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.     

Single Molecule Analysis Reveals Coexistence of Stable Serotonin Transporter Monomers and Oligomers in the Live Cell Plasma Membrane

The human serotonin transporter (hSERT) is responsible for the termination of synaptic serotonergic signaling. Although there is solid evidence that SERT forms oligomeric complexes, the exact stoichiometry of the complexes and the fractions of different coexisting oligomeric states still remain enigmatic. Here we used single molecule fluorescence microscopy to obtain the oligomerization state of the SERT via brightness analysis of single diffraction-limited fluorescent spots. Heterologously expressed SERT was labeled either with the fluorescent inhibitor JHC 1-64 or via fusion to monomeric GFP. We found a variety of oligomerization states of membrane-associated transporters, revealing molecular associations larger than dimers and demonstrating the coexistence of different degrees of oligomerization in a single cell; the data are in agreement with a linear aggregation model. Furthermore, oligomerization was found to be independent of SERT surface density, and oligomers remained stable over several minutes in the live cell plasma membrane. Together, the results indicate kinetic trapping of preformed SERT oligomers at the plasma membrane.     

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.     

From Endoplasmic Reticulum to Mitochondria: Absence of the Arabidopsis ATP Antiporter Endoplasmic Reticulum Adenylate Transporter1 Perturbs Photorespiration

The carrier Endoplasmic Reticulum Adenylate Transporter1 (ER-ANT1) resides in the endoplasmic reticulum (ER) membrane and acts as an ATP/ADP antiporter. Mutant plants lacking ER-ANT1 exhibit a dwarf phenotype and their seeds contain reduced protein and lipid contents. In this study, we describe a further surprising metabolic peculiarity of the er-ant1 mutants. Interestingly, Gly levels in leaves are immensely enhanced (26×) when compared with that of wild-type plants. Gly accumulation is caused by significantly decreased mitochondrial glycine decarboxylase (GDC) activity. Reduced GDC activity in mutant plants was attributed to oxidative posttranslational protein modification induced by elevated levels of reactive oxygen species (ROS). GDC activity is crucial for photorespiration; accordingly, morphological and physiological defects in er-ant1 plants were nearly completely abolished by application of high environmental CO₂ concentrations. The latter observation demonstrates that the absence of ER-ANT1 activity mainly affects photorespiration (maybe solely GDC), whereas basic cellular metabolism remains largely unchanged. Since ER-ANT1 homologs are restricted to higher plants, it is tempting to speculate that this carrier fulfils a plant-specific function directly or indirectly controlling cellular ROS production. The observation that ER-ANT1 activity is associated with cellular ROS levels reveals an unexpected and critical physiological connection between the ER and other organelles in plants.     

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     

Synucleins Antagonize Endoplasmic Reticulum Function to Modulate Dopamine Transporter Trafficking

Synaptic re-uptake of dopamine is dependent on the dopamine transporter (DAT), which is regulated by its distribution to the cell surface. DAT trafficking is modulated by the Parkinson''s disease-linked protein alpha-synuclein, but the contribution of synuclein family members beta-synuclein and gamma-synuclein to DAT trafficking is not known. Here we use SH-SY5Y cells as a model of DAT trafficking to demonstrate that all three synucleins negatively regulate cell surface distribution of DAT. Under these conditions the synucleins limit export of DAT from the endoplasmic reticulum (ER) by impairment of the ER-Golgi transition, leading to accumulation of DAT in this compartment. This mechanism for regulating DAT export indirectly through effects on ER and Golgi function represents a previously unappreciated role for the extended synuclein family that is likely applicable to trafficking of the many proteins that rely on the secretory pathway.     

