FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1987505890> ?p ?o ?g. }

• W1987505890 endingPage "2965" @default.
• W1987505890 startingPage "2953" @default.
• W1987505890 abstract "Article22 June 2006free access Sterols regulate ER-export dynamics of secretory cargo protein ts-O45-G Heiko Runz Corresponding Author Heiko Runz Cell Biology & Cell Biophysics Programme, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany Institute of Human Genetics, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Kota Miura Kota Miura Cell Biology & Cell Biophysics Programme, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany Search for more papers by this author Matthias Weiss Matthias Weiss Cellular Biophysics Group (BIOMS), German Cancer Research Center, Heidelberg, Germany Search for more papers by this author Rainer Pepperkok Corresponding Author Rainer Pepperkok Cell Biology & Cell Biophysics Programme, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany Search for more papers by this author Heiko Runz Corresponding Author Heiko Runz Cell Biology & Cell Biophysics Programme, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany Institute of Human Genetics, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Kota Miura Kota Miura Cell Biology & Cell Biophysics Programme, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany Search for more papers by this author Matthias Weiss Matthias Weiss Cellular Biophysics Group (BIOMS), German Cancer Research Center, Heidelberg, Germany Search for more papers by this author Rainer Pepperkok Corresponding Author Rainer Pepperkok Cell Biology & Cell Biophysics Programme, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany Search for more papers by this author Author Information Heiko Runz 1,2, Kota Miura1, Matthias Weiss3 and Rainer Pepperkok 1 1Cell Biology & Cell Biophysics Programme, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany 2Institute of Human Genetics, University of Heidelberg, Heidelberg, Germany 3Cellular Biophysics Group (BIOMS), German Cancer Research Center, Heidelberg, Germany *Corresponding authors: Cell Biology & Cell Biophysics Programme, European Molecular Biology Laboratory (EMBL), Meyerhofstr. 1, Heidelberg 69117, Germany. Tel.: +49 6221 387 332; Fax: +49 6221 387 306; E-mails: E-mail: [email protected] or [email protected] The EMBO Journal (2006)25:2953-2965https://doi.org/10.1038/sj.emboj.7601205 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Alterations in endoplasmic reticulum (ER) cholesterol are fundamental for a variety of cellular processes such as the regulation of lipid homeostasis or efficient protein degradation. We show that reduced levels of cellular sterols cause a delayed ER-to-Golgi transport of the secretory cargo membrane protein ts-O45-G and a relocation to the ER of an endogenous protein cycling between the ER and the Golgi complex. Transport inhibition is characterized by a delay in the accumulation of ts-O45-G in ER-exit sites (ERES) and correlates with a reduced mobility of ts-O45-G within ER membranes. A simple mathematical model describing the kinetics of ER-exit predicts that reduced cargo loading to ERES and not the reduced mobility of ts-O45-G accounts for the delayed ER-exit and arrival at the Golgi. Consistent with this, membrane turnover of the COPII component Sec23p is delayed in sterol-depleted cells. Altogether, our results demonstrate the importance of sterol levels in COPII mediated ER-export. Introduction Cholesterol is an important structural and functional component of cellular membranes in all animal cells. Already, minor disturbances of membrane cholesterol content result in severe changes in membrane physical properties, affecting a plethora of functions including intracellular signalling or transport events (Simons and Ikonen, 2000; Maxfield and Tabas, 2005). In contrast to other cellular membranes, cholesterol content of the endoplasmic reticulum (ER) has been estimated to be low, with cholesterol contributing not more than few percent of total membrane lipids (Colbeau et al, 1971; Lange et al, 1999). Nevertheless, the ER is a major sterol regulating organelle, harbouring the molecular machinery by which cellular cholesterol homeostasis is maintained. Smooth ER membranes are a source for lipid droplets as storage organelles of esterified