Inactivation of the Host Lipin Gene Accelerates RNA Virus Replication through Viral Exploitation of the Expanded Endoplasmic Reticulum Membrane

RNA viruses take advantage of cellular resources, such as membranes and lipids, to assemble viral replicase complexes (VRCs) that drive viral replication. The host lipins (phosphatidate phosphatases) are particularly interesting because these proteins play key roles in cellular decisions about membrane biogenesis versus lipid storage. Therefore, we examined the relationship between host lipins and tombusviruses, based on yeast model host. We show that deletion of PAH1 (phosphatidic acid phosphohydrolase), which is the single yeast homolog of the lipin gene family of phosphatidate phosphatases, whose inactivation is responsible for proliferation and expansion of the endoplasmic reticulum (ER) membrane, facilitates robust RNA virus replication in yeast. We document increased tombusvirus replicase activity in pah1Δ yeast due to the efficient assembly of VRCs. We show that the ER membranes generated in pah1Δ yeast is efficiently subverted by this RNA virus, thus emphasizing the connection between host lipins and RNA viruses. Thus, instead of utilizing the peroxisomal membranes as observed in wt yeast and plants, TBSV readily switches to the vastly expanded ER membranes in lipin-deficient cells to build VRCs and support increased level of viral replication. Over-expression of the Arabidopsis Pah2p in Nicotiana benthamiana decreased tombusvirus accumulation, validating that our findings are also relevant in a plant host. Over-expression of AtPah2p also inhibited the ER-based replication of another plant RNA virus, suggesting that the role of lipins in RNA virus replication might include several more eukaryotic viruses.     

Protein Translocation across the Rough Endoplasmic Reticulum

The rough endoplasmic reticulum is a major site of protein biosynthesis in all eukaryotic cells, serving as the entry point for the secretory pathway and as the initial integration site for the majority of cellular integral membrane proteins. The core components of the protein translocation machinery have been identified, and high-resolution structures of the targeting components and the transport channel have been obtained. Research in this area is now focused on obtaining a better understanding of the molecular mechanism of protein translocation and membrane protein integration.Protein translocation across the rough endoplasmic reticulum (RER) is an ancient and evolutionarily conserved process that is analogous to protein export across the cytoplasmic membranes of eubacterial and archaebacterial cells both with respect to the mechanism and core components. The RER membrane of eukaryotic cells is contiguous with the nuclear envelope and is morphologically composed of interconnected cisternae and tubules. Electron microscope images of mammalian cells and tissues revealed that the cisternal regions of the cytoplasmic surface of the endoplasmic reticulum are densely studded by membrane-bound ribosomes (Palade 1955a,b), giving rise to the term “rough ER.” The RER-bound ribosomes in en face images are often arranged in spirals or hairpins (Palade 1955a; Christensen and Bourne 1999), indicative of polyribosomes that are actively engaged in protein translation.Consistent with this high density of membrane-bound ribosomes, the RER is a major site of protein biosynthesis in eukaryotic cells. The nuclear envelope, the Golgi, lysosome, peroxisome, plasma membrane, and endosomes are biosynthetically derived from the rough ER. The three major groups of proteins that are synthesized by RER-bound ribosomes include secretory proteins, integral membrane proteins destined for ER-derived membranes, and the lumenal-resident proteins of the ER, Golgi, nuclear envelope, and lysosome. For those membranes that are not physically linked to the ER (e.g., the lysosome), integral membrane and lumenal proteins are delivered to their destination by vesicular transport pathways. Bioinformatics analysis of fully sequenced eukaryotic genomes indicates that roughly 30% of open reading frames encode integral membrane proteins (Wallin and von Heijne 1998); hence, a major role of the RER is the biosynthesis of membrane proteins. An important class of membrane proteins that are integrated into the RER has single carboxy-terminal TM spans and are known as tail-anchored (TA) membrane proteins. The posttranslational integration pathway for TA proteins has been a subject of several recent reviews (Borgese and Fasana 2011; Shao and Hegde 2011), thus we will not address the TA pathway in this article.     

