Visualization of cargo concentration by COPII minimal machinery in a planar lipid membrane
2009; Springer Nature; Volume: 28; Issue: 21 Linguagem: Inglês
10.1038/emboj.2009.269
ISSN1460-2075
AutoresKazuhito V. Tabata, Ken Sato, Toru Ide, Takayuki Nishizaka, Akihiko Nakano, Hiroyuki Noji,
Tópico(s)Lipid metabolism and biosynthesis
ResumoArticle17 September 2009free access Visualization of cargo concentration by COPII minimal machinery in a planar lipid membrane Kazuhito V Tabata Kazuhito V Tabata Department of Biomolecular Energetics, The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, Japan Search for more papers by this author Ken Sato Corresponding Author Ken Sato Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Meguro-ku, Tokyo, Japan Search for more papers by this author Toru Ide Toru Ide Network Center for Molecular and System Life Sciences, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan Search for more papers by this author Takayuki Nishizaka Takayuki Nishizaka Department of Physics, Gakushuin University, Toshima-ku, Tokyo, Japan Search for more papers by this author Akihiko Nakano Akihiko Nakano Molecular Membrane Biology Laboratory, RIKEN Advanced Science Institute, Wako, Saitama, Japan Department of Biological Sciences, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Hiroyuki Noji Hiroyuki Noji Department of Biomolecular Energetics, The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, Japan Search for more papers by this author Kazuhito V Tabata Kazuhito V Tabata Department of Biomolecular Energetics, The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, Japan Search for more papers by this author Ken Sato Corresponding Author Ken Sato Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Meguro-ku, Tokyo, Japan Search for more papers by this author Toru Ide Toru Ide Network Center for Molecular and System Life Sciences, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan Search for more papers by this author Takayuki Nishizaka Takayuki Nishizaka Department of Physics, Gakushuin University, Toshima-ku, Tokyo, Japan Search for more papers by this author Akihiko Nakano Akihiko Nakano Molecular Membrane Biology Laboratory, RIKEN Advanced Science Institute, Wako, Saitama, Japan Department of Biological Sciences, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Hiroyuki Noji Hiroyuki Noji Department of Biomolecular Energetics, The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, Japan Search for more papers by this author Author Information Kazuhito V Tabata1, Ken Sato 2, Toru Ide3, Takayuki Nishizaka4, Akihiko Nakano5,6 and Hiroyuki Noji1 1Department of Biomolecular Energetics, The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, Japan 2Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Meguro-ku, Tokyo, Japan 3Network Center for Molecular and System Life Sciences, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan 4Department of Physics, Gakushuin University, Toshima-ku, Tokyo, Japan 5Molecular Membrane Biology Laboratory, RIKEN Advanced Science Institute, Wako, Saitama, Japan 6Department of Biological Sciences, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo, Japan *Corresponding author. Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan. Tel.: +81 3 5454 6749; Fax: +81 3 5454 6730; E-mail: [email protected] The EMBO Journal (2009)28:3279-3289https://doi.org/10.1038/emboj.2009.269 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Selective protein export from the endoplasmic reticulum is mediated by COPII vesicles. Here, we investigated the dynamics of fluorescently labelled cargo and non-cargo proteins during COPII vesicle formation using single-molecule microscopy combined with an artificial planar lipid bilayer. Single-molecule analysis showed that the Sar1p–Sec23/24p-cargo complex, but not the Sar1p–Sec23/24p complex, undergoes partial dimerization before Sec13/31p recruitment. On addition of a complete COPII mixture, cargo molecules start to assemble into fluorescent spots and clusters followed by vesicle release from the planar membrane. We show that continuous GTPase cycles of Sar1p facilitate cargo concentration into COPII vesicle buds, and at the same time, non-cargo