cholesterol as well as the site of the enzyme HMG-CoA reductase, catalyzing the rate-limiting step in the biosynthesis of cholesterol and nonsterol isoprenoids. Reduction of the sterol pool in ER membranes triggers transport of the membrane protein SCAP and the transcription factor SREBP from the ER to the Golgi complex, where the latter one is proteolytically activated to its nuclear form, which initiates the expression of genes responsible for the synthesis and uptake of cholesterol and fatty acids (Goldstein et al, 2006). Apart from being a site for lipid synthesis and regulation, a pivotal function of the ER is its role in secretory protein trafficking. In mammalian cells, secretory cargo leaves the ER at distinct COPII coated ER-exit sites (ERES), which are stable for tens of minutes while the COPII components are rapidly turning over within seconds at these sites (Hammond and Glick, 2000; Stephens et al, 2000; Bevis et al, 2002; Soderholm et al, 2004; Forster et al, 2006). The vesicular coat complex COPII mediates cargo selection and concentration via an interaction of COPII components with cargo proteins or cargo receptors (Springer and Schekman, 1998; Aridor and Traub, 2002; Barlowe, 2003; Lee et al, 2005). The detailed molecular mechanisms of cargo accumulation in ERES as well as the mechanism how ERES are confined to the ER membrane remain to be fully understood. It has been suggested that in addition to interaction with proteins, interaction of COPII components with membrane lipids are critical for the formation of COPII coated transport carriers. The lipid composition of artificial liposomes has been demonstrated to be crucial for the efficient COPII vesicle formation in in vitro reconstitution experiments (Matsuoka et al, 1998) and treatment of semiintact cells with agents changing the lipid environment of the ER affect COPII vesicle formation and ER-export (Pathre et al, 2003). It is therefore tempting to speculate that because of its low prevalence, already slight alterations of cholesterol content in ER membranes might perturb protein export from the ER. To address this, we analyzed the ER-to-Golgi transport of YFP-tagged secretory marker protein ts-O45-G (Presley et al, 1997; Scales et al, 1997) in HeLa cells with different sterol levels. Our results show that ER-to-Golgi transport of ts-O45-G is significantly delayed in sterol-depleted cells. We further demonstrate by 4D time lapse microscopy that delayed arrival at the Golgi complex correlates with a marked delay in the accumulation of ts-O45-G in individual ERES. Consistent with this, the turnover kinetics of COPII components at ERES are reduced in sterol-depleted cells. Altogether, this suggests that a reduction in cellular sterol levels is associated with a reduced efficiency of COPII coated transport carrier formation at ERES. Results Exit of ts-O45-G-YFP from the ER is delayed in sterol-depleted cells Cellular sterols were depleted by exposing cells cultivated in lipoprotein-depleted serum (LDS) to 2-hydroxypropyl-β-cyclodextrin (HPCD). Several independent approaches have shown that this protocol allows efficient extraction of cholesterol from cultured cells (Kilsdonk et al, 1995b; Lange et al, 1999), thereby inducing a sterol-regulated transport of the SREBP cleavage activating protein SCAP from the ER to the Golgi complex where the proteolytic activation of SREBP is initiated (DeBose-Boyd et al, 1999; Nohturfft et al, 2000). In accordance with these data from the literature, our experimental conditions for sterol depletion induced the translocation of YFP-tagged SCAP from the ER to the Golgi complex (Supplementary Figure 1A) and initiated the proteolytic cleavage of SREBP-1 (Supplementary Figure 1B). Filipin staining demonstrated that under these conditions total cellular cholesterol levels were reduced to 62±0.2% of that in control cells (Supplementary Figure 1C). These data are consistent with previous work indicating that these treatments also drastically decrease the ER sterol levels (Lange et al, 1999). Sterol depletion was counteracted by addition of cholesterol and 25-hydroxycholesterol (25-HC) to the sterol-depleting culture medium (Kilsdonk et al, 1995a), a well-established procedure to potently suppress the activation of SREBP (Supplementary Figure 1A and B; see also DeBose-Boyd et al, 