Giant membrane vesicles as a model to study cellular substrate uptake dissected from metabolism

In order to use giant vesicles for substrate uptake studies in metabolically important tissues, we characterized giant vesicles isolated from heart, liver, skeletal muscle and adipose tissue. We investigated which cell types and which plasma membrane regions are involved in giant vesicle formation and we examined the presence of transporters for metabolic substrates. Analysis of giant vesicles with markers specific for distinct cell types and distinct domains of the plasma membrane reveals that the plasma membrane of parenchymal cells, but not endothelial cells, are the source of the vesicle membranes. In addition, plasma membrane regions enriched in caveolae and involved in docking of recycling vesicles from the endosomal compartment are retained in giant vesicles, indicating that KCl-induced alterations in recycling processes are involved in giant vesicle formation. Giant vesicles contain vesicular lumen consisting of the soluble constituents of the cytoplasm including, fatty-acid binding proteins. Furthermore, giant vesicles isolated from heart, liver, skeletal muscle and adipose tissue are similar in size (10–15 m) and shape and do not contain subcellular organelles, providing the advantage that substrate fluxes in the different organs can be studied independently of the surface/volume ratio but most importantly in the absence of intracellular metabolism.     

Intracellular trafficking and membrane targeting mechanisms of the human reduced folate carrier in Mammalian epithelial cells

The major pathway for cellular uptake of the water-soluble vitamin folic acid in mammalian cells is via a plasma membrane protein known as the reduced folate carrier (RFC). The molecular determinants that dictate plasma membrane expression of RFC as well as the cellular mechanisms that deliver RFC to the cell surface remain poorly defined. Therefore, we designed a series of fusion proteins of the human RFC (hRFC) with green fluorescent protein to image the targeting and trafficking dynamics of hRFC in living epithelial cells. We show that, in contrast to many other nutrient transporters, the molecular determinants that dictate hRFC plasma membrane expression reside within the hydrophobic backbone of the polypeptide and not within the cytoplasmic NH(2)- or COOH-terminal domains of the protein. Further, the integrity of the hRFC backbone is critical for export of the polypeptide from the endoplasmic reticulum to the cell surface. This trafficking is critically dependent on intact microtubules because microtubule disruption inhibits motility of hRFC-containing vesicles as well as final expression of hRFC in the plasma membrane. For the first time, these data define the mechanisms that control the intracellular trafficking and cell surface localization of hRFC within mammalian epithelia.     

PKCzeta is required for microtubule-based motility of vesicles containing the ntcp transporter

Intracellular trafficking regulates the abundance and therefore activity of transporters present at the plasma membrane. The transporter, Na+-taurocholate co-transporting polypeptide (ntcp), is increased at the plasma membrane upon treatment of cells with cAMP, for which microtubules (MTs) are required and the PI3K pathway and PKCzeta have been implicated. However, trafficking of ntcp on MTs has not been demonstrated directly and the regulation and intracellular localization of ntcp is not well understood. Here, we utilize in vitro and whole-cell immunofluorescence microscopy assays to demonstrate that ntcp is present on intracellular vesicles that bind MTs and move bidirectionally, using kinesin-1 and dynein. These vesicles co-localize with markers for recycling endosomes and early but not late endosomes. They frequently undergo fission, providing a mechanism for the exclusion of ntcp from late endosomes. PI(3,4,5)P3 activates PKCzeta and enhances motility of the ntcp vesicles and overcomes the partial inhibition produced by a PI3-kinase inhibitor. Specific inhibition of PKCzeta blocks the motility of ntcp-containing vesicles but has no effect on late vesicles as shown both in vitro and in living cells transfected with ntcp-GFP. These data indicate that PKCzeta is required specifically for the intracellular movement of vesicles that contain the ntcp transporter.     

