Role of ADP Ribosylation Factor 1 in the Assembly and Secretion of ApoB-100–Containing Lipoproteins
http://www.100md.com
动脉硬化血栓血管生物学 2005年第3期
From the Department of Medical Biochemistry, Wallenberg Laboratory for Cardiovascular Research, G?teborg University, G?teborg, Sweden.
Correspondence to Sven-Olof Olofsson, The Wallenberg Laboratory, Sahlgrenska University Hospital, SE-413 45 G?teborg, Sweden. E-mail Sven-Olof.Olofsson@medkem.gu.se
Abstract
Objective— We investigated the role of ADP ribosylation factor 1 (ARF1) in the assembly of very-low-density lipoproteins (VLDLs).
Methods and Results— The dominant-negative ARF1 mutant, T31N, decreased the assembly of apoB-100 VLDL 1 (Svedberg floatation units [Sf] 60 to 400) by 80%. The decrease coincided with loss of coatamer I (COPI) from the Golgi apparatus and inhibition of anterograde transport, as demonstrated by time-lapse studies of the vesicular stomatitis virus G protein. The VLDL 1 assembly was also completely inhibited at 15°C. Thus, the antegrade transport is essential for the assembly of VLDL 1. Intracellular localization of N-acetylgalactosaminyl transferase 2 indicated that the Golgi apparatus was at least partly intact when the VLDL assembly was inhibited. Transient transfection with phospholipase D 1 increased the assembly of VLDL 1 and VLDL 2 (Sf 20 to 60). Overexpression of ARF1 in stably transfected McA-RH7777 cells increased the secretion of VLDL 2 but not of VLDL 1, which was dependent on the availability of oleic acid. Secretion of VLDL 1 increased with increasing amounts of oleic acid, and VLDL 2 secretion decreased simultaneously.
Conclusions— Overexpression of ARF1 increased the assembly of VLDL 2 but not of VLDL 1, whose production was dependent on both anterograde transport and the availability of fatty acids.
The assembly of VLDL depends on the ARF1-driven anterograde transport. Overexpression of ARF1 increases the secretion of VLDL 2 but not of VLDL 1, which is highly dependent on of fatty acids. The secretion of both VLDL 1 and VLDL 2 are increased by phospholipase D 1.
Key Words: ADP ribosylation factor 1 ? apolipoprotein B ? coatamer 1 ? intracellular transport ? very-low-density lipoproteins
Introduction
Apolipoprotein B (apoB)-containing very-low-density lipoproteins (VLDLs) are formed in a 2-step process.1–3 In the first step, apoB forms a partially lipidated particle (a primordial lipoprotein or pre-VLDL) during its cotranslational translocation to the lumen of the endoplasmic reticulum (ER). This step involves the transfer of lipids to apoB catalyzed by the microsomal triglyceride transfer protein. In the absence of lipids or microsomal triglyceride transfer protein, the translocation of apoB is halted, and the protein is retracted through the translocon and degraded by proteasomes.4–8
The second step, in which the major amount of triglycerides is added to pre-VLDL, is less well-characterized. This step involves the formation of apoB-free lipid droplets in the smooth ER9 that associate with apoB-containing pre-VLDL in a compartment that is separate from the rough ER.9,10 Thus, transport and sorting processes involved in the transfer of proteins out of the rough ER may be important for the assembly of VLDL. Consistent with this possibility, the second step can be inhibited by brefeldin A11 and is dependent on ADP ribosylation factor 1 (ARF1) and phospholipase D (PLD) activity.12
ARF1, a member of the Ras superfamily of GTP-binding proteins, participates in the formation of coatamer I (COPI) secretory vesicles, which perform retrograde transport from the Golgi to the ER and within the Golgi stacks.13 ARF1 and COPI are important for the anterograde transport from the ER to the Golgi apparatus14–16, and ARF1 activates PLD1.17–19
In this study, we investigated the roles of ARF1 and PLD1 in the assembly of the buoyant VLDL 1 (Svedberg floatation units [Sf] 60 to 400) and the less buoyant VLDL 2 ( (Sf 20 to 60). Our results indicate that the second step of VLDL 1 assembly requires active ARF1, most likely reflecting the importance of anterograde transport.
Methods
Please see the online Methods, available at http://atvb.ahajournals.org.