1999; Nohturfft et al, 2000) and to rapidly deliver sterols to cultured cells (Supplementary Figure 1C; see also Christian et al, 1997; Lange et al, 1999). Under our sterol-depleting conditions, the translocation rate of SCAP-YFP from the ER to the Golgi was maximal at about 1 h after adding HPCD to the sterol-depleting medium (Supplementary Figure 1A). We therefore chose this time point for the analysis of ts-O45-G ER-export in sterol-depleted cells. HeLa cells expressing ts-O45-G-YFP at 39.5°C were shifted to the permissive temperature of 32°C (Scales et al, 1997), and transport from the ER to the Golgi complex was followed over a time period of 20 min (Figure 1). Confocal imaging of ts-O45-G-YFP in cells fixed at different time points after the temperature shift revealed a prominent difference in ts-O45-G-YFP trafficking to the Golgi. In control cells, an increase in the concentration of ts-O45-G-YFP at ERES could be observed almost instantly (<2 min) after the shift to the permissive temperature (Figure 1A, insets, upper panel; see also Presley et al, 1997; Scales et al, 1997) and juxta-nuclear Golgi labelling became apparent for the first time at 8–10 min after the temperature shift (Figure 1A, upper panel). In contrast, accumulation of ts-O45-G-YFP in ERES was significantly delayed under sterol depleted conditions (Figure 1A, insets, middle panel), becoming apparent only between 6 and 10 min after shifting to 32°C. Also, a clear localization of a prominent fraction of ts-O45-G-YFP to the Golgi could be seen only after about 15 min (Figure 1A, middle panel). In cells that received cholesterol and 25-HC to the sterol-depleting medium, ts-O45-G-YFP localization to ERES and subsequent Golgi labelling appeared similar as in nontreated control cells (Figure 1A, lower panel), indicating that the observed delay in ER to Golgi transport in sterol-depleted cells was specifically caused by the lack of sterols. Figure 1.Export of ts-O45-G from the ER is delayed in sterol-depleted cells. (A) HeLa-cells expressing ts-O45-G-YFP at 39.5°C under control conditions (upper panel) and in the absence (LDS/HPCD; medium panel) or presence of sterols (LDS/HPCD/+sterols; lower panel) were shifted to 32°C for the indicated time points before fixation. Insets show z-projections of areas in the periphery of represented cells. Arrows denote examples for ERES, arrowheads juxta-nuclear Golgi-like staining. Bars: 10 μm. (B) Numbers of ERES positive for ts-O45-G-YFP (nERESts-O45-G-YFP) or sec23 (nERESsec23) were counted and nERES/μm2 were plotted against time. Each time point represents the mean from 20 to 40 areas±s.d. from 2–4 independent experiments under control conditions (▪, full line) or in the absence (○, full line) or presence (•, dashed line) of sterols. (C) ts-O45-G-YFP fluorescence intensities in the Golgi [(Its-O45-G-YFP(Golgi)] and total cell area [Its-O45-G-YFP(cell)] plotted against time. Each time point represents the mean from 10–25 cells±s.d. from 2 to 4 independent experiments. Download figure Download PowerPoint At all time points investigated, the relative amount of ts-O45-G-YFP retained in reticular ER membranes was significantly higher in sterol-depleted cells than in controls. Quantification of the experiments showed that the number of ERES positive for ts-O45-G-YFP in control cells reached a maximum after 6 min following the temperature shift (Figure 1B). In contrast, in sterol-depleted cells, a comparable number of ts-O45-G-YFP-positive ERES was reached not before 15 min after shift to 32°C. Adding-back sterols could rescue this delay and ts-O45-G-YFP localization to ERES followed a similar dynamics as had been determined for control cells. No apparent difference in the number and distribution of ERES positive for the COPII marker Sec23 could be found between the three conditions tested (Figure 1B, inset). Quantification of ts-O45-G-YFP arrival at the Golgi in control cells and cells cultivated in the presence of sterols showed a steady increase during the course of the experiment (Figure 1C). In contrast, in sterol-depleted cells, the relative amounts of ts-O45-G in the Golgi compared to those in the ER remained at a basal level up to 15 min after shift to 32°C, indicative of an inhibition of ER to Golgi transport under these conditions. At later time points (>20 min) the amounts