A unique lysophospholipid acyltransferase (LPAT) antagonist, CI-976, affects secretory and endocytic membrane trafficking pathways

Kimberly Chambers, Bret Judson and William J. Brown*

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA

* Author for correspondence (e-mail: wjb5{at}cornell.edu)

Accepted 12 April 2005


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 Materials and Methods
 Results
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Previous studies have shown that inhibition of a Golgi-complex-associated lysophospholipid acyltransferase (LPAT) activity by the drug CI-976 stimulates Golgi tubule formation and subsequent redistribution of resident Golgi proteins to the endoplasmic reticulum (ER). Here, we show that CI-976 stimulates tubule formation from all subcompartments of the Golgi complex, and often these tubules formed independently, i.e. individual tubules usually did not contain markers from different subcompartments. Whereas the cis, medial and trans Golgi membranes redistributed to the ER, the trans Golgi network (TGN) collapsed back to a compact juxtanuclear position similar to that seen with brefeldin A (BFA) treatment. Also similar to BFA, CI-976 induced the formation of endosome tubules, but unlike BFA, these tubules did not fuse with TGN tubules. Finally, CI-976 produced an apparently irreversible block in the endocytic recycling pathway of transferrin (Tf) and Tf receptors (TfRs) but had no direct effect on Tf uptake from the cell surface. Tf and TfRs accumulated in centrally located, Rab11-positive vesicles indicating that CI-976 inhibits export of cargo from the central endocytic recycling compartment. These results, together with previous studies, demonstrate that CI-976 inhibits multiple membrane trafficking steps, including ones found in the endocytic and secretory pathways, and imply a wider role for lysophospholipid acyltransferases in membrane trafficking.

Key words: Golgi complex, Endosome, Membrane tubules, Lysophospholipid acyltransferase, CI-976


    Introduction
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Membrane tubules emanate from various regions of the Golgi complex and endosomes, and are believed to play a role in retrograde trafficking from the Golgi complex to the endoplasmic reticulum (ER) and recycling to the cell surface, respectively (Harter and Reinhard, 2000Go; Jackson, 2000Go; Lippincott-Schwartz et al., 2000Go; Presley et al., 1997Go; Sciaky et al., 1997Go). Recent studies revealed that a cytoplasmic phospholipase A2 (PLA2) enzyme is involved in tubule formation (de Figueiredo et al., 2001Go; de Figueiredo et al., 1998Go; de Figueiredo et al., 2000Go; de Figueiredo et al., 1999Go; Drecktrah and Brown, 1999Go; Polizotto et al., 1999Go). For example, brefeldin A (BFA)-stimulated Golgi tubulation is potently inhibited when cells are treated with membrane-permeant antagonists of PLA2 activity (de Figueiredo et al., 1998Go). Furthermore, stimulators of PLA2, melittin and PLA2 activating protein peptide (PLAPp), enhance cytosol-dependent Golgi tubulation in vitro and in a permeabilized cell system (Polizotto et al., 1999Go). PLA2 antagonists also inhibit constitutive retrograde trafficking from the Golgi complex to the ER (de Figueiredo et al., 2000Go). These results strongly suggest that membrane tubule formation is mediated in part by modification of membrane phospholipids (PLs).

