ER leads to the presence on the BCV of the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and the small GTPase Rab2 known to be located on secretory vesicles that traffic between the ER and the Golgi apparatus. GAPDH and the small GTPase Rab2 controls Brucella replication at late times post-infection. A specific interaction between the human small GTPase Rab2 and a Brucella spp. protein named RicA was identified. Altered kinetics of intracellular trafficking and faster proliferation of the Brucella abortus ΔricA mutant was observed compared with the wild-type strain. RicA is the first reported effector with a proposed function for B. abortus.

We have identified and characterized a cDNA encoding a novel Ca2+-binding protein named calumenin from mouse heart by the signal sequence trap method. The deduced amino acid sequence (315 residues) of calumenin contains an amino-terminal signal sequence and six Ca2+-binding (EF-hand) motifs and shows homology with reticulocalbin, Erc-55, and Cab45. These proteins seem to form a new subset of the EF-hand protein family expressed in the lumen of the endoplasmic reticulum (ER) and Golgi apparatus. Purified calumenin had Ca2+-binding ability. The carboxyl-terminal tetrapeptide His-Asp-Glu-Phe was shown to be responsible for retention of calumenin in ER by the retention assay, immunostaining with a confocal laser microscope, and the deglycosylation assay. This is the first report indicating that the Phe residue is included in the ER retention signal. Calumenin is expressed most strongly in heart of adult and 18.5-day embryos. The calumenin gene (Calu) was mapped at the proximal portion of mouse chromosome 7.

A chemical called Ilimaquinone (IQ) was identified based on its ability to convert the mammalian Golgi apparatus into small uniform size vesicles. We reasoned IQ hyper activates a membrane fission reaction and thus suitable for understanding this process. This reaction required a serine/threonine kinase called PKD. Surprisingly, PKD was found to control the biogenesis of transport carriers at the Trans Golgi Network (TGN) that are destined to cell surface. Other exit routes from the Golgi membranes were PKD independent. PKD binds diacylglycerol (DAG) and Arf1, and promotes the production of phosphatidylinositol‐4‐phosphate (PI4P). PI4P in turn recruits ceramide transfer protein (CERT) and the oxysterol binding protein (OSBP). The binding of Arfaptin1 to the TGN also requires PI4P. Arfaptin 1 contains a domain that has the ability to assemble into an amphipathic helix; PKD phosphorylates within this region and dissociates Arfaptin 1 from the TGN. PKD thus regulates membrane curvature by controlling the dynamics of the BAR domain containing Arfpatin 1 at the TGN. PKD also regulates the association of CERT and OSBP by phosphorylation. Altogether this suggests that PKD regulates the levels of PI4P, ceramide and sterols at the TGN, and important for transport carrier biogenesis. We have tested this hypothesis directly by affecting the levels of the ceramide product Sphingomyelin at the TGN. Perturbing the balance of Sphingomyelin inhibited transport carrier formation at the Golgi membranes without affecting the fusion of incoming carriers. These findings highlight the role of lipid homeostasis in transport carrier formation. Altogether, the discovery of IQ as a Golgi vesiculating compound has been valuable for revealing the mechanism of transport carrier biogenesis during protein secretion.

Sphingoid long-chain base 1-phosphates act as bioactive lipid molecules in eukaryotic cells. In budding yeast, long-chain base 1-phosphates are synthesized mainly by the long-chain base kinase Lcb4. We recently reported that, soon after yeast cells enter into the stationary phase, Lcb4 is rapidly degraded by being delivered to the vacuole in a palmitoylation- and phosphorylation-dependent manner. In this study, we investigated the complete trafficking pathway of Lcb4, from its synthesis to its degradation. After membrane anchoring by palmitoylation at the Golgi apparatus, Lcb4 is delivered to the plasma membrane (PM) through the late Sec pathway and then to the endoplasmic reticulum (ER). The yeast ER consists of a cortical network juxtaposed to the PM (cortical ER) with tubular connections to the nuclear envelope (nuclear ER). Remarkably, the localization of Lcb4 is restricted to the cortical ER. As the cells reach the stationary phase, G(1) cell cycle arrest initiates Lcb4 degradation and its delivery to the vacuole via the Golgi apparatus. The protein transport pathway from the PM to the ER found in this study has not been previously reported. We speculate that this novel pathway is mediated by the PM-ER contact.