of ts-O45-G in the Golgi became similar to those in control cells. It is likely that this equilibration of ts-O45-G in the Golgi to levels comparable to control cells is a result of an inhibition of constitutive post-TGN transport in sterol-depleted cells as it has been described previously (Wang et al, 2000). To analyze, if the observed delay in Golgi arrival of ts-O45-G in sterol-depleted cells could also be explained by a reduced velocity of ts-O45-G-containing vesicular-tubular clusters (VTCs), the long-range transport carriers moving ts-O45-G from the ER to the Golgi complex along microtubules (Presley et al, 1997; Scales et al, 1997; Watson et al, 2005), we performed live-cell imaging and tracked individual VTCs over time (Supplementary Figure 2; see also movies 1 and 2). No significant difference in the average velocity of VTCs between control and sterol-depleted cells could be observed (average velocity: 1.85±0.82 μm s−1 for control and 1.57±0.58 μm s−1 for sterol-depleted cells). These combined results clearly show that cargo trafficking along the early secretory pathway is delayed under sterol-depleted conditions and that particularly early events at the ER level might be sensitive to alterations in cellular sterol levels. To characterize this further, we analyzed by time-lapse microscopy the dynamics of ts-O45-G-YFP accumulation in individual ERES. To ensure that individual ERES could be followed during the entire time course of the experiment even when moving out of the focal plane, a 3D stack of optical sections was acquired every 3 s (Figure 2). In order to be able to follow and analyze a sufficient number of ERES becoming loaded with ts-O45-G even under sterol-depleted conditions, cells were shifted to 32°C and time-lapse imaging was started only 10 min after the temperature shift. In control cells, an accumulation of a major fraction of ts-O45-G-YFP in punctuate ERES and Golgi-like structures was already apparent at the beginning of the time-lapse analysis consistent with our results in fixed cells (Figure 2A, upper panel). In sterol-depleted cells, most ts-O45-G-YFP was still retained in reticular ER membranes at the beginning of the time-lapse analysis with only little Golgi-like labelling, and a frequent localization of ts-O45-G-YFP to ERES could only be observed at later time points of the time-lapse sequence (Figure 2B, upper panel). Figure 2.Cargo-loading to ERES is delayed in sterol-depleted cells. HeLa-cells expressing ts-O45-G-YFP were incubated at 39.5°C under control (A) or sterol depleted (B) conditions. Image acquisition was started between 5 and 10 min after shift to 32°C. Maximum intensity projections of frames of whole cells (top panels) or selected regions (lower 3 panels; outlined by box) from 4D-time-lapse sequences are shown. Arrows indicate ts-O45-G-YFP signal within selected ERES at the indicated time points after start of image acquisition. Arrowheads denote the release of vesicular carriers. Bars: 5 μm. Download figure Download PowerPoint In ERES of control cells, ts-O45-G-YFP fluorescence increased very rapidly after the first appearance of elevated levels of ts-O45-G-YFP in ERES and reached a plateau between 3 and 6 s thereafter (Figure 2A, lower panels; Figure 3A, upper panel; Supplementary movie 3). These ERES were then depleted of cargo by the release of a ts-O45-G containing VTC moving rapidly towards the Golgi complex (see Figure 2A arrowheads; see also Supplementary movies). In contrast, in sterol-depleted cells ts-O45-G-YFP loading to individual ERES occurred much more slowly (Figure 2B, lower panels; Supplementary movie 4), which was characterized by a steady increase in ts-O45-G-YFP fluorescence intensity that could last up to 300 s (Figure 3A, lower panel), indicating that cargo accumulation in individual ERES was delayed when sterols were reduced. Figure 3.Sterol-dependent cargo dynamics in individual ERES. (A) Relative fluorescence intensity changes of ts-O45-G-YFP in single ERES in HeLa-cells at 32°C (as described for Figure 2) cultivated under control (upper panel) or sterol-depleted conditions (lower panel). Three instances are shown for each condition. Fluorescence intensities within ERES were normalized to ts-O45-G-YFP background fluorescence in reticular ER membranes. Arrows indicate drops in signal intensity