Kinetic imaging of NPC1L1 and sterol trafficking between plasma membrane and recycling endosomes in hepatoma cells

Niemann-Pick C1-like 1 (NPC1L1) is a recently identified protein that mediates intestinal cholesterol absorption and regulates biliary cholesterol excretion. The itineraries and kinetics of NPC1L1 trafficking remain uncertain. In this study, we have visualized movement of NPC1L1-enhanced green fluorescent protein (NPC1L1-EGFP) and cholesterol analogs in hepatoma cells. At steady state, about 42% of NPC1L1 resided in the transferrin (Tf)-positive, sterol-enriched endocytic recycling compartment (ERC), whereas time-lapse microscopy demonstrated NPC1L1 traffic between the plasma membrane and the ERC. Fluorescence recovery after photobleaching revealed rapid recovery (half-time approximately 2.5 min) of about 35% of NPC1L1 in the ERC, probably replenished from peripheral sorting endosomes. Acute cholesterol depletion blocked internalization of NPC1L1-EGFP and Tf and stimulated recycling of NPC1L1-EGFP from the ERC to the plasma membrane. NPC1L1-EGFP facilitated transport of fluorescent sterols from the plasma membrane to the ERC. Insulin induced translocation of vesicles containing NPC1L1 and fluorescent sterol from the ERC to the cell membrane. Upon polarization of hepatoma cells, NPC1L1 resided almost exclusively in the canalicular membrane, where the protein is highly mobile. Our study demonstrates dynamic trafficking of NPC1L1 between the cell surface and intracellular compartments and suggests that this transport is involved in NPC1L1-mediated cellular sterol uptake.     

Membrane Orientation and Lateral Diffusion of BODIPY-Cholesterol as a Function of Probe Structure

Cholesterol tagged with the BODIPY fluorophore via the central difluoroboron moiety of the dye (B-Chol) is a promising probe for studying intracellular cholesterol dynamics. We synthesized a new BODIPY-cholesterol probe (B-P-Chol) with the fluorophore attached via one of its pyrrole rings to carbon-24 of cholesterol (B-P-Chol). Using two-photon fluorescence polarimetry in giant unilamellar vesicles and in the plasma membrane (PM) of living intact and actin-disrupted cells, we show that the BODIPY-groups in B-Chol and B-P-Chol are oriented perpendicular and almost parallel to the bilayer normal, respectively. B-Chol is in all three membrane systems much stronger oriented than B-P-Chol. Interestingly, we found that the lateral diffusion in the PM was two times slower for B-Chol than for B-P-Chol, although we found no difference in lateral diffusion in model membranes. Stimulated emission depletion microscopy, performed for the first time, to our knowledge, with fluorescent sterols, revealed that the difference in lateral diffusion of the BODIPY-cholesterol probes was not caused by anomalous subdiffusion, because diffusion of both analogs in the PM was free but not hindered. Our combined measurements show that the position and orientation of the BODIPY moiety in cholesterol analogs have a severe influence on lateral diffusion specifically in the PM of living cells.     

Ligand-induced trafficking of the sphingosine-1-phosphate receptor EDG-1

The endothelial-derived G-protein-coupled receptor EDG-1 is a high-affinity receptor for the bioactive lipid mediator sphingosine-1-phosphate (SPP). In the present study, we constructed the EDG-1-green fluorescent protein (GFP) chimera to examine the dynamics and subcellular localization of SPP-EDG-1 interaction. SPP binds to EDG-1-GFP and transduces intracellular signals in a manner indistinguishable from that seen with the wild-type receptor. Human embryonic kidney 293 cells stably transfected with the EDG-1-GFP cDNA expressed the receptor primarily on the plasma membrane. Exogenous SPP treatment, in a dose-dependent manner, induced receptor translocation to perinuclear vesicles with a tau1/2 of approximately 15 min. The EDG-1-GFP-containing vesicles are distinct from mitochondria but colocalize in part with endocytic vesicles and lysosomes. Neither the low-affinity agonist lysophosphatidic acid nor other sphingolipids, ceramide, ceramide-1-phosphate, or sphingosylphosphorylcholine, influenced receptor trafficking. Receptor internalization was completely inhibited by truncation of the C terminus. After SPP washout, EDG-1-GFP recycles back to the plasma membrane with a tau1/2 of approximately 30 min. We conclude that the high-affinity ligand SPP specifically induces the reversible trafficking of EDG-1 via the endosomal pathway and that the C-terminal intracellular domain of the receptor is critical for this process.     