Results
ARF1 T31N Interferes With the Second Step of VLDL Assembly
Sucrose gradient ultracentrifugation12 (for Methods see http://atvb.ahajournals.org) demonstrated heterogeneity of the apoB-100–containing lipoproteins secreted from McA-RH7777 cells (Figure 1A and 1B). Based on ultracentrifugation of purified lipoproteins, we concluded that VLDL 1 was confined to the 2 top fractions of the gradient and could not be detected in the more dense fractions of the gradient. The migration in the gradient of VLDL species (VLDL 1 and VLDL 2) with different assembly pathways has recently been reported.20
Figure 1. Overexpression of the dominant-negative ARF 1mutant T31N decreases the assembly of VLDL 1. Gradient ultracentrifugation of apoB-100–containing lipoproteins (filled squares) secreted from McA-RH7777 cells cultured in the absence (A) or presence (B) of oleic acid. McA-RH7777 cells were cultured in the absence or presence of 360 μmol/L oleic acid, pulse-labeled with [35S]-methionine-cysteine for 15 minutes and chased for 3 hours. The culture medium was collected and subjected to gradient ultracentrifugation. ApoB-100 was recovered from each fraction by immunoprecipitation and SDS-PAGE, and the radioactivity was determined. In parallel experiments, isolated low-density lipoprotein (LDL), very-low-density lipoprotein (VLDL), and VLDL 1 were ultracentrifuged in the gradient, and the fractions were analyzed for apoB, triglycerides, and cholesterol. The migration of the different lipoproteins is indicated. Filled triangles show the density gradient. C, Immunoblots of stably transfected McA-RH7777 cells T31N after induction of His-tagged T31N with tetracycline (1 μg/mL) for different times. After each period of induction, the cells were recovered, lysed, and subjected to SDS-PAGE. The gels were blotted against antibodies to the His-tag or to ARF1. The antibody to ARF 1 reacts with 2 bands; the expressed mutant (T31N ARF 1) and endogenous ARF 1. T31N ARF 1 is fused to a His-tag and will therefore migrate slower than endogenous ARF 1 on the gel. D, The effect of different lengths of induction of the ARF1 mutant T31N on the intracellular levels of apoB-100 VLDL 1 (filled), apoB-100 (shaded), and transferrin (open). McA-RH7777 cells stably transfected with T31N ARF 1 were cultured in the presence of oleic acid (360 μmol/L), incubated with 1 μg/mL tetracycline for the indicated period, labeled with [35S]-methionine-cysteine for 15 minutes, and chased for 15 minutes. Total apoB-100 and transferrin were recovered by immunoprecipitation and SDS-PAGE, and the radioactivity was determined. The total microsomal fraction was recovered, and the luminal content was recovered and subjected to gradient ultracentrifugation, and apoB-100 radioactivity present in VLDL 1 was determined. The results are given as mean and range of 2 experiments. Results are given as percent of the uninduced control.
When the cells were cultured in the absence of oleic acid, the majority of the apoB-100 formed bands in the VLDL 1 and VLDL 2 density ranges; a small amount banded in the low-density lipoprotein (LDL) density range and was included in the VLDL 2 fraction (Figure 1A). In the presence of oleic acid, the majority of apoB-100 was present in the VLDL 1 density region (Figure 1B).
Previously, we showed that VLDL assembly is dependent on ARF1.12 To investigate the mechanism of this dependence, we began by assessing the effect of the dominant-negative ARF1 mutant T31N on the expression of total apoB-100 and apoB-100–containing VLDL 1, and on transport through the secretory pathway. T31N ARF 1 was stably transfected to McA-RH 7777 cells using the inducible t-rex system. Expression of T31N ARF1 started 30 minutes after the start of the induction (by the addition of tetracycline) and was almost linear over the course of 24 hours; after 150 minutes, the amount of ARF1 T31N was similar to that of the total intracellular pool of ARF (Figure 1C).
To estimate the influence of T31N ARF1 on the second step of VLDL assembly, we followed the appearance of apoB-100–containing VLDL 1 in the secretory pathway after induction. Formation of apoB-100 VLDL 1 declined rapidly after 60 minutes of induction; at 240 minutes, it had decreased by 80% (Figure 1D). ApoB-100 and transferrin were also decreased at 240 minutes, although to a lesser extent than VLDL 1.
Inhibition of VLDL 1 Assembly by T31N ARF 1 Coincides With Dissociation of COPI From the Golgi Apparatus and Inhibition of Anterograde Transport
Next, we assessed the effects of T31N ARF 1 induction on the localization of COPI in McA-RH7777 cells using immunohistochemistry. Before and during the first 120 minutes of induction, the majority of ?-COP (a COPI protein) had a juxtanuclear localization (example indicated by arrow in Figure 2A), indicating that it was associated with the Golgi apparatus; however, after 240 minutes of induction, few cells had Golgi-associated ?-COP (Figure 2A). This dissociation of COPI from the Golgi apparatus coincided with the decrease in VLDL 1 assembly. In contrast, the localization of ERGIC 53, a marker for the ER–Golgi intermediate compartment (ERGIC), was unchanged at 240 minutes (Figure 2B; examples of reaction with the antibody to ERGIC 53 are indicated by arrows).
Figure 2. Overexpression of ARF1 T31N dissociates COP I from the Golgi apparatus (A), but ERGIC (B) and the Golgi apparatus remains largely intact. A, ARF 1 T31N was stably transfected to McA-RH7777 cells in the t-rex system. The cells were incubated with tetracycline for the indicated times, and ?-COP was detected with a monoclonal antibody (anti-?-COP). Bar=10 μm. B, Transfection as in (A). The cells were incubated with tetracycline for the indicated times, and ERGIC 53 was detected with a monoclonal antibody (anti-ERGIC 53). Bar=10 μm. C, McA-RH7777 cells were transfected as in (A) and induced with tetracycline for the indicated period of time. The cells were transiently transfected with the stalk region of GalNac-T2 fused to the N-terminus of yellow fluorescent protein (GalNac-T2-YFP) 48 hours before the cells were fixed. The cells were induced with tetracycline for 0 or 240 minutes, fixed in formaldehyde, mounted with FluorSave, and viewed at 63x magnification with a Zeiss LSM 510 Meta confocal microscope. Bar=10 μm.
To determine whether the decrease in VLDL 1 assembly was accompanied by disruption of the Golgi apparatus, we examined McA-RH7777 cells transiently expressing the stalk region of the Golgi enzyme N-acetylgalactosaminyl-transferase-2 (GalNac-T2) fused to yellow fluorescent protein (YFP; yellow staining in Figure 2C). After 240 minutes of T31N induction, Golgi structures containing GalNAc-T2 were still present, indicating that the Golgi apparatus was not completely disrupted and fused with the ER (Figure 2C).