proteins are excluded from cargo clusters. We propose that the minimal set of COPII components is required not only to concentrate cargo molecules, but also to mediate exclusion of non-cargo proteins from the COPII vesicles. Introduction Intracellular protein transport between the organelles of the secretory pathway is mediated by transport vesicles, which bud from a donor organelle and fuse with an appropriate acceptor membrane of a different compartment (Bonifacino and Glick, 2004). The formation of transport vesicles and sorting of cargo molecules into the emerging vesicles are mediated by coat protein complexes associated with the cytoplasmic face of the organelles (Kirchhausen, 2000; Bonifacino and Lippincott-Schwartz, 2003). A common feature of transport vesicles is that most of them use small GTPases to regulate coat assembly at the donor membranes. The COPII coat is responsible for direct capture of transmembrane cargo proteins and for the physical deformation of the endoplasmic reticulum (ER) membrane that drives the formation of COPII vesicles in anterograde transport from the ER to the Golgi (Lee et al, 2004; Sato and Nakano, 2007). COPII coat consists of the small GTPase Sar1p (Nakano and Muramatsu, 1989), the Sec23/24p complex, and the Sec13/31p complex that sequentially bind to the ER membrane (Barlowe et al, 1994). COPII vesicle formation is initiated by GDP–GTP exchange on Sar1p catalysed by the transmembrane guanine nucleotide exchange factor Sec12p (Barlowe and Schekman, 1993). GTP binding triggers the exposure of the N-terminal amphipathic α-helix element of Sar1p that inserts into the ER membrane (Huang et al, 2001; Bi et al, 2002). Membrane-bound Sar1p–GTP recruits the Sec23/24p complex by binding to the Sec23p portion (Bi et al, 2002), and the cytoplasmically exposed signal of the transmembrane cargo is captured by direct contact with Sec24p (Miller et al, 2003; Mossessova et al, 2003) to form a prebudding complex (Kuehn et al, 1998). The Sec23p subunit of the Sec23/24p complex is the GTPase-activating protein for Sar1p (Yoshihisa et al, 1993) and therefore stimulates Sar1p GTP hydrolysis on binding to Sar1p, which leads to disassembly of the prebudding complex (Antonny et al, 2001). However, the association of Sec23/24p on membranes can be maintained by interactions with cargo proteins and continual GTP loading of Sar1p by Sec12p (Futai et al, 2004; Sato and Nakano, 2005). Subsequently, the prebudding complex is thought to be polymerized by the Sec13/31p complex to generate COPII vesicles. At the final stage of vesicle formation, the neck of a COPII vesicle bud is not likely to break spontaneously and the N-terminal amphipathic helix of Sar1p seems to have an active role in membrane fission (Bielli et al, 2005; Lee et al, 2005). The molecular mechanisms of cargo sorting and transport carrier formation have been analysed in great detail mostly through in vitro reconstitution assays. COPII vesicles have been used as a model system to study the molecular mechanism of transport vesicle biogenesis as they can be generated from isolated organelles (Barlowe et al, 1994), synthetic liposomes (Matsuoka et al, 1998), and cargo-reconstituted proteoliposomes (Futai et al, 2004; Sato and Nakano, 2004). However, those earlier in vitro vesicle budding assays were unable to track cargo molecules in the spatially and temporally resolved manner required to determine the number and density of cargo molecules encapsulated in each vesicle. As we reported earlier, a horizontal planar lipid bilayer reconstituted with transmembrane proteins can be easily and reproducibly formed across a small hole in a partition between two aqueous compartments (Ide and Yanagida, 1999; Ide et al, 2002). We reconstituted fluorescently labelled transmembrane cargo protein into the planar lipid bilayer together with Sec12p, and visualized the spatiotemporal dynamics of cargo molecules induced by the minimum machinery required for COPII vesicle formation using a fluorescent microscope designed for single-molecule detection (Figure 1A). This system enables us to quantitatively resolve the assembly behaviour of cargo molecules in each step of the biochemically defined