reflecting events of cargo release. (B, C) Mean relative fluorescence signal intensities±s.d. from 13 control (full line) or 16 sterol depleted (dashed line) isolated ts-O45-G-YFP loading events to individual ERES. (B) Average fluorescence intensities 90 s around individual loading events were measured in ERES from three different ts-O45-G-expressing HeLa-cells per condition and plotted against time (in s). Note that we set the onset of increase in fluorescence intensity in each ERES as t=0. For sterol-depleted cells, successive 90 s were averaged. For control cells, we averaged fluorescence intensities ±45 s of the loading event. In (C), average fluorescence intensities over 120 s before an event of cargo release (arrow) were plotted against time. Download figure Download PowerPoint The average loading rate of ts-O45-G-YFP to individual ERES (kon) in control cells was 4.6-fold higher than that determined in sterol-depleted cells (kon (control)=0.146 versus kon (depleted)=0.032) (Figure 3B; see Material and methods for details on kon determination). The maximum fluorescence intensity of ts-O45-G-YFP in a single ERES relative to neighbouring ER membranes immediately before the VTC release was on average 3.47±0.27-fold (n=21 events) in control and 3.57±0.33-fold (n=23 events) in sterol-depleted cells, consistent with a cargo concentration step occurring prior to cargo export from the ER as it has been described previously (Balch et al, 1994; Martinez-Menarguez et al, 1999; Malkus et al, 2002). Most ERES released cargo one to four times during the course of an experiment, and irrespective of the sterol levels, cargo release was observed as a sharp drop in fluorescence intensity (Figure 3C; arrow). Ts-O45-G-YFP loaded ERES of control cells persisted for extended periods with little change in their fluorescence intensity before cargo release occurred abruptly (Figure 3A and C). In contrast, in sterol-depleted cells, discharge of cargo from ERES occurred almost instantly when the cargo load had reached an apparent upper limit (Figure 3A and C). Altogether, these results strongly support the hypothesis that sterols are an important factor regulating the accumulation of cargo proteins in ERES preceding ER-to-Golgi transport. Endogenous ERGIC-53 is relocated to ER-membranes in sterol-depleted cells We further tested if alterations in cellular sterol levels might also affect the ER to Golgi trafficking of endogenous cellular proteins. As candidate protein we chose ERGIC-53 a lectin-like transmembrane protein that is known to cycle between the ER and Golgi complex (Schweizer et al, 1988; Ben-Tekaya et al, 2005). If sterol depletion affected ER export of ERGIC-53 without compromising its recycling from the Golgi to the ER, one would assume a relocation of ERGIC-53 to the ER when cells are exposed to sterol depleting reagents for a sufficient period of time. Comparing cells cultivated under control conditions with those grown in the absence or presence of sterols revealed remarkable differences in the steady-state distribution of ERGIC-53. A major fraction of ERGIC-53 in control cells localized to ERES, with additional pools in the ER and juxtanuclear Golgi-like membranes (Figure 4A). In sterol-depleted cells, the punctuate ERES specific signal was reduced and an increase in diffuse ER-like reticular staining was observed (Figure 4A). After adding-back sterols, the ERES-specific ERGIC-53 signal increased to levels appearing even higher than in control cells (Figure 4A), indicating that the effect in sterol-depleted cells was sterol-specific. Also, a larger fraction of ERGIC-53 compared to controls appeared now associated with juxtanuclear Golgi-like membranes. Quantification of the number of ERGIC-53-positive ERES revealed a reduced fraction of the protein associated with ERES in sterol-depleted cells compared to controls (P<0.05) or cells cultivated in the presence of sterols (Figure 4B). More strikingly, quantification of the fluorescence intensities of ERGIC-53 in ERES relative to ERGIC-53 in ER-membranes showed that under sterol-depleted conditions significantly less ERGIC-53 was associated with individual ERES compared to controls or cells cultivated in the presence of sterols (P<0.001; Figure 4C). After adding back sterols, the ERGIC-53 specific signal in ERES relative to the ER was