To determine whether the inhibition of VLDL 1 assembly by ARF1 T31N coincides with inhibition of anterograde transport, we injected McA-RH7777 cells with a thermo-sensitive mutant of the vesicular stomatitis virus (VSV)-G protein (fused to GFP); after 4 to 6 hours at 39°C, the cells were placed in the temperature chamber (set at 32°C) of an inverted microscope and examined at 1-minute intervals after induction of T31N ARF 1 expression (Figure 3). At the start of the experiment, GFP fluorescence was diffuse and consistent with localization in the ER (Figure 3; 0 minutes induction of T31N and recording after 1 minute at the permissive temperature). In the uninduced control (Figure 3; 0 minutes induction), fluorescence appeared in the Golgi apparatus after 3 to 5 minutes (indicated by white arrows); at 30 minutes, the plasma membrane was clearly labeled (indicated by red arrow). Similar results were obtained after 60 and 120 minutes of induction. However, at 240 minutes, most of the fluorescence from the tagged VSV-G protein remained diffuse; virtually no protein had translocated to the Golgi apparatus (although small amounts could perhaps be seen after 30 minutes), indicating severe inhibition of anterograde transport from the ER to the Golgi apparatus.
Figure 3. Overexpression of the dominant-negative T31N ARF 1 inhibits the transport between ER and Golgi. The effect of T31N expression on the intracellular transport of a thermo-sensitive VSV-G mutant (ts 045) was investigated. The mutant protein was fused to GFP and microinjected into McA-RH7777 cells (stably transfected as in Figure 2A), which were kept at 39°C for 4 to 6 hours and transferred to an inverted microscope with the temperature chamber set at 32°C. Images were acquired at 1-minute intervals after addition of tetracycline to induce T31N expression (induction of T31N: 0 minutes, movie 1; 60 minutes, movie 2; 120 minutes, movie 3; 240 minutes, movie 4).
To further address the possibility that the VLDL 1 assembly was dependent on the anterograde transport through the secretory pathway, the effect of lowering the temperature to 15°C was investigated (Figure 4). At this temperature, the intracellular transport proceeds to the ERGIC but the proteins fail to reach cis-Golgi. The cells were pulsed with [35S]-methionine-cysteine for 10 minutes at 37°C and then chased for 20 minutes at 37°C or at 15°C, and the production of VLDL 1 was followed. The results (Figure 4) showed that the intracellular pool of radioactive apoB-100 VLDL 1 increased when the cells were chased at 37°C, whereas no increase could be seen when the chase was performed at 15°C. There was no effect of the lowering of the temperature on the expression of transferrin (not shown).
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Figure 4. The assembly of VLDL 1 is inhibited at 15°C. The effect on the VLDL 1 assembly of a lowering of the temperature to 15°C was investigated. Cells were pulse-labeled with [35S]-methionine-cysteine for 10 minutes (at 37°C) and chased for 20 minutes at 15°C or 37°C. After the pulse, as well as after each chase period, the content of the microsomal fraction was isolated and VLDL 1 recovered by gradient ultracentrifugation and the apoB-100 radioactivity was determined. Results are mean±SD; n=3.
PLD1 Increases the Secretion of ApoB-100–Containing Lipoproteins
Because PLD1 is activated by ARF117–19 and is important for anterograde transport,14 we assessed its effects on VLDL assembly in McA-RH7777 cells transiently transfected with vectors encoding wild-type PLD1 or an inactive mutant (K898R, control), both fused to GFP (see Figure IA, available online at http://atvb.ahajournals.org). Wild-type PLD1 induced a significant increase in the secretion of apoB-100 VLDL 1 (P=0.0016) and VLDL 2 (P=0.049) (Figure IA). Cells transfected with constructs encoding only the K898R mutant or GFP alone secreted similar amounts of apoB-100, indicating that the mutant did not have a dominant-negative effect (not shown).
Overexpression of Wild-Type ARF1 Increase the Secretion of VLDL 2 but not of VLDL1
To assess the effects of ARF1 overexpression on the secretion of apoB-100 and apoB-100–containing VLDL, ARF1 wild-type were stably transfected to McARH 7777 cells using the inducible t-rex system.12 Pulse-chase studies were performed 24 hours after induction of ARF1 expression (by the addition of tetracycline) in cells cultured in the presence or absence of oleic acid. Overexpression of ARF1 increased VLDL 2 secretion under both conditions but did not influence VLDL 1 secretion; however, total apoB-100 secretion increased only in cells cultured in the absence of oleic acid (Figure IB). Culturing the cells with different amounts of oleic acid showed a dose-dependent increase in VLDL 1 secretion (Figure IC) and a simultaneous decrease in VLDL 2 secretion (Figure ID). As a result, the VLDL 1/VLDL 2 ratio increased from 1.1±0.8 to 5.0±0.7 (mean±SD; n=4). Thus, the availability of fatty acids and the oleic acid-induced increase in the rate of the biosynthesis of triglycerides (not shown) appear to be major determinants of VLDL 1 formation.