Chlamydiae Assemble a Pathogen Synapse to Hijack the Host Endoplasmic Reticulum

Chlamydiae are obligate intracellular bacterial pathogens that replicate within a specialized membrane‐bound compartment, termed an ‘inclusion’. The inclusion membrane is a critical host–pathogen interface, yet the extent of its interaction with cellular organelles and the origin of this membrane remain poorly defined. Here we show that the host endoplasmic reticulum (ER) is specifically recruited to the inclusion, and that key rough ER (rER) proteins are enriched on and translocated into the inclusion. rER recruitment is a Chlamydia‐orchestrated process that occurs independently of host trafficking. Generation of infectious progeny requires an intact ER, since ER vacuolation early during infection stalls inclusion development, whereas disruption post ER recruitment bursts the inclusion. Electron tomography and immunolabelling of Chlamydia‐infected cells reveal ‘pathogen synapses’ at which ordered arrays of chlamydial type III secretion complexes connect to the inclusion membrane only at rER contact sites. Our data show a supramolecular assembly involved in pathogen hijack of a key host organelle.     

Peroxisomal Membrane Proteins Insert into the Endoplasmic Reticulum

We show that a comprehensive set of 16 peroxisomal membrane proteins (PMPs) encompassing all types of membrane topologies first target to the endoplasmic reticulum (ER) in Saccharomyces cerevisiae. These PMPs insert into the ER membrane via the protein import complexes Sec61p and Get3p (for tail-anchored proteins). This trafficking pathway is representative for multiplying wild-type cells in which the peroxisome population needs to be maintained, as well as for mutant cells lacking peroxisomes in which new peroxisomes form after complementation with the wild-type version of the mutant gene. PMPs leave the ER in a Pex3p-Pex19p–dependent manner to end up in metabolically active peroxisomes. These results further extend the new concept that peroxisomes derive their basic framework (membrane and membrane proteins) from the ER and imply a new functional role for Pex3p and Pex19p.     

Protein Translocation Across the Membrane of the Endoplasmic Reticulum

Saccharomyces cerevisiae and mammals concerning the mechanisms of the translocation step and discuss the roles of the proteins implicated in this process. Received: 5 June 1996/Revised: 20 September 1996     

A Cytosolic Chaperone Complexes with Dynamic Membrane J-Proteins and Mobilizes a Nonenveloped Virus out of the Endoplasmic Reticulum

Nonenveloped viruses undergo conformational changes that enable them to bind to, disrupt, and penetrate a biological membrane leading to successful infection. We assessed whether cytosolic factors play any role in the endoplasmic reticulum (ER) membrane penetration of the nonenveloped SV40. We find the cytosolic SGTA-Hsc70 complex interacts with the ER transmembrane J-proteins DnaJB14 (B14) and DnaJB12 (B12), two cellular factors previously implicated in SV40 infection. SGTA binds directly to SV40 and completes ER membrane penetration. During ER-to-cytosol transport of SV40, SGTA disengages from B14 and B12. Concomitant with this, SV40 triggers B14 and B12 to reorganize into discrete foci within the ER membrane. B14 must retain its ability to form foci and interact with SGTA-Hsc70 to promote SV40 infection. Our results identify a novel role for a cytosolic chaperone in the membrane penetration of a nonenveloped virus and raise the possibility that the SV40-induced foci represent cytosol entry sites.     

Murine Intracisternal A Type Particles Fail to Separate from the Membrane of the Endoplasmic Reticulum

Analysis of serial sections of murine cells containing intracisternal A particles revealed that over 99% of all A particles remain in a budding configuration. This indicates that these particles fail to detach from the membrane of the endoplasmic reticulum. This observation explains how, despite their intracellular abundance in certain murine tumors, no extracellular A-type particles can be found.     