Hydrolysis of PLs by a cytosolic PLA2 may cause a localized accumulation of lysophospholipids (LPLs) in the outer leaflet of the Golgi membrane bilayer (Brown et al., 2003Go). Such elevated LPL levels may increase membrane curvature leading to tubule formation (Christiansson et al., 1985Go; Fujii and Tamura, 1979Go). Therefore, Golgi tubulation might be mediated by controlling the lipid content of Golgi membranes. Lysophospholipid acyltransferases (LPATs) catalyze the reverse reaction of PLA2 by reacylating LPLs at the sn-2 position. In fact, an LPAT activity is known to be associated with the Golgi complex (Chambers and Brown, 2004Go; Lawrence et al., 1994Go), which could be involved in regulating the lipid composition and, thus, membrane structure, i.e., tubule formation. Interestingly, LPL acylation has been implicated as a common ingredient in several membrane fission events. The lysophosphatidic acid (LPA)-specific acyltransferase (LPAAT) activity of endophilin I has been reported to play a role in the formation of synaptic-like microvesicles (Schmidt et al., 1999Go), although subsequent studies question whether the catalytic activity is required (Farsad et al., 2001Go). An unrelated protein, C-terminal binding protein/BFA-ADP-ribosylated substrate (CtBP/BARS) was also found to exhibit LPAAT activity, and this enzyme is able to induce the fission of Golgi membrane tubules into vesicles during mitotic disassembly of the Golgi complex (Carcedo et al., 2004Go; Weigert et al., 1999Go). Again, however, the LPAAT activity of CtBP/BARS may facilitate but not be required for the fission reaction (Carcedo et al., 2004Go). So, although no definitive examples of LPAATs that are required for fission have been uncovered, mounting evidence suggests that alterations in membrane bilayer lipid composition could have a direct effect on membrane structure and trafficking events (Brown et al., 2003Go; Burger, 2000Go; Corda et al., 2002Go; Scales and Scheller, 1999Go).

Because our previous studies implicated a role for PLA2-generated LPL production in Golgi membrane tubulation, we were interested to see what effect, if any, inhibition of LPL metabolism would have on the Golgi complex. These studies led to the identification of a novel inhibitory activity for a previously characterized antagonist of acyl-CoA cholesterol acyltransferase (ACAT), CI-976 (Harte et al., 1995Go). We found that CI-976 was a potent inhibitor of a Golgi-associated LPAT activity that displayed a preference for lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) (Chambers and Brown, 2004Go; Drecktrah et al., 2003Go). Remarkably, CI-976 caused a dramatic stimulation of Golgi tubule formation and redistribution of resident enzymes back to the ER. Importantly, pretreatment of cells with PLA2 antagonists inhibited CI-976 from inducing tubules, a result which strongly suggests that the CI-976 effect is dependent on the accumulation of LPLs, i.e. the substrate for LPATs. These results are consistent with the idea that lipid modifying enzymes, specifically PLA2s and LPATs, can regulate membrane tubule formation by controlling the lipid composition on one-half of a lipid bilayer.



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Fig. 1. CI-976 induces tubule formation from all Golgi compartments. Clone 9 rat hepatocytes were treated with solvent control (A,D,G,J,M) or 20 µM CI-976 (B,C,E,F,H,I,K,L, N, O) for various amounts of time as indicated. Cells were then fixed and stained for immunofluorescence localization of various Golgi marker proteins: GPP130 (A-C), ManII (G-I), TGN38 (J-L) and M6PR (M-O). KDEL-R was visualized by transiently transfecting Clone 9 cells with a mutant form of the KDEL-R coupled to GFP (D-F). After 24 hours, the cells were then treated with 20 µM CI-976 and fixed.

 
To understand better the role that PL alterations play in the formation of membrane tubules from the Golgi complex, we have further characterized the effects of CI-976 on organelle morphology and membrane trafficking. We find that CI-976 has several novel properties including its ability to stimulate tubule formation independently from all regions of the Golgi complex, suggesting that a CI-976-sensitive LPAT is located throughout the Golgi stack. In the course of these studies we unexpectedly found that CI-976 also inhibits export of transferrin (Tf) and Tf receptors (TfRs) from centrally located recycling endosomes back to the cell surface. Together, the results show that CI-976 has the unique property of inhibiting both secretory and endocytic membrane trafficking.


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 Materials and Methods
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Materials and reagents
ACAT inhibitors CI-976 and DuP-128 were provided by GlaxoSmithKline Pharmaceuticals (Essex, UK) and PKF-058-035 was provided by Novartis Pharmaceuticals Corporation (Summit, NJ). All were stored as 5 mM stock solutions in dimethyl sulfoxide (DMSO) at 4°C. Alexa Fluor® 568-dextran (Alexa568-dextran), DiI-conjugated human low-density lipoprotein (DiI-LDL), and Alexa Fluor® 488 streptavidin-conjugated biotinylated epidermal growth factor (Alexa488-EGF) were purchased from Molecular Probes (Eugene, OR). Alexa568-dextran was stored as a 25 mg ml–1 stock solution in water at –20°C and then diluted into media as described below. Alexa Fluor® 568-transferrin (Alex568-Tf) and TRITC-transferrin (TRITC-Tf) were purchased from Molecular Probes (Eugene, OR). They were stored as 5 mg ml–1 stock solutions in PBS at 4°C and then diluted into media to 20 µg ml–1. All other chemicals were purchased from Sigma Chemical Co. (St Louis, MO).