ADP-ribosylation factors (Arf), a family of small GTP-binding proteins, play important roles in intracellular trafficking in animal and yeast cells. Here, we investigated the roles of two Arf homologs, Arf1 and Arf3 of Arabidopsis, in intracellular trafficking in plant cells. We generated dominant negative mutant forms of Arf 1 and Arf3 and examined their effect on trafficking of reporter proteins in protoplasts. Arf1[T31N] inhibited trafficking of H(+)-ATPase:green fluorescent protein (GFP) and sialyltransferase (ST):GFP to the plasma membrane and the Golgi apparatus. In addition, Arf1[T31N] caused relocalization of the Golgi reporter protein ST:GFP to the endoplasmic reticulum (ER). In protoplasts expressing Arf1[T31N], ST:red fluorescent protein remained in the ER, whereas H(+)-ATPase:GFP was mistargeted to another organelle. Also, expression of Arf1[T31N] in protoplasts resulted in profound changes in the morphology of the ER. The treatment of protoplasts with brefeldin A had exactly the same effect as Arf1[T31N] on various intracellular trafficking pathways. In contrast, Arf3[T31N] did not affect trafficking of any of these reporter proteins. Inhibition experiments using mutants with various domains swapped between Arf1 and Arf3 revealed that the N-terminal domain is interchangeable for trafficking inhibition. However, in addition to the T31N mutation, motifs in domains II, III, and IV of Arf1 were necessary for inhibition of trafficking of H(+)-ATPase:GFP. Together, these results strongly suggest that Arf1 plays a role in the intracellular trafficking of cargo proteins in Arabidopsis, and that Arf1 functions through a brefeldin A-sensitive factor.

BBF2H7-Mediated Sec23A Pathway Is Required for Endoplasmic Reticulum-to-Golgi Trafficking in Dermal Fibroblasts to Promote Collagen Synthesis

Protein kinase D (PKD) is a cytosolic protein, which upon binding to the trans-Golgi network (TGN) regulates the fission of transport carriers specifically destined to the cell surface. We have found that the first cysteine-rich domain (C1a), but not the second cysteine-rich domain (C1b), is sufficient for the binding of PKD to the TGN. Proline 155 in C1a is necessary for the recruitment of intact PKD to the TGN. Whereas C1a is sufficient to target a reporter protein to the TGN, mutation of serines 744/748 to alanines in the activation loop of intact PKD inhibits its localization to the TGN. Moreover, anti-phospho-PKD antibody, which recognizes only the activated form of PKD, recognizes the TGN-bound PKD. Thus, activation of intact PKD is important for binding to the TGN.

Most secretory proteins travel through a well-documented conventional secretion pathway involving the endoplasmic reticulum (ER) and the Golgi complex. However, recently, it has been shown that a significant number of proteins reach the plasma membrane or extracellular space via unconventional routes. Unconventional protein secretion (UPS) can be divided into two types: (i) the extracellular secretion of cytosolic proteins that do not bear a signal peptide (i.e. leaderless proteins) and (ii) the cell-surface trafficking of signal-peptide-containing transmembrane proteins via a route that bypasses the Golgi. Understanding the UPS pathways is not only important for elucidating the mechanisms of intracellular trafficking pathways but also has important ramifications for human health, because many of the proteins that are unconventionally secreted by mammalian cells and microorganisms are associated with human diseases, ranging from common inflammatory diseases to the lethal genetic disease of cystic fibrosis. Therefore, it is timely and appropriate to summarize and analyze the mechanisms of UPS involvement in disease pathogenesis, as they may be of use for the development of new therapeutic approaches. In this Review, we discuss the intracellular trafficking pathways of UPS cargos, particularly those related to human diseases. We also outline the disease mechanisms and the therapeutic potentials of new strategies for treating UPS-associated diseases.