African Swine Fever Virus Is Wrapped by the Endoplasmic Reticulum

African swine fever (ASF) virus is a large DNA virus that shares the striking icosahedral symmetry of iridoviruses and the genomic organization of poxviruses. Both groups of viruses have a complex envelope structure. In this study, the mechanism of formation of the inner envelope of ASF virus was investigated. Examination of thin cryosections by electron microscopy showed two internal membranes in mature intracellular virions and all structural intermediates. These membranes were in continuity with intracellular membrane compartments, suggesting that the virus gained two membranes from intracellular membrane cisternae. Immunogold electron microscopy showed the viral structural protein p17 and resident membrane proteins of the endoplasmic reticulum (ER) within virus assembly sites, virus assembly intermediates, and mature virions. Resident ER proteins were also detected by Western blotting of isolated virions. The data suggested the ASF virus was wrapped by the ER. Analysis of the published sequence of ASF virus (R. J. Yanez et al., Virology 208:249–278, 1995) revealed a reading frame, XP124L, that encoded a protein predicted to translocate into the lumen of the ER. Pulse-chase immunoprecipitation and glycosylation analysis of pXP124L, the product of the XP124L gene, showed that pXP124L was retained in the ER lumen after synthesis. When analyzed by immunogold electron microscopy, pXP124L localized to virus assembly intermediates and fully assembled virions. Western blot analysis detected pXP124L in virions isolated from Percoll gradients. The packaging of pXP124L from the lumen of the ER into the virion is consistent with ASF virus being wrapped by ER cisternae: a mechanism which explains the presence of two membranes in the viral envelope.African swine fever (ASF) virus is a large icosahedral enveloped DNA virus that causes a lethal hemorrhagic disease in domestic pigs. The virus is endemic in areas of southern Europe and in Africa where it causes major problems for the development of pig industries. At present there are no vaccines, and the disease is controlled through the slaughter of infected animals. The economic importance of ASF virus has made the virus the focus of much research since it was first described in 1921 (32). ASF virus is unique among animal viruses, and its classification has been controversial. ASF virus shares the striking icosahedral symmetry of iridoviruses (5, 8, 13, 34), while the presence of inverted terminal repeats and covalently linked ends in the 170-kDa genome suggests similarities with poxviruses (16). The ASF virus genome encoding at least 150 proteins has been sequenced (17, 51), and the amino acid sequences of at least 11 structural proteins are known. p73 is the major structural protein (14, 28) and has sequence similarities to the capsid protein of iridoviruses (39). The ordered proteolysis of pp220 produces p150, p37/p34 and p14 (40), which together comprise 25% of the viral proteins (3). These proteins localize to the interior of the virion (3). Three proteins, J13L/p54, I1L/p17, and p22, with membrane-spanning domains localize to the viral envelope (10, 37, 41, 43). Three other structural proteins, p14.5 encoded by E120R (30), p10 encoded by K78R (35), and p5AR encoded by A104R (7), have DNA-binding properties (51) and may be involved in DNA packaging. The virus has been the subject of several detailed electron microscopy studies (2–4, 8, 9, 11, 13, 34, 47). Electron micrographs of sections taken through ASF virus assembly sites reveal fully assembled virions as 200-nm hexagons and an ordered series of assembly intermediates with one to six sides of a hexagon. Close inspection of intracellular virions identifies multiple concentric layers of differing electron densities. According to recent models, the layers represent a central electron-dense nucleocapsid core, surrounded by an inner core shell, an inner envelope, and an outer capsid layer (3). The mechanism of formation of the inner envelope of ASF virus has not been resolved.Most viruses gain a single membrane envelope by budding into intracellular membrane compartments or from the plasma membrane, as reviewed in reference 21. When viruses bud into an intracellular compartment, the domains of the membrane proteins that are initially located in the lumen of membrane compartments are exposed on the outside of the virion after release from the cell (Fig. ​(Fig.1a).1a). A second mechanism of envelopment, described recently for poxviruses and herpesviruses (18, 20, 24, 38, 42, 46, 50), is more complex and involves the wrapping of virions by membrane cisternae derived from specific membrane compartments. Wrapping provides two membrane envelopes in one step and leaves the virion free in the cytoplasm. When compared with budding, wrapping reverses the orientation of membrane proteins within the virus such that the domains of membrane proteins located in the lumen of the wrapping organelle are confined to the interior of the virus after release from the cell, whereas cytoplasmic tails are exposed on the outside of the virus (Fig. ​(Fig.1b).1b). Given these important consequences for understanding the mechanism of assembly of the virus and for determining the final orientation of membrane proteins in virions, we have set out to determine whether ASF virus acquires its membranes by the conventional budding mechanism or whether the virus is wrapped by intracellular membrane compartments before release from the cell. Open in a separate windowFIG. 1Schematic comparison of budding and wrapping mechanisms of virus envelopment. (a) Budding. Viral nucleoprotein complexes bind to the cytoplasmic domains of virally encoded integral membrane proteins (|, membrane glycoproteins). Interactions between viral proteins lead to membrane curvature, and the virion gains a single membrane by budding into the lumen of the membrane compartment. When the virion is released from the cell, oligosaccharides () are exposed on the surface of the virus, and the cytoplasmic tail of the membrane glycoprotein is buried within the virion. (b) Wrapping. Viral nucleoprotein complexes bind to the cytoplasmic domains of virally encoded integral membrane proteins. The nucleoprotein complex is then wrapped by the membrane cisternae, and the virus gains two membranes. The particle remains in the cytosol. When the virion is released from the cell by cell lysis, oligosaccharides () are buried within the two membranes of the virion while the cytoplasmic tail of the membrane glycoprotein is exposed on the surface of the virus.In this study we have taken advantage of thin cryoelectron microscopic sections to enhance the definition of viral membranes. The micrographs show two membranes within mature intracellular virions and all structural intermediates. They also show assembly intermediates in continuity with cellular membrane compartments. Consistent with our earlier study showing that p73 was enveloped by the endoplasmic reticulum (ER) (15), immunogold labelling experiments show resident proteins of the ER within membranes found at assembly sites, in virus assembly intermediates, and in mature virions. Importantly, we have identified a protein (pXP124L) encoded by ASF virus that translocates completely into the lumen of the ER and is incorporated as a structural protein of the virus. The presence of two membranes within intracellular virions and structural intermediates and the packaging of a structural protein from the lumen of the ER into the virus, strongly suggest that ASF virus is wrapped by the ER.     