Discussion
In this study, we showed that the dominant-negative ARF1 mutant T31N inhibited the appearance of VLDL 1 in the secretory pathway of McA-RH7777 cells to a much greater extent than it inhibited the expression of total apoB-100 and transferrin, indicating that ARF1 is essential for the second step of the VLDL 1 assembly. However, overexpression of ARF1 wild-type did not increase the assembly of VLDL 1; instead, such an overexpression increased the production of VLDL 2. The secretion of VLDL 1 and VLDL 2 was stimulated by PLD 1. Finally, we observed that the assembly of VLDL 1 was increased by oleic acid while the secretion of VLDL 2 decreased.
ARF1 and COPI are important for the sorting and transport of proteins from the ER to the Golgi apparatus.15,16,21 Thus, ARF1 could promote the transfer of pre-VLDL from the site of synthesis in the rough ER to the smooth membrane compartment, where the second step occurs.10 Like other secreted proteins, apoB-100 in a non-VLDL form leaves the ER in Sar1/COPII vesicles,22 which participate in the formation of ERGIC. COPI (and ARF1) are essential for the anterograde transport of the ERGIC to the cis-Golgi. In the assembly of VLDL 1, ARF1 may promote the transfer of pre-VLDL to the cis-Golgi. Consistent with such a role, the decrease in VLDL 1 after 240 minutes of T31N ARF 1 induction coincided with the loss of ?- COP from the Golgi apparatus. Activation of ARF1 initiates the binding of COP I to the membrane of the secretory pathway, an important step in the formation of transport vesicles and ARF1/COPI-dependent sorting events.15,16,21 Because the ERGIC must interact with COPI before it can be transported to the cis-Golgi,23,24 the effect of T31N ARF 1 on VLDL 1 assembly may reflect inhibition of anterograde transport. Such a mechanism is supported by our observation that the inhibition of VLDL 1 assembly coincided with the loss of transfer of VSV-G protein from the ER to the Golgi. An important role of the anterograde transport in the assembly of VLDL 1 is supported by the total inhibition of the assembly at 15°C. At this temperature the anterograde transport is blocked and the proteins reach ERGIC but not cis-Golgi.25,26 Thus, together our results are consistent with that ARF1 promotes the anterograde transport of apoB into the second-step compartment. This could indicate that the second step occurs in a Golgi compartment, consistent with the results of subcellular fractionation studies.10
PLD1 also appears to participate in anterograde transport27 and is activated by ARF1. Overexpression of PLD1 gave rise to small but statistically significant increases in the secretion of both VLDL 1 and VLDL 2, confirming our results in a cell-free system,12 which suggested that PLD is important for the second step of VLDL assembly. The small effect of PLD 1 in intact cells may be caused by a dual role of PLD 1. We have recently presented evidence that PLD activity is involved in the assembly of cytosolic lipid droplets.28 Thus, PLD 1 may promote the formation of the lipoproteins, but this effect may be counteracted by a diversion of triglycerides into cytosolic lipid droplets.
Our findings in this study, together with previously published results, can be summarized in a hypothesis for the role of ARF1 in the assembly of VLDL 1 (Figure 5). The assembly process starts with the cotranslational lipidation of apoB-100, resulting in a primordial "pre-VLDL" particle.2,3 The amount of apoB-100 in pre-VLDL is determined, at least in part, by cotranslational degradation.5–8 The formed pre-VLDL is either converted to VLDL or sorted to post-translational degradation.2,3,8 The assembly of VLDL 1 is dependent on functional ARF1, most likely indicating that the transport through the secretory pathway must be intact for pre-VLDL to be transferred from its site of assembly in the rough ER to the smooth compartment of the second step10 (Figure 5). If ARF1 is inhibited, the conversion of pre-VLDL to VLDL 1 ceases and pre-VLDL is instead sorted to degradation. ARF1 is rate-limiting in the assembly and secretion of VLDL 2 but not of VLDL 1. This observation may be explained by an increased capacity to transfer pre-VLDL to the second-step compartment, resulting in increased assembly of VLDL 2 but not of VLDL 1, which was highly dependent on the availability of fatty acids. Thus, increased transport of pre-VLDL into the second-step compartment is not directly reflected in increased secretion of VLDL 1 unless sufficient amounts of fatty acids (and triglycerides) are available.
Figure 5. A model for the role of ARF1 and fatty acids in the assembly of VLDL 1 and VLDL 2. In the first step, which occurs cotranslationally, microsomal triglyceride transfer protein (MTP) catalyzes the partial lipidation of apoB-100, forming a pre-VLDL particle that is transported by an ARF1-dependent mechanism to the smooth membrane compartment, where the second step occurs. The secretion of VLDL 1 is determined by the availability of fatty acids (and the rate of triglyceride biosynthesis). Thus, in the presence of sufficient amounts of fatty acids and triglycerides, the majority of the pre-VLDL is converted to VLDL 1. An increase in the transport capacity (by overexpression of ARF 1) will increase the pre-VLDL in the second-step compartment above the VLDL 1 lipidation capacity resulting in a channeling of pre-VLDL into VLDL 2 and a subsequent increase in the secretion of this form of VLDL (A). During a shortage of fatty acids (B), VLDL 1 secretion decreases and pre-VLDL is channeled to VLDL 2. An increase of the transport capacity (by overexpression of ARF 1) results in a further increase in the formation of VLDL 2.
Acknowledgments
This study was supported by grant 7142 from the Swedish Medical Research Council and by the Swedish Heart and Lung Foundation, Novo Nordic Foundation, Swedish Strategic Funds (National Network and Graduate School for Cardiovascular Research), and S?derbergs Foundation. The time lapse investigations were performed at the Swegene center for cellular imaging in G?teborg
Received August 19, 2004; accepted December 1, 2004.