Titanium Dioxide Nanoparticles Induce Endoplasmic Reticulum Stress-Mediated Autophagic Cell Death via Mitochondria-Associated Endoplasmic Reticulum Membrane Disruption in Normal Lung Cells

Nanomaterials are used in diverse fields including food, cosmetic, and medical industries. Titanium dioxide nanoparticles (TiO₂-NP) are widely used, but their effects on biological systems and mechanism of toxicity have not been elucidated fully. Here, we report the toxicological mechanism of TiO₂-NP in cell organelles. Human bronchial epithelial cells (16HBE14o-) were exposed to 50 and 100 μg/mL TiO₂-NP for 24 and 48 h. Our results showed that TiO₂-NP induced endoplasmic reticulum (ER) stress in the cells and disrupted the mitochondria-associated endoplasmic reticulum membranes (MAMs) and calcium ion balance, thereby increasing autophagy. In contrast, an inhibitor of ER stress, tauroursodeoxycholic acid (TUDCA), mitigated the cellular toxic response, suggesting that TiO₂-NP promoted toxicity via ER stress. This novel mechanism of TiO₂-NP toxicity in human bronchial epithelial cells suggests that further exhaustive research on the harmful effects of these nanoparticles in relevant organisms is needed for their safe application.     

Endoplasmic Reticulum Targeting and Insertion of Tail-Anchored Membrane Proteins by the GET Pathway

Hundreds of eukaryotic membrane proteins are anchored to membranes by a single transmembrane domain at their carboxyl terminus. Many of these tail-anchored (TA) proteins are posttranslationally targeted to the endoplasmic reticulum (ER) membrane for insertion by the guided-entry of TA protein insertion (GET) pathway. In recent years, most of the components of this conserved pathway have been biochemically and structurally characterized. Get3 is the pathway-targeting factor that uses nucleotide-linked conformational changes to mediate the delivery of TA proteins between the GET pretargeting machinery in the cytosol and the transmembrane pathway components in the ER. Here we focus on the mechanism of the yeast GET pathway and make a speculative analogy between its membrane insertion step and the ATPase-driven cycle of ABC transporters.The mechanism of membrane protein insertion into the endoplasmic reticulum (ER) has been extensively studied for many years (Shao and Hegde 2011). From this work, the signal recognition particle (SRP)/Sec61 pathway has emerged as a textbook example of a cotranslational membrane insertion mechanism (Grudnik et al. 2009). The SRP binds a hydrophobic segment (either a cleavable amino-terminal signal sequence or a transmembrane domain) immediately after it emerges from the ribosomal exit tunnel. This results in a translational pause that persists until SRP engages its receptor in the ER and delivers the ribosome-nascent chain complex to the Sec61 channel. Last, the Sec61 channel enables protein translocation into the ER lumen along with partitioning of hydrophobic transmembrane domains into the lipid bilayer through the Sec61 lateral gate (Rapoport 2007).Approximately 5% of all eukaryotic membrane proteins have an ER targeting signal in a single carboxy-terminal transmembrane domain that emerges from the ribosome exit tunnel following completion of protein synthesis and is not recognized by the SRP (Stefanovic and Hegde 2007). Nonetheless, because hydrophobic peptides in the cytoplasm are prone to aggregation and subject to degradation by quality control systems (Hessa et al. 2011), these tail-anchored (TA) proteins still have to be specifically recognized, shielded from the aqueous environment, and guided to the ER membrane for insertion. In the past five years, the guided-entry of TA proteins (GET) pathway has come to prominence as the major machinery for performing these tasks and the enabler of many key cellular processes mediated by TA proteins including vesicle fusion, membrane protein insertion, and apoptosis. This research has rapidly yielded biochemical and structural insights (​and2)2) into many of the GET pathway components (Hegde and Keenan 2011; Chartron et al. 2012a; Denic 2012). In particular, Get3 is an ATPase that uses metabolic energy to bridge recognition of TA proteins by upstream pathway components with TA protein recruitment to the ER for membrane insertion. However, the precise mechanisms of nucleotide-dependent TA protein binding to Get3 and how the GET pathway inserts tail anchors into the membrane are still poorly understood. Here, we provide an overview of the budding yeast GET pathway with emphasis on mechanistic insights that have come from structural studies of its membrane-associated steps and make a speculative juxtaposition with the ABC transporter mechanism.

Table 1.