subreactions of COPII vesicle formation. Figure 1.Imaging of Bet1p-Cy3 molecules reconstituted in a planar lipid bilayer. (A) Overview of the horizontal planar lipid bilayer formation apparatus. The apparatus consists of two chambers. On the bottom of the upper chamber, a thin plastic film with a small hole (∼100 μm) was attached and an artificial planar lipid bilayer was formed horizontally across the hole on an agarose-coated coverslip. The upper chamber could be moved vertically using a micromanipulator. (B) A typical fluorescent spot of Bet1p-Cy3 in the bilayer membrane was photobleached in a single step (red) to the background level (blue). (C–G) Distribution of the fluorescence intensity of Bet1p-Cy3 in the presence of the COPII component(s). To the bilayer membranes reconstituted with Bet1p-Cy3 (0.28 molecules μm−2) and Sec12Δlum (C) was added Sar1p (70 ng) (D), Sar1p (70 ng) and Sec23/24p (410 ng) (E), or Sar1p (70 ng), Sec23/24p (410 ng), and Sec13/31p (1.4 μg) (F) in molar excess of Bet1p-Cy3 in the presence of GTP, except for experiments in (G), in which GDP was added in place of GTP. Histograms of the fluorescence intensity were fit to either single or double Gaussian distributions (lines) and the percentages of fractions of each component are indicated (numbers). Download figure Download PowerPoint Results The behaviour of Bet1p molecules in the early stages of COPII vesicle formation We chose yeast ER–Golgi v-SNARE Bet1p as a model cargo for this study. As the wild-type yeast Bet1p has no cysteine, a cysteine addition to the C-terminal end allows Cy3-maleimide to be specifically attached so that a single Bet1p molecule possesses a single molecule of Cy3 (Bet1p-Cy3). Bet1p-Cy3 was reconstituted in a planar lipid bilayer with a major–minor mix formulation (Matsuoka et al, 1998) optimized for COPII assembly. Approximately 90% of the N-terminal elements of the reconstituted Bet1p-Cy3 were oriented towards the upper surface of the bilayer membrane (Sato and Nakano, 2005). When Bet1p-Cy3 was visualized with evanescent field illumination, each dot moved in the membrane laterally with a diffusion coefficient (D) of D=4.5±2.0 μm2 s−1, indicating that Bet1p-Cy3 molecules were able to move freely within the membrane, even though the membrane was in contact with the agarose layer on the coverslip. We examined the fluorescent intensity and photobleaching characteristics of every dot to clarify whether the single fluorescent dots in the membrane represented a single Bet1p molecule. Time-dependent changes in fluorescent intensity for individual dots in the membrane showed single-step photobleaching, which is characteristic of single fluorophores (Figure 1B). The distribution of the fluorescent intensities of the dots fit well with a single Gaussian distribution with a peak intensity similar to that of the step size for photobleaching (Figure 1C). These results indicated that Bet1p-Cy3 alone existed as a monomer in the bilayer membrane. We next examined the behaviour of Bet1p molecules in the presence of a subset of COPII components. The binding of Sar1p to Bet1p has been shown to precede recruitment of Sec23/24p (Springer and Schekman, 1998). We reconstituted both Bet1p-Cy3 and Sec12Δlum (Sec12p without its luminal domain, but containing the transmembrane region) (Sato and Nakano, 2005) in the bilayer membrane and added Sar1p and Sec23/24p in molar excess of Bet1p-Cy3 to the membrane in the presence of GTP. The diffusion coefficient of Bet1p-Cy3 was decreased by the addition of Sar1p (D=2.6±1.4 μm2 s−1) or by both Sar1p and Sec23/24p (D=2.8±1.8 μm2 s−1), most likely as a result of the association of Bet1p-Cy3 with Sar1p and Sec23/24p. The distribution of the fluorescence intensity of Bet1p-Cy3 in the presence of Sar1p-GTP showed a single peak, which fit well with a single Gaussian distribution (Figure 1D), and the fluorescence intensity from each Bet1p-Cy3 decayed in a single step (data not shown), suggesting that the Bet1p molecules bound to Sar1p existed as monomers. In contrast, the distribution of the fluorescence intensity of Bet1p-Cy3 in the presence of both Sar1p–GTP and Sec23/24p was fitted to a sum of two Gaussian distribution functions (Figure 1E). The fluorescent dots in the first component showed single-step-bleaching characteristics, suggesting that the first component contained single Bet1p-Cy3 molecules (data not shown). The second component (15.8% of the fraction), which emitted a signal twice as intense as the first component, should represent dots containing two Bet1p-Cy3 molecules. This component most likely represents self-dimerization of the prebudding complexes rather than two Bet1p-Cy3 molecules captured within a single Sec23/24p coat, because Sec23/24p has been shown to be associated with a single Sar1p and Bet1p (Bi et al, 2002; Mossessova et al, 2003). We also visualized prebudding complexes labelled with Sar1p-Cy3 (Supplementary Figure S1). A portion of Sar1p-Cy3 dots also shifted from the monomeric region to the dimeric region on the addition of Sec23/24p in the presence of nonlabelled Bet1p. In contrast, Sar1p-Cy3 that associated with Sec23/24p in the absence of Bet1p remained a monomer. These results suggest that partial dimerization occurs only between prebudding complexes but not within Sar1p–Sec23/24p complexes. Visualization of cargo recruitment into forming COPII vesicles and vesicle release from the membrane We next traced Bet1p-Cy3 molecules in the presence of the complete set of COPII components to visualize cargo recruitment into forming COPII vesicles followed by release of the vesicles from the planar bilayer membrane. Bet1p-Cy3 accumulation into nascent COPII vesicles should be observed as a formation of spots with increased fluorescence intensity. In addition, as the evanescent field illumination images only those fluorescently labelled molecules within ∼150 nm above the surface of the coverslip, the disappearance of these fluorescent spots should represent the release of COPII vesicles away from the membrane. On addition of COPII components, however, the intensity distribution of the resulting spots showed that most of these spots contained less than two molecules, whereas Bet1p-Cy3 remained as a monomer with GDP (Figures 1F and G; Supplementary Movie S1). We also found that the accumulation of such fluorescent spots in the presence of the complete COPII coat was also accompanied by a constraint in their lateral mobility. These results suggest that the Sec13/31p-driven prebudding complex assembly is extremely inefficient under this condition. One possible explanation for this is that the loss of lateral mobility of prebudding complexes, as observed in Supplementary Movie S1, could limit the potential for Sec13/31p-mediated clustering. Alternatively, the interaction between the bilayer membrane and the agarose layer on the coverslip limited spherical polymerization of the prebudding complexes. To overcome this problem, the upper chamber was placed upward from the coverslip, where the distance between the bilayer and the coverslip was about 10–20 μm, so as to position both sides of the membrane in an aqueous environment (Figure 2A). Under this condition, we acquired video images of the fluorescence from the membrane with epifluorescence illumination instead of evanescent field illumination. When COPII components were added onto the membrane, Bet1p-Cy3 was found to accumulate into mobile fluorescent spots with increased fluorescence intensity, followed by the formation of clusters in the membrane (Figure 2A; Supplementary Movie S2). The dynamics observed here confirmed the widely accepted hypothesis that the prebudding complexes are clustered by Sec13/31p. Figure 2.Clustering of Bet1p-Cy3 molecules and downward COPII vesicle budding with the bilayer membrane formed in an aqueous environment. (A) Sequential images of the Bet1p-Cy3 clustering in the bilayer membrane (Supplementary Movie S2 online). Sar1p (70 ng), Sec23/24p (410 ng), and Sec13/31p (1.4 μg) were added to the bilayer membrane reconstituted with Bet1p-Cy3 (95.6 molecules μm−2) and Sec12Δlum at t=0 s in the presence of GTP or GDP as illustrated in the upper panel. Epifluorescence images were taken with exposure times of 66 ms. (B) Schematic illustration of downward COPII vesicle budding. (C) Time course of the increase in the number of fluorescent particles that appeared on the