A Cluster of Thin Tubular Structures Mediates Transformation of the Endoplasmic Reticulum to Autophagic Isolation Membrane

Recent findings have suggested that the autophagic isolation membrane (IM) might originate from a domain of the endoplasmic reticulum (ER) called the omegasome. However, the morphological relationships between ER, omegasome, and IM remain unclear. In the present study, we found that hybrid structures composed of a double FYVE domain-containing protein 1 (DFCP1)-positive omegasome and the IM accumulated in Atg3-deficient mouse embryonic fibroblasts (MEFs). Moreover, correlative light and electron microscopy and immunoelectron microscopy revealed that green fluorescent protein (GFP)-tagged DFCP1 was localized on tubular or vesicular elements adjacent to the IM rims. Through detailed morphological analyses, including optimization of a fixation method and electron tomography, we observed a cluster of thin tubular structures between the IM edges and ER, part of which were continuous with IM and/or ER. The formation of these thin tubular clusters was observed in several cell lines and MEFs deficient for Atg5, Atg7, or Atg16L1 but not in FIP200-deficient cells, suggesting that they were relevant to the earlier events in autophagosome formation. Taken together, our findings indicate that these tubular profiles represent a part of the omegasome that links the ER with the IM.     

Rabies Virus Hijacks and Accelerates the p75NTR Retrograde Axonal Transport Machinery

Rabies virus (RABV) is a neurotropic virus that depends on long distance axonal transport in order to reach the central nervous system (CNS). The strategy RABV uses to hijack the cellular transport machinery is still not clear. It is thought that RABV interacts with membrane receptors in order to internalize and exploit the endosomal trafficking pathway, yet this has never been demonstrated directly. The p75 Nerve Growth Factor (NGF) receptor (p75NTR) binds RABV Glycoprotein (RABV-G) with high affinity. However, as p75NTR is not essential for RABV infection, the specific role of this interaction remains in question. Here we used live cell imaging to track RABV entry at nerve terminals and studied its retrograde transport along the axon with and without the p75NTR receptor. First, we found that NGF, an endogenous p75NTR ligand, and RABV, are localized in corresponding domains along nerve tips. RABV and NGF were internalized at similar time frames, suggesting comparable entry machineries. Next, we demonstrated that RABV could internalize together with p75NTR. Characterizing RABV retrograde movement along the axon, we showed the virus is transported in acidic compartments, mostly with p75NTR. Interestingly, RABV is transported faster than NGF, suggesting that RABV not only hijacks the transport machinery but can also manipulate it. Co-transport of RABV and NGF identified two modes of transport, slow and fast, that may represent a differential control of the trafficking machinery by RABV. Finally, we determined that p75NTR-dependent transport of RABV is faster and more directed than p75NTR-independent RABV transport. This fast route to the neuronal cell body is characterized by both an increase in instantaneous velocities and fewer, shorter stops en route. Hence, RABV may employ p75NTR-dependent transport as a fast mechanism to facilitate movement to the CNS.     

Coupled Translocation Events Generate Topological Heterogeneity at the Endoplasmic Reticulum Membrane

Topogenic determinants that direct protein topology at the endoplasmic reticulum membrane usually function with high fidelity to establish a uniform topological orientation for any given polypeptide. Here we show, however, that through the coupling of sequential translocation events, native topogenic determinants are capable of generating two alternate transmembrane structures at the endoplasmic reticulum membrane. Using defined chimeric and epitope-tagged full-length proteins, we found that topogenic activities of two C-trans (type II) signal anchor sequences, encoded within the seventh and eighth transmembrane (TM) segments of human P-glycoprotein were directly coupled by an inefficient stop transfer (ST) sequence (TM7b) contained within the C-terminus half of TM7. Remarkably, these activities enabled TM7 to achieve both a single- and a double-spanning TM topology with nearly equal efficiency. In addition, ST and C-trans signal anchor activities encoded by TM8 were tightly linked to the weak ST activity, and hence topological fate, of TM7b. This interaction enabled TM8 to span the membrane in either a type I or a type II orientation. Pleiotropic structural features contributing to this unusual topogenic behavior included 1) a short, flexible peptide loop connecting TM7a and TM7b, 2) hydrophobic residues within TM7b, and 3) hydrophilic residues between TM7b and TM8.     