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Correspondence to Sven-Olof Olofsson, The Wallenberg Laboratory, Sahlgrenska University Hospital, SE-413 45 G?teborg, Sweden. E-mail Sven-Olof.Olofsson@medkem.gu.se
Abstract
Objective— We investigated the role of ADP ribosylation factor 1 (ARF1) in the assembly of very-low-density lipoproteins (VLDLs).
Methods and Results— The dominant-negative ARF1 mutant, T31N, decreased the assembly of apoB-100 VLDL 1 (Svedberg floatation units [Sf] 60 to 400) by 80%. The decrease coincided with loss of coatamer I (COPI) from the Golgi apparatus and inhibition of anterograde transport, as demonstrated by time-lapse studies of the vesicular stomatitis virus G protein. The VLDL 1 assembly was also completely inhibited at 15°C. Thus, the antegrade transport is essential for the assembly of VLDL 1. Intracellular localization of N-acetylgalactosaminyl transferase 2 indicated that the Golgi apparatus was at least partly intact when the VLDL assembly was inhibited. Transient transfection with phospholipase D 1 increased the assembly of VLDL 1 and VLDL 2 (Sf 20 to 60). Overexpression of ARF1 in stably transfected McA-RH7777 cells increased the secretion of VLDL 2 but not of VLDL 1, which was dependent on the availability of oleic acid. Secretion of VLDL 1 increased with increasing amounts of oleic acid, and VLDL 2 secretion decreased simultaneously.
Conclusions— Overexpression of ARF1 increased the assembly of VLDL 2 but not of VLDL 1, whose production was dependent on both anterograde transport and the availability of fatty acids.
The assembly of VLDL depends on the ARF1-driven anterograde transport. Overexpression of ARF1 increases the secretion of VLDL 2 but not of VLDL 1, which is highly dependent on of fatty acids. The secretion of both VLDL 1 and VLDL 2 are increased by phospholipase D 1.
Key Words: ADP ribosylation factor 1 ? apolipoprotein B ? coatamer 1 ? intracellular transport ? very-low-density lipoproteins
Introduction
Apolipoprotein B (apoB)-containing very-low-density lipoproteins (VLDLs) are formed in a 2-step process.1–3 In the first step, apoB forms a partially lipidated particle (a primordial lipoprotein or pre-VLDL) during its cotranslational translocation to the lumen of the endoplasmic reticulum (ER). This step involves the transfer of lipids to apoB catalyzed by the microsomal triglyceride transfer protein. In the absence of lipids or microsomal triglyceride transfer protein, the translocation of apoB is halted, and the protein is retracted through the translocon and degraded by proteasomes.4–8
The second step, in which the major amount of triglycerides is added to pre-VLDL, is less well-characterized. This step involves the formation of apoB-free lipid droplets in the smooth ER9 that associate with apoB-containing pre-VLDL in a compartment that is separate from the rough ER.9,10 Thus, transport and sorting processes involved in the transfer of proteins out of the rough ER may be important for the assembly of VLDL. Consistent with this possibility, the second step can be inhibited by brefeldin A11 and is dependent on ADP ribosylation factor 1 (ARF1) and phospholipase D (PLD) activity.12
ARF1, a member of the Ras superfamily of GTP-binding proteins, participates in the formation of coatamer I (COPI) secretory vesicles, which perform retrograde transport from the Golgi to the ER and within the Golgi stacks.13 ARF1 and COPI are important for the anterograde transport from the ER to the Golgi apparatus14–16, and ARF1 activates PLD1.17–19
In this study, we investigated the roles of ARF1 and PLD1 in the assembly of the buoyant VLDL 1 (Svedberg floatation units [Sf] 60 to 400) and the less buoyant VLDL 2 ( (Sf 20 to 60). Our results indicate that the second step of VLDL 1 assembly requires active ARF1, most likely reflecting the importance of anterograde transport.
Methods
Please see the online Methods, available at http://atvb.ahajournals.org.