Probing the dynamics of intact cells and nuclear envelope precursor membrane vesicles by deuterium solid state NMR spectroscopy

Membrane dynamics is an essential part of many cellular mechanisms such as intracellular trafficking, membrane fusion/fission and mitotic organelle reconstitution. The dynamics of membranes is dependent primarily on their phospholipid and cholesterol composition and how these molecules are ordered in relation to one another. To determine the physical status of membranes in whole cells or purified membranes of subcellular compartments we have developed a novel application exploiting solid-state ²H-NMR spectroscopy. We utilise this method to probe the dynamics of intact sperm and nuclear envelope precursor membranes. We show, using mass spectrometry, that either multilamellar or small unilamellar vesicles of deuterium-labelled palmitoyl-oleoylphosphatidylcholine can be used to probe the dynamics of sperm cells or nuclear envelope precursor membrane vesicles, respectively. Using ²H-NMR we determine the order parameters of sperm cells and nuclear envelope precursor membrane vesicles. We demonstrate that whole sperm membranes are more dynamic than nuclear envelope precursor membranes due to the higher cholesterol levels of the latter. Our new application can be exploited as a generic method for monitoring membrane dynamics in whole cells, various subcellular membrane compartments and membrane domains in subcellular compartments.     

Vasopressin regulated trafficking of a green fluorescent protein-aquaporin 2 chimera in LLC-PK1 cells

 Aquaporin 2 (AQP2) transfected into LLC-PK₁ cells functions as a vasopressin-regulated water channel that recycles between intracellular vesicles and the plasma membrane upon vasopressin stimulation. The green fluorescent protein (GFP) of the jellyfish, Aequorea victoria, was used as an autofluorescent tag to monitor AQP2 trafficking in transfected LLC-PK₁ cells. Two chimeras were constructed, one in which GFP was fused to the amino-terminus of AQP2 [GFP-AQP2(NT)] and the second in which it was fused to the carboxyl-terminus [AQP2-GFP(CT)]. The GFP-AQP2(NT) chimera trafficked in a regulated pathway from intracellular vesicles to the basolateral plasma membrane in response to vasopressin or forskolin stimulation of cells. In contrast, the AQP2-GFP(CT) chimera expressed in LLC-PK₁ cells was localized constitutively on both apical and basolateral plasma membranes. The cellular location of this chimera was not modified by vasopressin or forskolin. Thus, while the GFP-AQP2(NT) chimera will be useful to study AQP2 trafficking in vitro, the abnormal, constitutive membrane localization of the AQP2-GFP(CT) chimera suggests that one or more trafficking signals exist on the carboxyl-terminus of the AQP2 protein. Accepted: 8 April 1998     

Organelle-specific isoenzymes of plant V-ATPase as revealed by in vivo-FRET analysis

Background

The ability to specifically label proteins within living cells can provide information about their dynamics and function. To study a membrane protein, we fused a multi-functional reporter protein, HaloTag^®, to the extracellular domain of a truncated integrin.

Results

Using the HaloTag technology, we could study the localization, trafficking and processing of an integrin-HaloTag fusion, which we showed had cellular dynamics consistent with native integrins. By labeling live cells with different fluorescent impermeable and permeable ligands, we showed spatial separation of plasma membrane and internal pools of the integrin-HaloTag fusion, and followed these protein pools over time to study bi-directional trafficking. In addition to combining the HaloTag reporter protein with different fluorophores, we also employed an affinity tag to achieve cell capture.

Conclusion

The HaloTag technology was used successfully to study expression, trafficking, spatial separation and real-time translocation of an integrin-HaloTag fusion, thereby demonstrating that this technology can be a powerful tool to investigate membrane protein biology in live cells.     