Retention of the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein in the Endoplasmic Reticulum Does Not Redirect Virus Assembly from the Plasma Membrane

The envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) has been shown to redirect the site of virus assembly in polarized epithelial cells. To test whether localization of the glycoprotein exclusively to the endoplasmic reticulum (ER) could redirect virus assembly to that organelle in nonpolarized cells, an ER -retrieval signal was engineered into an epitope-tagged variant of Env. The epitope tag, attached to the C terminus of Env, did not affect the normal maturation and transport of the glycoprotein or the incorporation of Env into virions. The epitope-tagged Env was also capable of mediating syncytium formation and virus entry with a similar efficiency to that of wild-type Env. When the epitope was modified to contain a consensus K(X)KXX ER retrieval signal, however, the glycoprotein was no longer proteolytically processed into its surface and transmembrane subunits and Env could not be detected at the cell surface by biotinylation. Endoglycosidase H analysis revealed that the modified Env was not transported to the Golgi apparatus. Immunofluorescent staining patterns were also consistent with the exclusion of Env from the Golgi. As expected, cells expressing the modified Env failed to form syncytia with CD4⁺ permissive cells. Despite this tight localization of Env to the ER, when the modified Env was expressed in the context of virus, virions continued to be produced efficiently from the plasma membrane of transfected cells. However, these virions contained no detectable glycoprotein and were noninfectious. Electron microscopy revealed virus budding from the plasma membrane of these cells, but no virus was seen assembling at the ER membrane and no assembled virions were found within the cell. These results suggest that the accumulation of Env in an intracellular compartment is not sufficient to redirect the assembly of HIV Gag in nonpolarized cells.     

Tracking Single Serotonin Transporter Molecules at the Endoplasmic Reticulum and Plasma Membrane