A catalog of GET pathway component structures

Reticulomics: Protein-Protein Interaction Studies with Two Plasmodesmata-Localized Reticulon Family Proteins Identify Binding Partners Enriched at Plasmodesmata,Endoplasmic Reticulum,and the Plasma Membrane

The endoplasmic reticulum (ER) is a ubiquitous organelle that plays roles in secretory protein production, folding, quality control, and lipid biosynthesis. The cortical ER in plants is pleomorphic and structured as a tubular network capable of morphing into flat cisternae, mainly at three-way junctions, and back to tubules. Plant reticulon family proteins (RTNLB) tubulate the ER by dimerization and oligomerization, creating localized ER membrane tensions that result in membrane curvature. Some RTNLB ER-shaping proteins are present in the plasmodesmata (PD) proteome and may contribute to the formation of the desmotubule, the axial ER-derived structure that traverses primary PD. Here, we investigate the binding partners of two PD-resident reticulon proteins, RTNLB3 and RTNLB6, that are located in primary PD at cytokinesis in tobacco (Nicotiana tabacum). Coimmunoprecipitation of green fluorescent protein-tagged RTNLB3 and RTNLB6 followed by mass spectrometry detected a high percentage of known PD-localized proteins as well as plasma membrane proteins with putative membrane-anchoring roles. Förster resonance energy transfer by fluorescence lifetime imaging microscopy assays revealed a highly significant interaction of the detected PD proteins with the bait RTNLB proteins. Our data suggest that RTNLB proteins, in addition to a role in ER modeling, may play important roles in linking the cortical ER to the plasma membrane.The endoplasmic reticulum (ER) is a multifunctional organelle (Hawes et al., 2015) and is the site of secretory protein production, folding, and quality control (Brandizzi et al., 2003) and lipid biosynthesis (Wallis and Browse, 2010), but it is also involved in many other aspects of day-to-day plant life, including auxin regulation (Friml and Jones, 2010) and oil and protein body formation (Huang, 1996; Herman, 2008). The cortical ER network displays a remarkable polygonal arrangement of motile tubules that are capable of morphing into small cisternae, mainly at the three-way junctions of the ER network (Sparkes et al., 2009). The cortical ER network of plants has been shown to play multiple roles in protein trafficking (Palade, 1975; Vitale and Denecke, 1999) and pathogen responses (for review, see Pattison and Amtmann, 2009; Beck et al., 2012).In plants, the protein family of reticulons (RTNLBs) contributes significantly to tubulation of the ER (Tolley et al., 2008, 2010; Chen et al., 2012). RTNLBs are integral ER membrane proteins that feature a C-terminal reticulon homology domain (RHD) that contains two major hydrophobic regions. These regions form two V-shaped transmembrane wedges joined together via a cytosolic loop, with the C and N termini of the protein facing the cytosol. RTNLBs can dimerize or oligomerize, creating localized tensions in the ER membrane, inducing varying degrees of membrane curvature (Sparkes et al., 2010). Hence, RTNLBs are considered to be essential in maintaining the tubular ER network.The ability of RTNLBs to constrict membranes is of interest in the context of cell plate development and the formation of primary plasmodesmata (PD; Knox et al., 2015). PD formation involves extensive remodeling of the cortical ER into tightly furled tubules to form the desmotubules, axial structures that run through the PD pore (Overall and Blackman, 1996; Ehlers and Kollmann, 