Membrane Orientation and Lateral Diffusion of BODIPY-Cholesterol as a Function of Probe Structure

Cholesterol tagged with the BODIPY fluorophore via the central difluoroboron moiety of the dye (B-Chol) is a promising probe for studying intracellular cholesterol dynamics. We synthesized a new BODIPY-cholesterol probe (B-P-Chol) with the fluorophore attached via one of its pyrrole rings to carbon-24 of cholesterol (B-P-Chol). Using two-photon fluorescence polarimetry in giant unilamellar vesicles and in the plasma membrane (PM) of living intact and actin-disrupted cells, we show that the BODIPY-groups in B-Chol and B-P-Chol are oriented perpendicular and almost parallel to the bilayer normal, respectively. B-Chol is in all three membrane systems much stronger oriented than B-P-Chol. Interestingly, we found that the lateral diffusion in the PM was two times slower for B-Chol than for B-P-Chol, although we found no difference in lateral diffusion in model membranes. Stimulated emission depletion microscopy, performed for the first time, to our knowledge, with fluorescent sterols, revealed that the difference in lateral diffusion of the BODIPY-cholesterol probes was not caused by anomalous subdiffusion, because diffusion of both analogs in the PM was free but not hindered. Our combined measurements show that the position and orientation of the BODIPY moiety in cholesterol analogs have a severe influence on lateral diffusion specifically in the PM of living cells.     

Cell biology of the human thiamine transporter-1 (hTHTR1). Intracellular trafficking and membrane targeting mechanisms

The human thiamine transporter hTHTR1 is involved in the cellular accumulation of thiamine (vitamin B1) in many tissues. Thiamine deficiency disorders, such as thiamine-responsive megaloblastic anemia (TRMA), which is associated with specific mutations within hTHTR1, likely impairs the functionality and/or intracellular targeting of hTHTR1. Unfortunately, nothing is known about the mechanisms that control the intracellular trafficking or membrane targeting of hTHTR1. To identify molecular determinants involved in hTHTR1 targeting, we generated a series of hTHTR1 truncations fused with the green fluorescent protein and imaged the targeting and trafficking dynamics of each construct in living duodenal epithelial cells. Whereas the full-length fusion protein was functionally expressed at the plasma membrane, analysis of the truncated mutants demonstrated an essential role for both NH(2)-terminal sequence and the integrity of the backbone polypeptide for cell surface expression. Most notably, truncation of hTHTR1 within a region where several TRMA truncations are clustered resulted in intracellular retention of the mutant protein. Finally, confocal imaging of the dynamics of intracellular hTHTR1 vesicles revealed a critical role for microtubules, but not microfilaments, in hTHTR1 trafficking. Taken together, these results correlate hTHTR1 structure with cellular expression profile and reveal a critical dependence on hTHTR1 backbone integrity and microtubule-based trafficking processes for functional expression of hTHTR1.     

Adaptor protein Ruk/CIN85 is associated with a subset of COPI-coated membranes of the Golgi complex

Regulator of ubiquitous kinase/Cbl-interacting protein of 85 kDa (Ruk/CIN85) and CD2-associated protein/Cas ligand with multiple SH3 domains (CD2AP/CMS) comprise a family of vertebrate adaptor proteins involved in several important cellular processes, including downregulation of activated receptor tyrosine kinases, regulation of cytoskeletal rearrangements, phosphatidylinositol 3-kinase (PI 3-kinase) signalling and apoptosis. The role of Ruk/CIN85 as a scaffold protein involved in membrane trafficking processes has been demonstrated in model cell systems. However, intracellular localization of endogenous Ruk/CIN85 has never been comprehensively assessed. We carried out detailed studies of subcellular distribution of Ruk/CIN85 in adherent cultured human cells using antibodies that recognize distinct epitopes of the protein and revealed a punctate immunostaining pattern, common for proteins involved in intracellular trafficking processes. Our data indicate that Ruk/CIN85 is distributed between several different membrane trafficking compartments, but the major pool of Ruk/CIN85 is associated with the Golgi complex, mainly with a subpopulation of COPI-coated vesicles involved in retrograde endoplasmic reticulum-Golgi and intra-Golgi transport. This localization pattern is dependent on the integrity of Golgi complex and intact microtubular network. Only a small pool of Ruk/CIN85 is present in compartments involved in clathrin-mediated endocytosis and sorting. These results suggest that endogenous Ruk/CIN85 may be involved in regulation of specific membrane trafficking processes.     