Transmembrane proteins are synthesized and folded in the endoplasmic reticulum (ER), an interconnected network of flattened sacs or tubes. Up to now, this organelle has eluded a detailed analysis of the dynamics of its constituents, mainly due to the complex three-dimensional morphology within the cellular cytosol, which precluded high-resolution, single-molecule microscopy approaches. Recent evidences, however, pointed out that there are multiple interaction sites between ER and the plasma membrane, rendering total internal reflection microscopy of plasma membrane proximal ER regions feasible. Here we used single-molecule fluorescence microscopy to study the diffusion of the human serotonin transporter at the ER and the plasma membrane. We exploited the single-molecule trajectories to map out the structure of the ER close to the plasma membrane at subdiffractive resolution. Furthermore, our study provides a comparative picture of the diffusional behavior in both environments. Under unperturbed conditions, the majority of proteins showed similar mobility in the two compartments; at the ER, however, we found an additional 15% fraction of molecules moving with 25-fold faster mobility. Upon degradation of the actin skeleton, the diffusional behavior in the plasma membrane was strongly influenced, whereas it remained unchanged in the ER.Live-cell microscopy and three-dimensional electron tomography has boosted our understanding of endoplasmic reticulum (ER) dynamics and morphology. Proteins have been identified which regulate the formation of cisternae versus tubelike membranes, and the contacts between ER and the various cellular organelles have been studied in detail (1). Little information, however, is available when it comes to protein dynamics and organization within the ER membrane. Its complex three-dimensional topology hampers standard diffraction-limited fluorescence microscopy approaches: in fluorescence recovery after photobleaching, for example, the obtained diffusion coefficients can be several-folds off, if the ER morphology is not correctly taken into account (2). A method is therefore needed which allows for resolving molecular movements on length scales below the typical dimensions of the ER structures.In principle, single-molecule tracking would provide the required spatial resolution due to the high precision in localizing the moving point emitters: localization errors of <40 nm can be easily achieved (3). This technique has given rise to multiple studies, in which the paths of the diffusing objects were used to make conclusions on the properties of the environment; particularly, the plasma membrane has become a favorite target for such investigations, yielding precise determinations of the diffusion coefficients of a variety of membrane proteins or lipids (4).Here, we report what is, to our knowledge, the first application of single-molecule tracking for a comparative study of the diffusion dynamics of a membrane protein at the ER versus the plasma membrane. As the protein of interest, we chose the human serotonin transporter (SERT): it is a polytopic membrane protein containing 12 transmembrane domains, with both C- and N-termini residing in the cytoplasm. Stable SERT oligomers of various degrees were observed to coexist in the plasma membrane (5). Functionally, SERT (6) is a pivotal element in shaping serotonergic neurotransmission: SERT-mediated high-affinity uptake of released serotonin clears the synaptic cleft and supports refilling of vesicular stores (7). Wild-type SERT (SERT-wt) is efficiently targeted to the presynaptic plasma membrane, whereas the truncation of its C-terminus (SERT-ΔC30) retains the mutant protein in the ER (8). The N-terminal mGFP- and eYFP-fusion constructs of the two versions of SERT thus allowed us to specifically address SERT located at the ER (eYFP-SERT-ΔC30) or at the plasma membrane (mGFP-SERT-wt (7)).Our experiments were performed at 37°C on proteins heterologously expressed in CHO cells. Total internal reflection (TIR) illumination afforded a reduction in background fluorescence and allowed for selective imaging of single mGFP-SERT-wt molecules at the cells’ plasma membrane or single eYFP-SERT-ΔC30 molecules at plasma membrane-proximal ER (Fig. 1 and see the Supporting Material). TIR was particularly crucial for single-molecule imaging of the ER-retained mutant, where out-of-focus background would surpass the weak single-molecule signals in epi-illumination.Open in a separate windowFigure 1Schematics of the plasma membrane (PM) and a part of the ER containing mGFP-SERT-wt or the ER-retained eYFP-SERT-ΔC30 mutant, respectively. Both can be excited by total internal reflection fluorescence (TIRF) excitation. Experiments were carried out either on cells expressing mGFP-SERT-wt or eYFP-SERT-ΔC30.For both mutants, the majority of molecules were mobile: in fluorescence-recovery-after-photobleaching experiments we observed a mobile fraction of 82 ± 8% for mGFP-SERT-wt and 91 ± 4% for eYFP-SERT-ΔC30. For single-molecule tracking, the high surface density of signals was reduced by completely photobleaching a rectangular part of the cell in epi-illumination; after a brief recovery period, a few single-molecule signals had entered the bleached area and could be monitored and tracked at high signal/noise using TIR excitation. Samples were illuminated stroboscopically for t_ill = 2 ms, and movies of 500 frames were recorded with a delay of t_del = 6 ms; the short delay times ensured that even rapidly diffusing molecules hardly reached the