2001). At only 15 nm in diameter, the desmotubule is one of the most constricted membrane structures found in nature, with no animal counterparts (Tilsner et al., 2011). PD are membrane-rich structures characterized by a close association of the plasma membrane (PM) with the ER. The forces that model the ER into desmotubules, however, are poorly understood. RTNLBs are excellent candidates for this process and can constrict fluorescent protein-labeled ER membranes into extremely fine tubules (Sparkes et al., 2010). We showed recently that two of the RTNLBs present in the PD proteome, RTNLB3 and RTNLB6 (Fernandez-Calvino et al., 2011), are present in primary PD at cytokinesis (Knox et al., 2015). However, nothing is known of the proteins that interact with RTNLBs identified in the PD proteome or that may link RTNLBs to the PM. To date, the only protein shown to bind to plant RTNLBs is RHD3-LIKE2, the plant homolog of the ER tubule fusion protein ATLASTIN (Lee et al., 2013).Here, we used a dual approach to identify interacting partners of RTNLB3 and RTNLB6 (Fernandez-Calvino et al., 2011; Knox et al.., 2015). First, we used GFP immunoprecipitation assays coupled to mass spectrometry (MS) to identify proteins potentially binding to RTNLB3 and RTNLB6. Second, from the proteins we identified, we conducted a detailed Förster resonance energy transfer by fluorescence lifetime imaging microscopy (FRET-FLIM) analysis to confirm prey-bait interactions in vivo.The application of time-resolved fluorescence spectroscopy to imaging biological systems has allowed the design and implementation of fluorescence lifetime imaging microscopy (FLIM). The technique allows measuring and determining the space map of picosecond fluorescence decay at each pixel of the image through confocal single and multiphoton excitation. The general fluorescence or Förster resonance energy transfer (FRET) to determine the colocalization of two color chromophores can now be improved to determine physical interactions using FRET-FLIM and protein pairs tagged with appropriate GFP fluorophores and monomeric red fluorescent protein (mRFP). FRET-FLIM measures the reduction in the excited-state lifetime of GFP (donor) fluorescence in the presence of an acceptor fluorophore (e.g. mRFP) that is independent of the problems associated with steady-state intensity measurements. The observation of such a reduction is an indication that the two proteins are within a distance of 1 to 10 nm, thus indicating a direct physical interaction between the two protein fusions (Osterrieder et al., 2009; Sparkes et al., 2010; Schoberer and Botchway, 2014). It was shown previously that a reduction of as little as approximately 200 ps in the excited-state lifetime of the GFP-labeled protein represents quenching through a protein-protein interaction (Stubbs et al., 2005).Our interaction data identified a large percentage (40%) of ER proteins, including other RTNLB family members. However, we also found a relatively large number (25%) of proteins present in the published PD proteome (Fernandez-Calvino et al., 2011) and a surprisingly high proportion (35%) of PM proteins. Of the PD-resident proteins we identified, a significant number were shown previously to be targets of viral movement proteins (MPs) or proteins present within lipid rafts, consistent with the view that PD are lipid-rich microdomains (Bayer et al., 2014). Additional proteins identified suggested roles for RTNLBs in transport and pathogen defense. We suggest that RTNLBs may play key roles in anchoring and/or signaling between the cortical ER and PM.     