Role of cytoplasmic termini in sorting and shuttling of the aquaporin-2 water channel

In mammals, the regulation of water homeostasis is mediated by the aquaporin-1 (AQP1) water channel, which localizes to the basolateral and apical membranes of the early nephron segment, and AQP2, which is translocated from intracellular vesicles to the apical membrane of collecting duct cells after vasopressin stimulation. Because a similar localization and regulation are observed in transfected Madin-Darby Canine Kidney (MDCK) cells, we investigated which segments of AQP2 are important for its routing to forskolin-sensitive vesicles and the apical membrane through analysis of AQP1-AQP2 chimeras. AQP1 with the entire COOH tail of AQP2 was constitutively localized in the apical membrane, whereas chimeras with shorter COOH tail segments of AQP2 were localized in the apical and basolateral membrane. AQP1 with the NH₂ tail of AQP2 was constitutively localized in both plasma membranes, whereas AQP1 with the NH₂ and COOH tail of AQP2 was sorted to intracellular vesicles and translocated to the apical membrane with forskolin. These data indicate that region N220-S229 is essential for localization of AQP2 in the apical membrane and that the NH₂ and COOH tail of AQP2 are essential for trafficking of AQP2 to intracellular vesicles and its shuttling to and from the apical membrane. routing signals; chimera; Madin-Darby canine kidney cells; regulated trafficking     

Protein kinase A-regulated membrane trafficking of a green fluorescent protein-aquaporin 5 chimera in MDCK cells

The green fluorescent protein (GFP) of the jellyfish, Aeqorea victoria, was used as an autofluorescent tag to track the trafficking of aquaporin 5 (AQP5), an exocrine gland-type water channel. Two groups of chimeric proteins were constructed; one in which GFP was fused to the amino-terminus of AQP5 (GFP-AQP5) and the other, in which it was fused to the carboxyl terminus of it (AQP5-GFP). In each group, 2 chimeras were produced, a wild-type AQP5 with its normal sequence and a mutant AQP5 having a mutated amino acid at 259, i.e., GFP-AQP5-T259A and AQP5-GFP-T259A. They were used to transfect Madin-Darby canine kidney (MDCK) cells. The GFP-AQP5 chimera was localized in the intracellular vesicles, which trafficked to the plasma membrane in response to N(6), 2'-O-dibutyryladenosine 3', 5'-cyclic monophosphate (dbcAMP). Membrane trafficking was inhibited by N-[2-(p-bromocinnamylamino)ethyl]-5-isoquimolinesulfonamide (H-89) but not by palmitoyl-dl-carnitine chloride (PCC). In contrast, the AQP5-GFP chimera expressed in MDCK cells was localized constitutively on the plasma membrane. The cellular localization of the latter chimera was not affected by stimulation with dbcAMP in the presence or absence of H-89 or PCC. Replacement of Thr-259 with Ala-259 did not affect the dbcAMP-induced translocation of the chimeric protein, suggesting that phosphorylation of Thr-259 was not necessary for AQP5 trafficking under the present experimental conditions. Thus, the GFP-AQP5 chimera will be a useful tool to study AQP5 trafficking in vitro, whereas the constitutive membrane localization of the AQP5-GFP chimera suggests the importance of the carboxyl terminus of the AQP5 protein for its sorting, whether it is translocated to intracellular vesicles or to the plasma membrane.     