borders of the ER tubes between two consecutive frames. This illumination protocol was run for 20 times per cell, yielding ∼2500 trajectories per cell.The single-molecule localizations were first used to map those areas that are accessible to the diffusing proteins. eYFP-SERT-ΔC30 showed distinct hotspots, representing plasma membrane-proximal ER, excitable by the evanescent field (Fig. 2 A). These hotspots hardly moved within the timescale of an experiment (tens of minutes, see Fig. S1 in the Supporting Material); indeed, remarkable ER stability was previously observed using superresolution microscopy (9). In contrast, a rather homogeneous distribution was observed for mGFP-SERT-wt in the plasma membrane (Fig. 2 B).Open in a separate windowFigure 2Superresolution and tracking data at the ER and the plasma membrane. Superresolution images are shown for the ER-retained SERT mutant eYFP-SERT-ΔC30 (A) and for mGFP-SERT-wt in the plasma membrane (B). (C and D) Diffusion coefficients of eYFP-SERT-ΔC30 (C) and mGFP-SERT-wt (D) are shown as normalized histograms before (blue) and after (red) Cytochalasin D treatment. Data were fitted by Gaussian mobility distributions (see Table S1 in the Supporting Material for the fit results).Next, we compared the mobility of the observed proteins. Single-molecule localizations were linked to trajectories as described in Gao and Kilfoil (10), and the apparent diffusion coefficient, D, of each molecule was estimated from the first two points of the mean-square displacement membrane. The distribution of log₁₀ D showed a pronounced single peak (Fig. 2 D). It could be well fitted by a linear combination of two Gaussian functions, with the major fraction (85%) characterized by D_wt = 0.30 μm²/s; a broad shoulder to the left indicates the presence of proteins that are immobilized during the observation period. In contrast, the mobility of the ER-retained mutant showed a substantially different distribution, containing two clearly visible peaks (Fig. 2 C). We fitted the data with a three-component Gaussian model: the main fraction (82%) behaved similar to SERT at the plasma membrane, with D_ΔC30 = 0.32 μm²/s. In addition, a large fraction (15%) with high mobility of D_ΔC30 = 7.8 μm²/s and a minor fraction (3%) with low mobility was observed. The proteins responded as expected to degradation of the actin membrane skeleton (red bars in Fig. 2, C and D): at the plasma membrane, the mobility of mGFP-SERT-wt increased 4.6-fold (mean values), whereas at the ER membrane there was only a minor change for eYFP-SERT-ΔC30 mobility (1.06-fold increase; note that the ER is not connected to actin filaments (11)).The observation of a high mobility subfraction at the ER membrane is surprising. In general, the presence of obstacles—irrespective of whether randomly distributed or clustered, mobile or immobile—reduces the diffusivity of mobile tracers in a membrane (12). It is generally assumed that the high protein density in cell membranes is responsible for the rather low fluidity when compared to synthetic membranes (compare, e.g., Saxton and Jacobson (13) with Weiss et al. (14)). Interestingly, the observed diffusion constant of 7.8 μm²/s is of similar order as the mobility determined for various proteins in synthetic lipid membranes (14). It is thus tempting to hypothesize the presence of extended protein-depleted regions of higher fluidity within the ER membrane; such membrane domains were indeed observed already at the plasma membrane (15). We were also concerned, however, that protein degradation fragments could have contributed to our data: the three-dimensional mobility of an 85-kDa protein is ∼10 μm²/s (16), similar to the high mobility diffusion constant of eYFP-SERT-ΔC30.We tested the two explanations by analyzing the spatial distribution of fast (D_ΔC30 > 1 μm²/s) versus slow trajectories (D_ΔC30 < 1 μm²/s) of eYFP-SERT-ΔC30 (Fig. 3). Both types of trajectories clustered in the same regions, and no segregation into ER subdomains was observable at the resolved length scales. This finding—on the one hand—disfavors freely diffusing protein fragments as the origin of the high mobility fraction. On the other hand, it calls for further experiments to identify the origin of the fast and the slow mobility subfraction. Interestingly, when analyzing all eYFP-SERT-ΔC30 trajectories we found that 80% of the molecules showed diffusion confined to domains of 230-nm radius (see Fig. S2). This size is clearly smaller than the lateral extensions of the visible ER regions observed in Fig. 3. The finding indicates domain formation at the ER membrane; domains are averaged out in Fig. 3 due to the long recording times. Note that free diffusion was observed for mGFP-SERT-wt at the plasma membrane (5).Open in a separate windowFigure 3Ripley’s K function analysis of the different mobility fractions in the ER. For the cell presented in Fig. 2, the first position of every slow (D < 1 μm²/s; red) and fast (D > 1 μm²/s; blue) trajectory was plotted in panel A. Contour lines indicate regions of ER attachment to the plasma membrane. In panel B, the point-correlation function L(r)−r is plotted for the slow (red) and fast (blue) fraction. Furthermore, the correlation between fast versus slow is plotted (green). All three curves show a peak at ∼450 nm, which agrees with the extensions of the ER attachment zones.In conclusion, we have shown that single-molecule tracking is feasible for constituents of the ER membrane. We found a surprising diffusion behavior of SERT resulting in the following:

• 1.A slow fraction showing mobility reminiscent of protein diffusion in the plasma membrane, likely reflecting SERT diffusing in protein-crowded regions of the ER membrane; and
• 2.A fast fraction showing 25-fold faster diffusion kinetics.

This likely represents diffusion in altered ER membrane environments, possibly of different lipid or protein composition. Given the fact that synthesis of virtually all membrane proteins and most lipids proceeds at the ER membrane, ER heterogeneity at the nanoscale due to the continuous synthesis activity and selection for correct folding appears highly plausible.     

Tracking Single Serotonin Transporter Molecules at the Endoplasmic Reticulum and Plasma Membrane

Transmembrane proteins are synthesized and folded in the endoplasmic reticulum (ER), an interconnected network of flattened sacs or tubes. Up to now, this organelle has eluded a detailed analysis of the dynamics of its constituents, mainly due to the complex three-dimensional morphology within the cellular cytosol, which precluded high-resolution, single-molecule microscopy approaches. Recent evidences, however, pointed out that there are multiple interaction sites between ER and the plasma membrane, rendering total internal reflection microscopy of plasma membrane proximal ER regions feasible. Here we used single-molecule fluorescence microscopy to study the diffusion of the human serotonin transporter at the ER and the plasma membrane. We exploited the single-molecule trajectories to map out the structure of the ER close to the plasma membrane at subdiffractive resolution. Furthermore, our study provides a comparative picture of the diffusional behavior in both environments. Under unperturbed conditions, the majority of proteins showed similar mobility in the two compartments; at the ER, however, we found an additional 15% fraction of molecules moving with 25-fold faster mobility. Upon degradation of the actin skeleton, the diffusional behavior in the plasma membrane was strongly influenced, whereas it remained unchanged in the ER.     

An Endoplasmic Reticulum Retrieval Signal Partitions Human Foamy Virus Maturation to Intracytoplasmic Membranes

Among all retroviruses, foamy viruses (FVs) are unique in that they regularly mature at intracytoplasmic membranes. The envelope glycoprotein of FV encodes an endoplasmic reticulum (ER) retrieval signal, the dilysine motif (KKXX), that functions to localize the human FV (HFV) glycoprotein to the ER. This study analyzed the function of the dilysine motif in the context of infectious molecular clones of HFV that encoded mutations in the dilysine motif. Electron microscopy (EM) demonstrated virion budding both intracytoplasmically and at the plasma membrane for the wild-type and mutant viruses. Additionally, mutant viruses retained their infectivity, but viruses lacking the dilysine signal budded at the plasma membrane to a greater extent than did wild-type viruses. Interestingly, this relative increase in budding across the plasma membrane did not increase the overall release of viral particles into cell culture media as measured by protein levels in viral pellets or infectious virus titers. We conclude that the dilysine motif of HFV imposes a partial restriction on the site of viral maturation but is not necessary for viral infectivity.     

A Conserved Endoplasmic Reticulum Membrane Protein Complex (EMC) Facilitates Phospholipid Transfer from the ER to Mitochondria

Mitochondrial membrane biogenesis and lipid metabolism require phospholipid transfer from the endoplasmic reticulum (ER) to mitochondria. Transfer is thought to occur at regions of close contact of these organelles and to be nonvesicular, but the mechanism is not known. Here we used a novel genetic screen in S. cerevisiae to identify mutants with defects in lipid exchange between the ER and mitochondria. We show that a strain missing multiple components of the conserved ER membrane protein complex (EMC) has decreased phosphatidylserine (PS) transfer from the ER to mitochondria. Mitochondria from this strain have significantly reduced levels of PS and its derivative phosphatidylethanolamine (PE). Cells lacking EMC proteins and the ER–mitochondria tethering complex called ERMES (the ER–mitochondria encounter structure) are inviable, suggesting that the EMC also functions as a tether. These defects are corrected by expression of an engineered ER–mitochondrial tethering protein that artificially tethers the ER to mitochondria. EMC mutants have a significant reduction in the amount of ER tethered to mitochondria even though ERMES remained intact in these mutants, suggesting that the EMC performs an additional tethering function to ERMES. We find that all Emc proteins interact with the mitochondrial translocase of the outer membrane (TOM) complex protein Tom5 and this interaction is important for PS transfer and cell growth, suggesting that the EMC forms a tether by associating with the TOM complex. Together, our findings support that the EMC tethers ER to mitochondria, which is required for phospholipid synthesis and cell growth.     

Localization of Rabies Virus Glycoprotein into the Endoplasmic Reticulum Produces Immunoprotective Antigen

Rabies virus surface glycoprotein (rabies G-protein) with (G+RS) and without (G?RS) endoplasmic reticulum retrieval signal was expressed and characterized in tobacco plants. Transgenically expressed rabies G-protein was estimated at 0.015?C0.38?% of total leaf protein. The relative migration of the rabies G-protein on SDS-PAGE was at the position, as anticipated for the viral coat protein (~66?kDa). Immunolocalization by confocal microscopy established that immunoprotective G+RS expressed in tobacco was primarily confined to ER. G+RS showed binding to Con A lectin and was susceptible to N-glycosidase F activity similar to native rabies G-protein. However, the G?RS transgenically expressed in tobacco leaves was glycosylated differently and was resitant to N-glycosidase F. Immunological studies and Rapid Fluorescent Foci Inhibition Test (RFFIT) showed that G+RS was immunogenic and immunoprotective, whereas G?RS was moderately immunogenic but non-protective against live virus challenge. Hence, plants can express the antigenic component of rabies virus with suitable glycosylation, which is important to give protection against rabies virus infection.