Results
ARF1 T31N Interferes With the Second Step of VLDL Assembly
Sucrose gradient ultracentrifugation12 (for Methods see http://atvb.ahajournals.org) demonstrated heterogeneity of the apoB-100–containing lipoproteins secreted from McA-RH7777 cells (Figure 1A and 1B). Based on ultracentrifugation of purified lipoproteins, we concluded that VLDL 1 was confined to the 2 top fractions of the gradient and could not be detected in the more dense fractions of the gradient. The migration in the gradient of VLDL species (VLDL 1 and VLDL 2) with different assembly pathways has recently been reported.20
Figure 1. Overexpression of the dominant-negative ARF 1mutant T31N decreases the assembly of VLDL 1. Gradient ultracentrifugation of apoB-100–containing lipoproteins (filled squares) secreted from McA-RH7777 cells cultured in the absence (A) or presence (B) of oleic acid. McA-RH7777 cells were cultured in the absence or presence of 360 μmol/L oleic acid, pulse-labeled with [35S]-methionine-cysteine for 15 minutes and chased for 3 hours. The culture medium was collected and subjected to gradient ultracentrifugation. ApoB-100 was recovered from each fraction by immunoprecipitation and SDS-PAGE, and the radioactivity was determined. In parallel experiments, isolated low-density lipoprotein (LDL), very-low-density lipoprotein (VLDL), and VLDL 1 were ultracentrifuged in the gradient, and the fractions were analyzed for apoB, triglycerides, and cholesterol. The migration of the different lipoproteins is indicated. Filled triangles show the density gradient. C, Immunoblots of stably transfected McA-RH7777 cells T31N after induction of His-tagged T31N with tetracycline (1 μg/mL) for different times. After each period of induction, the cells were recovered, lysed, and subjected to SDS-PAGE. The gels were blotted against antibodies to the His-tag or to ARF1. The antibody to ARF 1 reacts with 2 bands; the expressed mutant (T31N ARF 1) and endogenous ARF 1. T31N ARF 1 is fused to a His-tag and will therefore migrate slower than endogenous ARF 1 on the gel. D, The effect of different lengths of induction of the ARF1 mutant T31N on the intracellular levels of apoB-100 VLDL 1 (filled), apoB-100 (shaded), and transferrin (open). McA-RH7777 cells stably transfected with T31N ARF 1 were cultured in the presence of oleic acid (360 μmol/L), incubated with 1 μg/mL tetracycline for the indicated period, labeled with [35S]-methionine-cysteine for 15 minutes, and chased for 15 minutes. Total apoB-100 and transferrin were recovered by immunoprecipitation and SDS-PAGE, and the radioactivity was determined. The total microsomal fraction was recovered, and the luminal content was recovered and subjected to gradient ultracentrifugation, and apoB-100 radioactivity present in VLDL 1 was determined. The results are given as mean and range of 2 experiments. Results are given as percent of the uninduced control.
When the cells were cultured in the absence of oleic acid, the majority of the apoB-100 formed bands in the VLDL 1 and VLDL 2 density ranges; a small amount banded in the low-density lipoprotein (LDL) density range and was included in the VLDL 2 fraction (Figure 1A). In the presence of oleic acid, the majority of apoB-100 was present in the VLDL 1 density region (Figure 1B).
Previously, we showed that VLDL assembly is dependent on ARF1.12 To investigate the mechanism of this dependence, we began by assessing the effect of the dominant-negative ARF1 mutant T31N on the expression of total apoB-100 and apoB-100–containing VLDL 1, and on transport through the secretory pathway. T31N ARF 1 was stably transfected to McA-RH 7777 cells using the inducible t-rex system. Expression of T31N ARF1 started 30 minutes after the start of the induction (by the addition of tetracycline) and was almost linear over the course of 24 hours; after 150 minutes, the amount of ARF1 T31N was similar to that of the total intracellular pool of ARF (Figure 1C).
To estimate the influence of T31N ARF1 on the second step of VLDL assembly, we followed the appearance of apoB-100–containing VLDL 1 in the secretory pathway after induction. Formation of apoB-100 VLDL 1 declined rapidly after 60 minutes of induction; at 240 minutes, it had decreased by 80% (Figure 1D). ApoB-100 and transferrin were also decreased at 240 minutes, although to a lesser extent than VLDL 1.
Inhibition of VLDL 1 Assembly by T31N ARF 1 Coincides With Dissociation of COPI From the Golgi Apparatus and Inhibition of Anterograde Transport
Next, we assessed the effects of T31N ARF 1 induction on the localization of COPI in McA-RH7777 cells using immunohistochemistry. Before and during the first 120 minutes of induction, the majority of ?-COP (a COPI protein) had a juxtanuclear localization (example indicated by arrow in Figure 2A), indicating that it was associated with the Golgi apparatus; however, after 240 minutes of induction, few cells had Golgi-associated ?-COP (Figure 2A). This dissociation of COPI from the Golgi apparatus coincided with the decrease in VLDL 1 assembly. In contrast, the localization of ERGIC 53, a marker for the ER–Golgi intermediate compartment (ERGIC), was unchanged at 240 minutes (Figure 2B; examples of reaction with the antibody to ERGIC 53 are indicated by arrows).
Figure 2. Overexpression of ARF1 T31N dissociates COP I from the Golgi apparatus (A), but ERGIC (B) and the Golgi apparatus remains largely intact. A, ARF 1 T31N was stably transfected to McA-RH7777 cells in the t-rex system. The cells were incubated with tetracycline for the indicated times, and ?-COP was detected with a monoclonal antibody (anti-?-COP). Bar=10 μm. B, Transfection as in (A). The cells were incubated with tetracycline for the indicated times, and ERGIC 53 was detected with a monoclonal antibody (anti-ERGIC 53). Bar=10 μm. C, McA-RH7777 cells were transfected as in (A) and induced with tetracycline for the indicated period of time. The cells were transiently transfected with the stalk region of GalNac-T2 fused to the N-terminus of yellow fluorescent protein (GalNac-T2-YFP) 48 hours before the cells were fixed. The cells were induced with tetracycline for 0 or 240 minutes, fixed in formaldehyde, mounted with FluorSave, and viewed at 63x magnification with a Zeiss LSM 510 Meta confocal microscope. Bar=10 μm.
To determine whether the decrease in VLDL 1 assembly was accompanied by disruption of the Golgi apparatus, we examined McA-RH7777 cells transiently expressing the stalk region of the Golgi enzyme N-acetylgalactosaminyl-transferase-2 (GalNac-T2) fused to yellow fluorescent protein (YFP; yellow staining in Figure 2C). After 240 minutes of T31N induction, Golgi structures containing GalNAc-T2 were still present, indicating that the Golgi apparatus was not completely disrupted and fused with the ER (Figure 2C).