increased when compared to controls. It is possible that this may have been caused by an overcompensation when adding back sterols. Similar data were obtained when cells were kept at 37°C or 39.5°C (data not shown). These results suggest that the inhibition of ER-exit in sterol-depleted cells is not restricted to ts-O45-G only and point towards a more general role for sterols in cargo exit from the ER. Figure 4.Steady-state localization of ERGIC-53 to ERES is affected by cellular sterol levels. (A) Immunostaining against ERGIC-53 in HeLa-cells cultivated under control conditions and in the absence (LDS/HPCD) or presence of sterols (LDS/HPCD/+sterols). Insets show z-projections of areas in the periphery of represented cells. Arrows denote examples for ERES. Arrowheads denote examples of juxta-nuclear Golgi-like staining. Bars: 10 μm. (B) Numbers (n) of ERGIC-53 positive ERES (nERES ERGIC-53) were counted in an area representing about 20% of total cell area. Each column represents the mean from 20 areas±s.d. from three independent experiments. (C) Ratios±s.d. of mean relative fluorescence signal intensities of ERGIC-53 in individual ERES [IERGIC-53(ERES)] compared to the area surrounding the respective ERES [IERGIC-53(ER)]. Each column represents the mean from 30 areas from three independent experiments. Download figure Download PowerPoint The mobility of ts-O45-G in the ER is reduced in sterol-depleted cells One mechanism by which cargo proteins might fail to be efficiently released from the ER could be by a restriction of their free diffusion in membranes. To examine a possible role of sterols on ts-O45-G dynamics within the ER, we determined its mobility by using fluorescence recovery after photobleaching (FRAP) (Nehls et al, 2000; Stephens et al, 2000; Forster et al, 2006). At 39.5°C, ts-O45-G-YFP showed a rapid mobility comparable to other FP-tagged proteins residing in ER membranes (Nehls et al, 2000). Fluorescence recovery reached a plateau within few seconds after photobleaching (Figure 5A, upper panel and B), indicating a rapid exchange of bleached ts-O45-G-YFP with unbleached ts-O45-G-YFP. However, when cellular sterols were depleted, a delay of fluorescence recovery compared to control cells could be observed (Figure 5A, lower panel and B). Quantitative analysis showed an increase in the half-time of maximal fluorescence recovery (τ1/2) of ts-O45-G-YFP in sterol-depleted cells compared to controls or cells exposed to sterol-depleting agents in the presence of sterols (Table I). Independent of the ER sterol levels only a small fraction (about 10%) of ts-O45-G-YFP was immobile. Figure 5.Lateral mobility of ts-O45-G-YFP in the ER is reduced upon sterol depletion. (A) FRAP analysis of ER-localized ts-O45-G-YFP in HeLa-cells incubated at 39.5°C under control (upper panel) or sterol-depleted conditions (lower panel). Images were obtained before photobleaching and at the indicated time points thereafter. The cell area acquired during the time course of the experiment is outlined by a box with the photobleached area (circle) in its centre. Bars: 5 μm. (B–D) FRAP analysis of ts-O45-G-YFP in the ER at 39.5°C (B), or ss-YFP (C) and Insig1-YFP (D) at 37°C under control (•, curve fit: full line) or sterol-depleted conditions (▴, curve fit: dashed line). Relative fluorescence intensities from one representative cell per condition were plotted against time (in s). Images were taken at 500 ms intervals (ss-YFP: 250 ms). For details on recovery curve analysis see Supplementary data. Download figure Download PowerPoint Table 1. Dynamics of ts-O45-G-YFP in the ER is reduced no matter of how sterols are depleted Construct Temp (°C) Condition τ1/2±s.d. (s)a fmob±s.d.a ncells ts-O45-G-YFP 39.5 Control 2.511±0.107 0.878±0.115 45 −Sterols (LDS/HPCD) 4.262±0.146 0.881±0.140 49 (LDS/HPCD/+sterols) 2.998±0.226 0.835±0.160 25 ts-O45-G-YFP 39.5 +Sterols (25-HC) 2.489±0.099 0.888±0.096 46 LDS 2.910±0.174 0.873±0.107 11 U18666A 3.180±0.318 0.918±0.102 21 Lovastatin 3.273±0.382 0.863±0.124 9 LDS/Lovastatin 3.534±0.336 0.869±0.120 18 MCD 3.360±0.272 0.936±0.068 13 LDS/MCD 5.187±0.520 0.931±0.100 12 ss-YFP 37 Control 1.294±0.197 0.964±0.095 18 −Sterols (LDS/HPCD) 1.315±0.149 0.928±0.143 17 Insig1-YFP 37 Control 3.086±0.152 0.929±0.080 21 −Sterols (LDS/HPCD) 3.170±0.136 0.910±0.095 17 SCAP-YFP 37 