Subcellular Distribution of Homer 1b/c in Relation to Endoplasmic Reticulum and Plasma Membrane Proteins in Purkinje Neurons

The subcellular distribution of endoplasmic reticulum proteins (IP₃R1 and RYR), plasma membrane(PM) proteins (mGluR1 and PMCA Ca²⁺-pump), and scaffolding proteins, such as Homer 1b/c, was assessed by laser scanning confocal microscopy of rat cerebellum parasagittal sections. There appeared to be two classes of Ca²⁺ stores, nonjunctional Ca²⁺ stores and junctional Ca²⁺ stores, possibly referable to central cisternae/tubules and sub-PM cisternae, respectively, in soma, dendrites, and dendritic spines. Only some IP₃R1s appeared to be part of multimeric, junctional Ca²⁺ signaling networks, whose composition is shown to include PMCA, mGluR1, Homer 1b/c and, not always, RYR1.     

The Endoplasmic Reticulum Provides the Membrane Platform for Biogenesis of the Flavivirus Replication Complex

The cytoplasmic replication of positive-sense RNA viruses is associated with a dramatic rearrangement of host cellular membranes. These virus-induced changes result in the induction of vesicular structures that envelop the virus replication complex (RC). In this study, we have extended our previous observations on the intracellular location of West Nile virus strain Kunjin virus (WNV_KUN) to show that the virus-induced recruitment of host proteins and membrane appears to occur at a pre-Golgi step. To visualize the WNV_KUN replication complex, we performed three-dimensional (3D) modeling on tomograms from WNV_KUN replicon-transfected cells. These analyses have provided a 3D representation of the replication complex, revealing the open access of the replication complex with the cytoplasm and the fluidity of the complex to the rough endoplasmic reticulum. In addition, we provide data that indicate that a majority of the viral RNA species housed within the RC is in a double-stranded RNA (dsRNA) form.West Nile virus (WNV) belongs to the Flaviviridae, which is a large family of enveloped, positive-strand RNA viral pathogens that are responsible for causing severe disease and mortality in humans and animals each year. The Australian WNV strain Kunjin virus (WNV_KUN) is a relatively low-pathogenic virus that is closely related to the pathogenic WNV strain New York 99 (WNV_NY99), the causative agent of the 1999 epidemic of encephalitis in New York City (11).It has become increasingly known that the replication of most, if not all, positive-sense RNA viruses, whether they infect plants, insects, or humans, is associated with dramatic membrane alterations resulting in the formation of membranous microenvironments that facilitate efficient virus replication. In most cases the induced membrane structures house the actively replicating viral RNA and comprise 70- to 100-nm membrane “vesicles” (sometimes referred to as spherules). Although this distinct morphology is shared across virus families, the cellular origins of these membranes is diverse: the endoplasmic reticulum (ER), mitochondria, peroxisomes, and trans-Golgi membranes have been implicated in different viral systems (1, 8, 13, 23, 31, 38, 41, 45). This diversity implies that the processes involved in inducing the membrane vesicles/spherules are shared, rather than the composition of the membrane itself, although the exact purpose for utilizing membranes derived from different cellular compartments is still not completely resolved or understood.The replication of the flavivirus WNV_KUN is associated with the induction of morphologically distinct membrane structures that have defined roles during the WNV_KUN replication cycle. Three well-defined structures can be seen as large convoluted membranes (CM), paracrystalline arrays (PC), or membrane sacs containing small vesicles, termed vesicle packets (VP) (18, 20, 48). Based on localization studies with viral proteins of specific functions, we observed that components of the virus protease complex (namely, nonstructural protein 3 [NS3] with cofactor NS2B) localize specifically to the CM/PC, whereas viral double-stranded RNA (dsRNA) and the viral RNA-dependent RNA polymerase (RdRp) NS5 localized primarily to VP (20-22, 47, 48). Additionally, we observed that the CM and PC originate from and are modified membranes of the intermediate compartment (IC) and rough endoplasmic reticulum (RER), whereas the VP appear to be derived from trans-Golgi network (TGN) membranes (19). Recently, we have found that the WNV_KUN NS4A protein by itself has the intrinsic capacity to induce the CM and PC structures (35), a property also subsequently shown for Dengue virus (DENV) NS4A (29). Additionally, we have shown that upon WNV infection cellular cholesterol and cholesterol-synthesizing proteins are redistributed to the virus-induced membranes and that this redistribution severely disrupted the formation of cholesterol-rich microdomains (23). Furthermore, we have shown that the membranous structures induced during WNV replication provide partial protection of the WNV replication components from the interferon (IFN)-induced antiviral MxA protein, suggesting that the distinct compartmentalization of viral replication and components of the cellular antiviral response may be an evolutionary mechanism by which flaviviruses can protect themselves from host surveillance (6).In this study we focused on three-dimensional (3D) modeling to give insight into the 3D structure of the VP and provide evidence of how these complexes are organized and formed within the RER membrane. These results add valuable information to our understanding of how the WNV replication complex (RC) functions.     

Rate of Retrograde Transport of Cholera Toxin from the Plasma Membrane to the Golgi Apparatus and Endoplasmic Reticulum Decreases During Neuronal Development

Abstract: Various glycolipid-binding toxins are internalized from the cell surface to the Golgi apparatus. Prominent among these is cholera toxin (CT), which consists of a pentameric B subunit that binds to ganglioside GM1 and an A subunit that mediates toxicity. We now demonstrate that rhodamine (Rh)-CT can be further internalized from the Golgi apparatus to the endoplasmic reticulum (ER) in cultured hippocampal neurons and in neuroblastoma N18TG-2 cells and that the A subunit is essential for retrograde transport to the ER. In addition, the rate of internalization of Rh-CT to the Golgi apparatus and ER decreases dramatically as hippocampal neurons mature. The Golgi apparatus was labeled in almost all 1-day-old neurons after <1 h of incubation with Rh-CT but was labeled in <10% of 14-day-old neurons after 1 h. During the first 14 days in culture, there was a 15-fold increase in the number of ¹²⁵I-CT-binding sites per cell, indicating that the decrease in the rate of internalization of Rh-CT is not due to reduced levels of cell surface GM1 in older neurons. These results imply that the rate of retrograde transport of CT from the plasma membrane to the Golgi apparatus and ER is regulated during neuronal development and differentiation.     