coverslip after the addition of the COPII components. Each thin trace corresponds to an individual experiment. The numbers of fluorescent particles were normalized so that the initial number of fluorescent particles on the coverslip was 100%. Thick traces with standard error deviations represent the averages of all thin traces. Download figure Download PowerPoint To ensure that the bilayer membrane with this setting can support COPII vesicle generation, a complete set of COPII components was added to the lower chamber to induce vesicle budding in a downward direction towards the surface of the coverslip, and the fluorescence signals that appeared on the coverslip surface were detected using evanescent field illumination (Figure 2B). As only ∼10% of the N-terminal elements of the reconstituted Bet1p were oriented towards the coverslip, we used Bet1p-4Cy3 at a concentration 300-fold higher than that used in Figure 1 to increase the signal intensity for these experiments. After 30 min incubation, small fluorescent particles undergoing rapid Brownian motion were observed on the surface of the coverslip, whereas no signals were observed immediately after COPII addition (Supplementary Movie S3). These fluorescent particles may represent the COPII vesicles incorporated with Bet1p-4Cy3. By counting the number of fluorescent particles at different time points after the addition of COPII components, we obtained a time course of COPII vesicle production (Figure 2C). The particle number increased in proportion to the incubation time and reached a plateau after about 30 min of incubation. Fluorescent signals were infrequently observed in the presence of GDP, indicating specific accumulation of vesicle formation. We conclude that the COPII components can generate COPII vesicles from the bilayer membrane under this condition. Incorporation of Bet1p molecules into a COPII vesicle bud is saturable To quantify the number of Bet1p molecules in a COPII vesicle bud immediately before the vesicle pinch-off, we needed to arrest the vesicle release from the membrane. To achieve this, we used a thick planar membrane. The planar bilayers of physiological thickness used in Figures 1 and 2 were formed with lipids dissolved in squalene (8.4 nm thickness). As the molecular environment between the monolayers in a bilayer changes with the solvent, the thickness of the planar bilayer increases approximately two-fold when decane is substituted for squalene (13.6 nm thickness) (data not shown). Bet1p-Cy3 reconstituted in the thick membrane behaved similarly to that shown in Figures 1 and 2A; that is, partial dimerization of prebudding complexes (data not shown) followed by accumulation into fluorescent spots (Supplementary Movie S4) except for the vesicle pinch-off as observed in Figure 2C (data not shown). Thus, the COPII components can collect cargo molecules in the thick membrane, but cannot induce vesicle pinch-off, possibly because of the membrane thickness (Supplementary Figure S2). To determine the number of Bet1p-Cy3 molecules contained in each COPII bud, we first acquired fluorescent spots in the thick membrane by the addition of the COPII components, and then the upper chamber was positioned down so that the membrane was attached to the coverslip. This procedure restricted the diffusion movements of only the assembled Bet1p-Cy3 in the membrane, but not those of monomeric Bet1p-Cy3 and the prebudding complex. Owing to the interaction between the membrane and the agarose layer, visualization of only the assembled Bet1p-Cy3 as immobile fluorescent spots became possible. Inspection of the fluorescent image arising from the presence of complete COPII components and GTP showed that the small fluorescent spots were scattered among several large asymmetric aggregates (Figure 3A, upper panel). According to their size, these aggregates are likely to correspond to the fluorescent clusters observed in Supplementary Movies S2 and S4, whereas the small spots are not visible in the movies because of rapid lateral diffusion. The distribution of the small spots appears relatively evenly spaced, which is similar to the results of a computational model for the process of COPII assembly (Heinzer