Control 3.343±0.117 0.868±0.020 34 −Sterols (LDS/HPCD) 4.473±0.399 0.795±0.034 28 Procollagen-YFP 37 Control 3.899±0.259 0.870±0.026 16 −Sterols (LDS/HPCD) 5.177±0.289 0.831±0.033 20 a Means±s.d. from 2 to 5 independent experiments. Ts-O45-G-YFP dynamics were also reduced by exposing cells to alternative sterol-depleting agents (for details, see Supplementary data), but essentially unchanged in cells saturated with sterols by an extended exposure of cells to 25-HC (Table I). No apparent morphological differences in ER structure could be observed in sterol-depleted cells compared to controls (Figure 5A, data not shown). However, to exclude the possibility that the reduced mobility of ts-O45-G-YFP was a consequence of a putative disintegration of the ER induced by sterol-depleting agents, we performed FRAP on soluble YFP-protein carrying the ER-retention signal KDEL (ss-YFP) (Figure 5C) as well as on the ER-resident integral membrane protein Insig1-YFP (Figure 5D) (Yang et al, 2002). However, no difference in τ1/2 could be seen between sterol depleted and control cells (Table I), indicating that the overall ER environment was unaffected by our sterol-depletion conditions. To test, if sterol depletion could also affect the mobility of other cargo proteins, we performed FRAP on the YFP-tagged cargo proteins SCAP and procollagen-I (Stephens and Pepperkok, 2002). Most notably, similar to ts-O45-G, these alternative cargo proteins also showed an increased τ1/2 in ER membranes of sterol-depleted cells relative to controls (Table I). This suggested that a reduced mobility in ER membranes of sterol-depleted cells might not be restricted to ts-O45-G, but also affects further cargo molecules in the ER. Dynamics of cargo accumulation within ERES and not the diffusional mobility is critical for cargo export dynamics from the ER Our experiments showed thus far that delayed arrival of ts-O45-G at the Golgi in sterol-depleted cells is accompanied by an inhibition of cargo loading to ERES and a reduction of ts-O45-G mobility in ER membranes. This raised the possibility that the reduced diffusional mobility could account for the delayed loading of cargo to ERES and thus inhibit ER-export of ts-O45-G. We therefore wanted to analyze if at all and how diffusional mobility and cargo loading to ERES are interrelated and whether these events could account for the delayed ER export of ts-O45-G in sterol-depleted cells. To address this, these processes were simulated and tested which parameters are critical to reproduce our experimental findings such as the delayed Golgi arrival of ts-O45-G in sterol-depleted cells (Figure 1). We assumed that export of a given cargo protein from the ER is influenced by two major parameters (Figure 6A; see Supplementary Data for details). First, by its mobility within the ER which is reflected by its apparent diffusion constant Dapp, and second, by its loading to ERES, characterized by the binding constants kon and koff. The export rate of ts-O45-G from the ER could also be affected by the distance between neighbouring ERES (dERES), the diameter of each ERES (AERES) or the extent of cargo accumulation in ERES (Ithreshold) preceding cargo release and subsequent transport to the Golgi. Since our experiments showed that these parameters were not affected by the cellular sterol levels (Figure 1B, inset, and 2 and 3), they" @default.
• W1987505890 created "2016-06-24" @default.
• W1987505890 creator A5017298007 @default.
• W1987505890 creator A5048342905 @default.
• W1987505890 creator A5054742160 @default.
• W1987505890 creator A5089969264 @default.
• W1987505890 date "2006-06-22" @default.
• W1987505890 modified "2023-10-14" @default.
• W1987505890 title "Sterols regulate ER-export dynamics of secretory cargo protein ts-O45-G" @default.
• W1987505890 cites W1499112972 @default.
• W1987505890 cites W1522741331 @default.
• W1987505890 cites W1601012886 @default.
• W1987505890 cites W1914535079 @default.
• W1987505890 cites W1943315667 @default.
• W1987505890 cites W1969963064 @default.
• W1987505890 cites W1970541648 @default.
• W1987505890 cites W1971050831 @default.
• W1987505890 cites W1974747471 @default.
• W1987505890 cites W1986849641 @default.
• W1987505890 cites W1990394752 @default.
• W1987505890 cites W1995193554 @default.
• W1987505890 cites W1998822364 @default.