Monitoring of the Cytoskeleton-Dependent Intracellular Trafficking of Fluorescent Iron Oxide Nanoparticles by Nanoparticle Pulse-Chase Experiments in C6 Glioma Cells

Iron oxide nanoparticles (IONPs) are used for various biomedical and therapeutic approaches. To investigate the uptake and the intracellular trafficking of IONPs in neural cells we have performed nanoparticle pulse-chase experiments to visualize the internalization and the fate of fluorescent IONPs in C6 glioma cells and astrocyte cultures. Already a short exposure to IONPs for 10 min at 4 °C (nanoparticle pulse) allowed binding of substantial amounts of nanoparticles to the cells, while internalization of IONPs into the cell was prevented. The uptake of bound IONPs and the intracellular trafficking was started by increasing the temperature to 37 °C (chase period). While hardly any cellular fluorescence nor any iron staining was detectable directly after the nanoparticle pulse, dotted cellular fluorescence and iron patterns appeared already within a few minutes after start of the chase incubation and became intensified in the perinuclear region during further incubation for up to 90 min. Longer chase incubations resulted in separation of the fluorescent coat from the core of the internalized IONPs. Disruption of actin filaments in C6 cells strongly impaired the internalization of IONPs, whereas destabilization of microtubules traped IONP-containing vesicles to the plasma membrane. In conclusion, nanoparticle pulse-chase experiments allowed to synchronize the cellular uptake of fluorescent IONPs and to identify for C6 cells an actin-dependent early and a microtubule-dependent later process in the intracellular trafficking of fluorescent IONPs.     

Cellular localization and trafficking of the human ABCA1 transporter

ABCA1, the ATP-binding cassette protein mutated in Tangier disease, mediates the efflux of excess cellular sterol to apoA-I and thereby the formation of high density lipoprotein. The intracellular localization and trafficking of ABCA1 was examined in stably and transiently transfected HeLa cells expressing a functional human ABCA1-green fluorescent protein (GFP) fusion protein. The fluorescent chimeric ABCA1 transporter was found to reside on the cell surface and on intracellular vesicles that include a novel subset of early endosomes, as well as late endosomes and lysosomes. Studies of the localization and trafficking of ABCA1-GFP in the presence of brefeldin A or monensin, agents known to block intracellular vesicular trafficking, as well as apoA-I-mediated cellular lipid efflux, showed that: (i) ABCA1 functions in lipid efflux at the cell surface, and (ii) delivery of ABCA1 to lysosomes for degradation may serve as a mechanism to modulate its surface expression. Time-lapse fluorescence microscopy revealed that ABCA1-GFP-containing early endosomes undergo fusion, fission, and tubulation and transiently interact with one another, late endocytic vesicles, and the cell surface. These studies establish a complex intracellular trafficking pathway for human ABCA1 that may play important roles in modulating ABCA1 transporter activity and cellular cholesterol homeostasis.     

Vesicular and nonvesicular transport of phosphatidylcholine in polarized HepG2 cells

We have investigated the transport and canalicular enrichment of fluorescent phosphatidylcholine (PC) in HepG2 cells using the fluorescent analogs of PC C6-NBD-PC and β-BODIPY-PC. Fluorescent PC was efficiently transported to the biliary canaliculus (BC) and became enriched on the lumenal side of the canalicular membrane as shown for C6-NBD-PC. Some fluorescent PC was transported in vesicles to a subapical compartment (SAC) or apical recycling compartment (ARC) in polarized HepG2 cells as shown by colocalization with fluorescent sphingomyelin (C6-NBD-SM) and fluorescent transferrin, respectively. Extensive trafficking of vesicles containing fluorescent PC between the basolateral domain, the SAC/ARC and the BC as well as endocytosis of PC analogs from the canalicular membrane were found. Evidence for nonvesicular transport included enrichment of the PC-analog β-BODIPY-PC in the BC (t_1/2 = 3.54 min) prior to its accumulation in the SAC/ARC (t_1/2 = 18.5 min) at 37 °C. Transport of fluorescent PC to the canalicular membrane also continued after disruption of the actin or microtubule cytoskeleton and at 2 °C. These results indicate that: (i) a nonvesicular transport pathway significantly contributes to the canalicular enrichment of PC in hepatocytic cells, and (ii) vesicular transport of fluorescent PC occurs from both membrane domains via the SAC/ARC.     