et al, 2008). We selected these small spots, and the distribution of fluorescence intensity was analysed under several different concentrations of Bet1p-Cy3 in the membrane (Figure 3B, left panel). In all cases, the fluorescence intensity showed a single maximum. When the mean value of the fluorescence intensity was plotted against the Bet1p-Cy3 concentration, the fluorescence intensity increased in proportion to the concentration of Bet1p-Cy3 in the membrane and reached a plateau (Figure 3C). These results indicate that these small spots consisted of uniformly assembled Bet1p-Cy3 and that the incorporation of Bet1p-Cy3 into the spots is saturable. The saturation number of Bet1p-Cy3 molecules per spot was estimated to be about 70 on the basis of fluorescent intensity. On the basis of recent structural data on the cuboctahedral or icosidodecahedral cage assembled from Sec13/31p (Stagg et al, 2006, 2008), and because Bet1p and Sec24p form a 1:1 complex (Mossessova et al, 2003), the theoretical maximum number of Bet1p molecules that can be captured by Sec23/24p into a single COPII vesicle is calculated to be 48 or 120. This value is close to the estimated saturation number of Bet1p-Cy3 in each spot. Therefore, it is likely that these small spots correspond to the structures of the COPII vesicle buds immediately before vesicle pinch-off. An important question is whether the large aggregates observed among the small spots were also active in vesicle generation, but it was not clear because of the difficulty in resolving the movement of individual COPII vesicles away from the membrane with our detection system. Figure 3.COPII-induced Bet1p-Cy3 assembly in the decane bilayer membrane. (A) COPII components (70 ng Sar1p, 410 ng Sec23/24p, and 1.4 μg Sec13/31p) were added from the upper chamber to the decane bilayer membrane formed in an aqueous environment and fluorescent spots were acquired (as shown in Supplementary Movie S4 online) in the presence of GTP (upper panel) or GMP-PNP (lower panel). The bilayer membrane was then attached to the coverslip. Fluorescent images were taken under evanescent field illumination. (B) Distribution of the fluorescence intensity of the small spots observed in (A) under different concentrations of Bet1p-Cy3 in the membrane. Insets show the same histograms plotted with a narrower bin size. (C) A correlation plot of the Bet1p-Cy3 concentration versus the mean fluorescent intensity of the fractions obtained from (B). Download figure Download PowerPoint Sar1p GTPase cycles are required for efficient cargo recruitment into COPII vesicle buds Although Sar1p GTP hydrolysis is dispensable for COPII vesicle formation (Barlowe et al, 1994), we have shown earlier that Sar1p GTPase cycles selectively dissociate Sec23/24p molecules that are bound to anionic phospholipids, but not those associated with cargo proteins, which promotes cargo concentration (Sato and Nakano, 2005). To directly observe the effect of GTP hydrolysis on cargo concentration into the nascent COPII vesicle, we also investigated Bet1p-Cy3 assembly in the presence of a nonhydrolysable GTP analog, GMP-PNP, in the same manner as described for the measurement with GTP (Figure 3A, lower panel and , right panel). On addition of COPII components in the presence of GMP-PNP, fluorescent spots as well as large aggregates were monitored. However, the fluorescence intensity of spots and aggregates was significantly lower than that observed with GTP, and the images were associated with high background fluorescence (Figure 3C). These results indicate that cargo recruitment into nascent vesicles is inefficient in the absence of Sar1p GTPase cycles, and that significant amounts of Bet1p-Cy3 molecules remain outside the spots and aggregates. Moreover, the sizes of the large aggregates induced by GMP-PNP were similar to those of GTP, but displayed a speckled fluorescent pattern. As Sec23/24p also binds to anionic phospholipids in the membrane, cargo is not a requisite for membrane recruitment for Sec23/24p (Matsuoka et al, 1998). Thus, the presence of GMP-PNP results in the generation of stable complexes of both the Sar1p–Sec23/24p-cargo and Sar1p–Sec23/24p form. Therefore, the