To determine whether the inhibition of VLDL 1 assembly by ARF1 T31N coincides with inhibition of anterograde transport, we injected McA-RH7777 cells with a thermo-sensitive mutant of the vesicular stomatitis virus (VSV)-G protein (fused to GFP); after 4 to 6 hours at 39°C, the cells were placed in the temperature chamber (set at 32°C) of an inverted microscope and examined at 1-minute intervals after induction of T31N ARF 1 expression (Figure 3). At the start of the experiment, GFP fluorescence was diffuse and consistent with localization in the ER (Figure 3; 0 minutes induction of T31N and recording after 1 minute at the permissive temperature). In the uninduced control (Figure 3; 0 minutes induction), fluorescence appeared in the Golgi apparatus after 3 to 5 minutes (indicated by white arrows); at 30 minutes, the plasma membrane was clearly labeled (indicated by red arrow). Similar results were obtained after 60 and 120 minutes of induction. However, at 240 minutes, most of the fluorescence from the tagged VSV-G protein remained diffuse; virtually no protein had translocated to the Golgi apparatus (although small amounts could perhaps be seen after 30 minutes), indicating severe inhibition of anterograde transport from the ER to the Golgi apparatus.
Figure 3. Overexpression of the dominant-negative T31N ARF 1 inhibits the transport between ER and Golgi. The effect of T31N expression on the intracellular transport of a thermo-sensitive VSV-G mutant (ts 045) was investigated. The mutant protein was fused to GFP and microinjected into McA-RH7777 cells (stably transfected as in Figure 2A), which were kept at 39°C for 4 to 6 hours and transferred to an inverted microscope with the temperature chamber set at 32°C. Images were acquired at 1-minute intervals after addition of tetracycline to induce T31N expression (induction of T31N: 0 minutes, movie 1; 60 minutes, movie 2; 120 minutes, movie 3; 240 minutes, movie 4).
To further address the possibility that the VLDL 1 assembly was dependent on the anterograde transport through the secretory pathway, the effect of lowering the temperature to 15°C was investigated (Figure 4). At this temperature, the intracellular transport proceeds to the ERGIC but the proteins fail to reach cis-Golgi. The cells were pulsed with [35S]-methionine-cysteine for 10 minutes at 37°C and then chased for 20 minutes at 37°C or at 15°C, and the production of VLDL 1 was followed. The results (Figure 4) showed that the intracellular pool of radioactive apoB-100 VLDL 1 increased when the cells were chased at 37°C, whereas no increase could be seen when the chase was performed at 15°C. There was no effect of the lowering of the temperature on the expression of transferrin (not shown).
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Figure 4. The assembly of VLDL 1 is inhibited at 15°C. The effect on the VLDL 1 assembly of a lowering of the temperature to 15°C was investigated. Cells were pulse-labeled with [35S]-methionine-cysteine for 10 minutes (at 37°C) and chased for 20 minutes at 15°C or 37°C. After the pulse, as well as after each chase period, the content of the microsomal fraction was isolated and VLDL 1 recovered by gradient ultracentrifugation and the apoB-100 radioactivity was determined. Results are mean±SD; n=3.
PLD1 Increases the Secretion of ApoB-100–Containing Lipoproteins
Because PLD1 is activated by ARF117–19 and is important for anterograde transport,14 we assessed its effects on VLDL assembly in McA-RH7777 cells transiently transfected with vectors encoding wild-type PLD1 or an inactive mutant (K898R, control), both fused to GFP (see Figure IA, available online at http://atvb.ahajournals.org). Wild-type PLD1 induced a significant increase in the secretion of apoB-100 VLDL 1 (P=0.0016) and VLDL 2 (P=0.049) (Figure IA). Cells transfected with constructs encoding only the K898R mutant or GFP alone secreted similar amounts of apoB-100, indicating that the mutant did not have a dominant-negative effect (not shown).
Overexpression of Wild-Type ARF1 Increase the Secretion of VLDL 2 but not of VLDL1
To assess the effects of ARF1 overexpression on the secretion of apoB-100 and apoB-100–containing VLDL, ARF1 wild-type were stably transfected to McARH 7777 cells using the inducible t-rex system.12 Pulse-chase studies were performed 24 hours after induction of ARF1 expression (by the addition of tetracycline) in cells cultured in the presence or absence of oleic acid. Overexpression of ARF1 increased VLDL 2 secretion under both conditions but did not influence VLDL 1 secretion; however, total apoB-100 secretion increased only in cells cultured in the absence of oleic acid (Figure IB). Culturing the cells with different amounts of oleic acid showed a dose-dependent increase in VLDL 1 secretion (Figure IC) and a simultaneous decrease in VLDL 2 secretion (Figure ID). As a result, the VLDL 1/VLDL 2 ratio increased from 1.1±0.8 to 5.0±0.7 (mean±SD; n=4). Thus, the availability of fatty acids and the oleic acid-induced increase in the rate of the biosynthesis of triglycerides (not shown) appear to be major determinants of VLDL 1 formation.
Discussion
In this study, we showed that the dominant-negative ARF1 mutant T31N inhibited the appearance of VLDL 1 in the secretory pathway of McA-RH7777 cells to a much greater extent than it inhibited the expression of total apoB-100 and transferrin, indicating that ARF1 is essential for the second step of the VLDL 1 assembly. However, overexpression of ARF1 wild-type did not increase the assembly of VLDL 1; instead, such an overexpression increased the production of VLDL 2. The secretion of VLDL 1 and VLDL 2 was stimulated by PLD 1. Finally, we observed that the assembly of VLDL 1 was increased by oleic acid while the secretion of VLDL 2 decreased.