Membrane processes and biophysical characterization of living cells decorated with chromatic polydiacetylene vesicles

The structural complexity of the cell membrane makes analysis of membrane processes in living cells, as compared to model membrane systems, highly challenging. Living cells decorated with surface-attached colorimetric/fluorescent polydiacetylene patches might constitute an effective platform for analysis and visualization of membrane processes in situ. This work examines the biological and chemical consequences of plasma membrane labeling of promyelocytic leukemia cells with polydiacetylene. We show that the extent of fusion between incubated lipid/diacetylene vesicles and the plasma membrane is closely dependent upon the lipid composition of both vesicles and cell membrane. In particular, we find that cholesterol presence increased bilayer fusion between the chromatic vesicles and the plasma membrane, suggesting that membrane organization plays a significant role in the fusion process. Spectroscopic data and physiological assays show that decorating the cell membrane with the lipid/diacetylene patches reduces the overall lateral diffusion within the membrane bilayer, however polydiacetylene labeling does not adversely affect important cellular metabolic pathways. Overall, the experimental data indicate that the viability and physiological integrity of the surface-engineered cells are retained, making possible utilization of the platform for studying membrane processes in living cells. We demonstrate the use of the polydiacetylene-labeled cells for visualizing and discriminating among different membrane interaction mechanisms of pharmaceutical compounds.     

The role of membranes and membrane trafficking in RNA localization

Eukaryotic cells possess highly sophisticated membrane trafficking pathways that define specific membrane domains and provide a means for moving vesicles between them (Mostov, Su, and ter Beest, 2003, Nat. Cell Biol. 5, 287-293). Here, I review recent data that indicate a role for membrane trafficking in mRNA localization. Specifically, I review evidence that some localized mRNAs are anchored to specific membrane domains and/or transported on membranous organelles or vesicles to specific subcellular sites. This review is not intended as a discussion on indirect influences of membrane trafficking on mRNA localization. I will not, for example, discuss the role of membrane trafficking in the regulation of extracellular signalling events that could indirectly influence mRNA localization through polarization of the actin or microtubule cytoskeleton (for examples, see reviews by Drubin and Nelson, 1996, Cell 84, 335-344; Shulman and St Johnston, 1999, Trends Cell Biol. 9, M60-M64).     

Norwalk virus nonstructural protein p48 forms a complex with the SNARE regulator VAP-A and prevents cell surface expression of vesicular stomatitis virus G protein

Norwalk virus (NV), a reference strain of human calicivirus in the Norovirus genus of the family Caliciviridae, contains a positive-strand RNA genome with three open reading frames. ORF1 encodes a 1,789-amino-acid polyprotein that is processed into nonstructural proteins that include an NTPase, VPg, protease, and RNA-dependent RNA polymerase. The N-terminal protein p48 of ORF1 shows no significant sequence similarity to viral or cellular proteins, and its function in the human calicivirus replication cycle is not known. The lack of sequence similarity to any protein in the public databases suggested that p48 may have a unique function in the NV replication cycle or, alternatively, may perform a characterized function in replication by a unique mechanism. In this report, it is shown that p48 displays a vesicular localization pattern in transfected cells when fused to the fluorescent reporter EYFP. A predicted transmembrane domain at the C terminus of p48 was not necessary for the observed localization pattern, but this domain was sufficient to redirect localization of EYFP to a fluorescent pattern consistent with the Golgi apparatus. A yeast two-hybrid screen identified the SNARE regulator vesicle-associated membrane protein-associated protein A (VAP-A) as a binding partner of p48. Biochemical assays confirmed that p48 and VAP-A interact and form a stable complex in mammalian cells. Furthermore, expression of the vesicular stomatitis virus G glcyoprotein on the cell surface was inhibited when cells coexpressed p48, suggesting that p48 disrupts intracellular protein trafficking.