• W1987505890 cites W2002426370 @default.
• W1987505890 cites W2003123776 @default.
• W1987505890 cites W2005733184 @default.
• W1987505890 cites W2008149441 @default.
• W1987505890 cites W2012275595 @default.
• W1987505890 cites W2017902030 @default.
• W1987505890 cites W2018415306 @default.
• W1987505890 cites W2032261439 @default.
• W1987505890 cites W2040148851 @default.
• W1987505890 cites W2045571454 @default.
• W1987505890 cites W2059158034 @default.
• W1987505890 cites W2072024204 @default.
• W1987505890 cites W2082621348 @default.
• W1987505890 cites W2087596465 @default.
• W1987505890 cites W2092296126 @default.
• W1987505890 cites W2097632838 @default.
• W1987505890 cites W2104152706 @default.
• W1987505890 cites W2115112794 @default.
• W1987505890 cites W2115420718 @default.
• W1987505890 cites W2129177835 @default.
• W1987505890 cites W2129224672 @default.
• W1987505890 cites W2130388575 @default.
• W1987505890 cites W2134403233 @default.
• W1987505890 cites W2138059603 @default.
• W1987505890 cites W2149743336 @default.
• W1987505890 cites W2160509178 @default.
• W1987505890 cites W2161458516 @default.
• W1987505890 cites W2167150730 @default.
• W1987505890 cites W2188056684 @default.
• W1987505890 cites W2299406270 @default.
• W1987505890 cites W2341412675 @default.
• W1987505890 doi "https://doi.org/10.1038/sj.emboj.7601205" @default.
• W1987505890 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/1500972" @default.
• W1987505890 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16794576" @default.
• W1987505890 hasPublicationYear "2006" @default.
• W1987505890 type Work @default.
• W1987505890 sameAs 1987505890 @default.
• W1987505890 citedByCount "58" @default.
• W1987505890 countsByYear W19875058902012 @default.
• W1987505890 countsByYear W19875058902013 @default.
• W1987505890 countsByYear W19875058902014 @default.
• W1987505890 countsByYear W19875058902015 @default.
• W1987505890 countsByYear W19875058902016 @default.
• W1987505890 countsByYear W19875058902017 @default.
• W1987505890 countsByYear W19875058902018 @default.
• W1987505890 countsByYear W19875058902019 @default.
• W1987505890 countsByYear W19875058902020 @default.
• W1987505890 countsByYear W19875058902021 @default.
• W1987505890 countsByYear W19875058902022 @default.
• W1987505890 countsByYear W19875058902023 @default.
• W1987505890 crossrefType "journal-article" @default.
• W1987505890 hasAuthorship W1987505890A5017298007 @default.
• W1987505890 hasAuthorship W1987505890A5048342905 @default.
• W1987505890 hasAuthorship W1987505890A5054742160 @default.
• W1987505890 hasAuthorship W1987505890A5089969264 @default.
• W1987505890 hasBestOaLocation W19875058901 @default.
• W1987505890 hasConcept C121332964 @default.
• W1987505890 hasConcept C145912823 @default.
• W1987505890 hasConcept C156399914 @default.
• W1987505890 hasConcept C24890656 @default.
• W1987505890 hasConcept C49039625 @default.
• W1987505890 hasConcept C55493867 @default.
• W1987505890 hasConcept C70721500 @default.
• W1987505890 hasConcept C86803240 @default.
• W1987505890 hasConcept C95444343 @default.
• W1987505890 hasConceptScore W1987505890C121332964 @default.
• W1987505890 hasConceptScore W1987505890C145912823 @default.
• W1987505890 hasConceptScore W1987505890C156399914 @default.
• W1987505890 hasConceptScore W1987505890C24890656 @default.
• W1987505890 hasConceptScore W1987505890C49039625 @default.
• W1987505890 hasConceptScore W1987505890C55493867 @default.
• W1987505890 hasConceptScore W1987505890C70721500 @default.
• W1987505890 hasConceptScore W1987505890C86803240 @default.
• W1987505890 hasConceptScore W1987505890C95444343 @default.
• W1987505890 hasIssue "13" @default.
• W1987505890 hasLocation W19875058901 @default.

• next