ARF1 and COPI are important for the sorting and transport of proteins from the ER to the Golgi apparatus.15,16,21 Thus, ARF1 could promote the transfer of pre-VLDL from the site of synthesis in the rough ER to the smooth membrane compartment, where the second step occurs.10 Like other secreted proteins, apoB-100 in a non-VLDL form leaves the ER in Sar1/COPII vesicles,22 which participate in the formation of ERGIC. COPI (and ARF1) are essential for the anterograde transport of the ERGIC to the cis-Golgi. In the assembly of VLDL 1, ARF1 may promote the transfer of pre-VLDL to the cis-Golgi. Consistent with such a role, the decrease in VLDL 1 after 240 minutes of T31N ARF 1 induction coincided with the loss of ?- COP from the Golgi apparatus. Activation of ARF1 initiates the binding of COP I to the membrane of the secretory pathway, an important step in the formation of transport vesicles and ARF1/COPI-dependent sorting events.15,16,21 Because the ERGIC must interact with COPI before it can be transported to the cis-Golgi,23,24 the effect of T31N ARF 1 on VLDL 1 assembly may reflect inhibition of anterograde transport. Such a mechanism is supported by our observation that the inhibition of VLDL 1 assembly coincided with the loss of transfer of VSV-G protein from the ER to the Golgi. An important role of the anterograde transport in the assembly of VLDL 1 is supported by the total inhibition of the assembly at 15°C. At this temperature the anterograde transport is blocked and the proteins reach ERGIC but not cis-Golgi.25,26 Thus, together our results are consistent with that ARF1 promotes the anterograde transport of apoB into the second-step compartment. This could indicate that the second step occurs in a Golgi compartment, consistent with the results of subcellular fractionation studies.10
PLD1 also appears to participate in anterograde transport27 and is activated by ARF1. Overexpression of PLD1 gave rise to small but statistically significant increases in the secretion of both VLDL 1 and VLDL 2, confirming our results in a cell-free system,12 which suggested that PLD is important for the second step of VLDL assembly. The small effect of PLD 1 in intact cells may be caused by a dual role of PLD 1. We have recently presented evidence that PLD activity is involved in the assembly of cytosolic lipid droplets.28 Thus, PLD 1 may promote the formation of the lipoproteins, but this effect may be counteracted by a diversion of triglycerides into cytosolic lipid droplets.
Our findings in this study, together with previously published results, can be summarized in a hypothesis for the role of ARF1 in the assembly of VLDL 1 (Figure 5). The assembly process starts with the cotranslational lipidation of apoB-100, resulting in a primordial "pre-VLDL" particle.2,3 The amount of apoB-100 in pre-VLDL is determined, at least in part, by cotranslational degradation.5–8 The formed pre-VLDL is either converted to VLDL or sorted to post-translational degradation.2,3,8 The assembly of VLDL 1 is dependent on functional ARF1, most likely indicating that the transport through the secretory pathway must be intact for pre-VLDL to be transferred from its site of assembly in the rough ER to the smooth compartment of the second step10 (Figure 5). If ARF1 is inhibited, the conversion of pre-VLDL to VLDL 1 ceases and pre-VLDL is instead sorted to degradation. ARF1 is rate-limiting in the assembly and secretion of VLDL 2 but not of VLDL 1. This observation may be explained by an increased capacity to transfer pre-VLDL to the second-step compartment, resulting in increased assembly of VLDL 2 but not of VLDL 1, which was highly dependent on the availability of fatty acids. Thus, increased transport of pre-VLDL into the second-step compartment is not directly reflected in increased secretion of VLDL 1 unless sufficient amounts of fatty acids (and triglycerides) are available.
Figure 5. A model for the role of ARF1 and fatty acids in the assembly of VLDL 1 and VLDL 2. In the first step, which occurs cotranslationally, microsomal triglyceride transfer protein (MTP) catalyzes the partial lipidation of apoB-100, forming a pre-VLDL particle that is transported by an ARF1-dependent mechanism to the smooth membrane compartment, where the second step occurs. The secretion of VLDL 1 is determined by the availability of fatty acids (and the rate of triglyceride biosynthesis). Thus, in the presence of sufficient amounts of fatty acids and triglycerides, the majority of the pre-VLDL is converted to VLDL 1. An increase in the transport capacity (by overexpression of ARF 1) will increase the pre-VLDL in the second-step compartment above the VLDL 1 lipidation capacity resulting in a channeling of pre-VLDL into VLDL 2 and a subsequent increase in the secretion of this form of VLDL (A). During a shortage of fatty acids (B), VLDL 1 secretion decreases and pre-VLDL is channeled to VLDL 2. An increase of the transport capacity (by overexpression of ARF 1) results in a further increase in the formation of VLDL 2.
Acknowledgments
This study was supported by grant 7142 from the Swedish Medical Research Council and by the Swedish Heart and Lung Foundation, Novo Nordic Foundation, Swedish Strategic Funds (National Network and Graduate School for Cardiovascular Research), and S?derbergs Foundation. The time lapse investigations were performed at the Swegene center for cellular imaging in G?teborg
Received August 19, 2004; accepted December 1, 2004.
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