observed speckled aggregates, most likely representing these two forms of prebudding complexes, are clustered by Sec13/31p. These findings confirm the idea that the GTPase cycles of Sar1p are required for efficient cargo concentration into COPII vesicles. COPII minimal machinery is able to exclude non-cargo proteins form cargo clusters It is now quite evident that most transmembrane cargo molecules are actively sorted into the COPII vesicles by direct interactions with the coat proteins, whereas transmembrane ER-resident proteins can be retained in the ER either by static retention or by continuous signal-mediated retrieval from post-ER compartments (Sato and Nakano, 2007). Although transmembrane ER-resident proteins that have accidentally escaped by bulk flow are retrieved by COPI-coated retrograde vesicles, many ER-resident proteins seem to avoid bulk flow by static retention mechanisms that exclude them from entering COPII vesicles (Bonifacino and Glick, 2004; Lee et al, 2004). However, no components have been so far identified that are required for static ER retention. To investigate whether the minimal set of COPII components can mediate exclusion of ER-resident proteins from COPII vesicles, we further studied the dynamics of fluorescently labelled ‘non-cargo’ proteins together with the dynamics of Bet1p-Cy3 during cargo assembly mediated by COPII components. For these experiments, we used ATTO647N-labelled MBP-Ufe1p (Sato and Nakano, 2004) (Ufe1p-ATTO647N), a model for resident ER proteins. Both Bet1p-Cy3 and Ufe1p-ATTO647N were reconstituted into the bilayer membrane together with Sec12Δlum and we analysed their mutual localization in the presence of the COPII components. Fluorescence signals representing Ufe1p-ATTO647N and Bet1p-Cy3 molecules excited at each wavelength are shown in Figure 4A. In a control experiment with GDP, Ufe1p-ATTO647N and Bet1p-Cy3 exhibited equal mixing of ATTO647N and Cy3 fluorescence, indicating that these molecules were randomly distributed relative to one another. However, when Ufe1p-ATTO647N molecules were visualized in the ATTO647N channel, the addition of the COPII components with GTP yielded large clusters observed as dark contrasting regions that were relatively devoid of fluorescent signals compared with the background (Figure 4A). These clusters are likely to represent COPII-mediated Bet1p-Cy3 clusters that are segregated from Ufe1p-ATTO647N because (1) as apparent from a merged image, such regions overlapped to a great extent with the distribution of Bet1p-Cy3 clusters observed in the Cy3 channel and (2) such dark contrasting regions were not observed in the absence of Bet1p (Figure 4A). Without exclusion mechanisms, transmembrane proteins without export signals should be neither enriched nor depleted in Bet1p clusters, but the density of Ufe1p-ATTO647N included in Bet1p clusters was significantly lower than that observed outside the clusters (68.0% of background fluorescence) (Figure 4B). Furthermore, the exclusion was less effective (83.0% of background fluorescence) in the presence of GMP-PNP. These results show that exclusion of non-cargo proteins from cargo clusters is at least partly mediated by the COPII minimal machinery, and that the process of exclusion requires Sar1p GTPase cycles. We further traced Cy3-labelled MBP-Ufe1p (Ufe1p-Cy3) co-reconstituted with nonlabelled Bet1p and Sec12Δlum in the presence of the COPII components, as shown in Supplementary Figure S3. The addition of the COPII components yielded large clusters observed as dark contrasting regions that were relatively low levels of fluorescent signals (Supplementary Figure S3A), as also observed in Figure 4A. These are likely to represent unlabelled Bet1p clusters that are segregated from Ufe1p-Cy3, because such regions were not observed in the absence of Bet1p (Supplementary Figure S3B). Figure 4.The mutual localization of Ufe1p-ATTO647N and Bet1p-Cy3 in the presence of the COPII components. (A) Sar1p (70 ng), Sec23/24p (410 ng), and Sec13/31p (1.4 μg) were added from the upper chamber to the decane bilayer membrane (formed in an aqueous environment) reconstituted with Ufe1
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