Environmental Transition of Signal-Anchor Sequences during Membrane Insertion via the Endoplasmic Reticulum Translocon

In biogenesis of membrane proteins on the endoplasmic reticulum, a protein-conducting channel called the translocon functions in both the membrane translocation of lumenal domains and the integration of transmembrane segments. Here we analyzed the environments of polypeptide chains during the processes by water-dependent alkylation of N-ethylmaleimide at site-directed Cys residues. Using the technique, the region embedded in the hydrophobic portion of the membrane within a signal-anchor sequence and its shortening by insertion of a Pro residue could be detected. When translocation of the N-terminal domain of the signal-anchor was arrested by trapping an N-terminally fused affinity tag sequence, the signal-anchor was susceptible to alkylation, indicating that its migration into the hydrophobic environment was also arrested. Furthermore, when the tag sequence was separated from the signal-anchor by insertion of a hydrophilic sequence, the signal-anchor became inaccessible to alkylation even in the N-terminally trapped state. This suggests that membrane integration of the signal-anchor synchronizes with partial translocation of its N-terminal domain. Additionally, in an integration intermediate of a membrane protein, both of the two translocation-arrested hydrophilic chains were in an aqueous environment flanking the translocon, suggesting that the translocon provides the hydrophilic pathway capable of at least two translocating chains.     

Detection of Transient In Vivo Interactions between Substrate and Transporter during Protein Translocation into the Endoplasmic Reticulum

The split-ubiquitin technique was used to detect transient protein interactions in living cells. N_ub, the N-terminal half of ubiquitin (Ub), was fused to Sec62p, a component of the protein translocation machinery in the endoplasmic reticulum of Saccharomyces cerevisiae. C_ub, the C-terminal half of Ub, was fused to the C terminus of a signal sequence. The reconstitution of a quasi-native Ub structure from the two halves of Ub, and the resulting cleavage by Ub-specific proteases at the C terminus of C_ub, serve as a gauge of proximity between the two test proteins linked to N_ub and C_ub. Using this assay, we show that Sec62p is spatially close to the signal sequence of the prepro-α-factor in vivo. This proximity is confined to the nascent polypeptide chain immediately following the signal sequence. In addition, the extent of proximity depends on the nature of the signal sequence. C_ub fusions that bore the signal sequence of invertase resulted in a much lower Ub reconstitution with N_ub-Sec62p than otherwise identical test proteins bearing the signal sequence of prepro-α-factor. An inactive derivative of Sec62p failed to interact with signal sequences in this assay. These in vivo findings are consistent with Sec62p being part of a signal sequence-binding complex.     

A Non-enveloped Virus Hijacks Host Disaggregation Machinery to Translocate across the Endoplasmic Reticulum Membrane

Mammalian cytosolic Hsp110 family, in concert with the Hsc70:J-protein complex, functions as a disaggregation machinery to rectify protein misfolding problems. Here we uncover a novel role of this machinery in driving membrane translocation during viral entry. The non-enveloped virus SV40 penetrates the endoplasmic reticulum (ER) membrane to reach the cytosol, a critical infection step. Combining biochemical, cell-based, and imaging approaches, we find that the Hsp110 family member Hsp105 associates with the ER membrane J-protein B14. Here Hsp105 cooperates with Hsc70 and extracts the membrane-penetrating SV40 into the cytosol, potentially by disassembling the membrane-embedded virus. Hence the energy provided by the Hsc70-dependent Hsp105 disaggregation machinery can be harnessed to catalyze a membrane translocation event.