The following antibodies were generously supplied to us: rabbit polyclonal anti-{alpha}-mannosidase II (Kelly Moremen, University of Georgia); mouse monoclonal anti-GS28 (Dr Wanjin Hong, Institute of Molecular and Cell Biology, Singapore); and rabbit polyclonal anti-GPP130 (Adam Linstedt, Carnegie Mellon University). Mouse monoclonal anti-TGN38 antibody was purchased from Affinity BioReagents (Golden, CO). All secondary fluorescent antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Jennifer Lippincott-Schwartz (NICHD, Bethesda, MD) kindly provided the expression vector that encodes a cis Golgi-restricted mutant form of the KDEL-R coupled to GFP (Sciaky et al., 1997Go). Mouse monoclonal anti-transferrin receptor (TfR) antibody was purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN). Marino Zerial kindly provided the expression vector that encodes Rab11 coupled to GFP (Sonnichsen et al., 2000Go).

Cell culture, transfection and fluorescence microscopy
Clone 9 rat hepatocytes and HeLa cells were maintained in minimal essential medium (MEM) with 10% Nu-Serum and 1% penicillin/streptomycin at 37°C in an atmosphere of 95% air and 5% CO2. For some experiments, Clone 9 cells were transfected with CLONfectin from CLONTECH (Palo Alto, CA) as described by the manufacturer. Clone 9 cells were grown on glass coverslips for 2 days before experiments were performed. Cells were washed three times with serum-free MEM and incubated in serum-free MEM containing inhibitors at the concentrations and for the times indicated in the Results. In previous studies, we found that the IC50 of CI-976 for a rat liver Golgi membrane-associated LPAT is ~15 µM and that at 50 µM the activity was completely inhibited (Drecktrah et al., 2003Go). Also, treatment of cells with CI-976 between 20-50 µM induced reversible Golgi tubulation. Therefore, for experiments here we used concentrations between 20-50 µM. We note that CI-976 was not active in media containing serum.

For immunofluorescence microscopy, cells were fixed with either 3.7% formaldehyde or 100% methanol at –20°C (for TGN38). Primary antibodies used were: mouse anti-GS28 diluted 1:100, mouse 10E6 diluted 1:100, rabbit anti-GPP130 diluted 1:500, rabbit anti-ManII diluted 1:1000, mouse anti-TGN38 diluted 1:100, rabbit anti-M6PR diluted 1:2000. Secondary antibodies used were: goat anti-mouse FITC diluted 1:100, donkey anti-rabbit TRITC diluted 1:100, and goat anti-mouse rhodamine diluted 1:100. When staining for TGN38, 1% BSA was added to the primary and secondary antibodies. For most experiments, cells were viewed by wide-field epifluorescence (Zeiss Axioskop 2). In other experiments, cells were examined with a Perkin-Elmer UltraView spinning disc confocal microscope.

For CI-976 dose-response experiments on Tf recycling, HeLa cells were plated on glass coverslips 2 days prior to the experiment. Cells were briefly washed in MEM minus serum and then incubated for 45 minutes in the presence of Alexa568-Tf. Following the transferrin uptake, cells were washed as above and incubated in various concentrations of CI-976 for 1 hour. Cells were then fixed and processed for immunofluorescence as above. To quantify the effects of CI-976 on the recycling of Tf, cells were imaged and classified for brightness using Openlab (Improvision, Lexington, MA) software. Using the Region of Interest (ROI function), a region was drawn around the perimeter of each cell (a minimum of 30 cells per experiment) and the mean pixel intensity was calculated. To correct for background fluorescence a ROI of an area devoid of cells was measured for pixel intensity. The background pixel intensity was then subtracted from all of the means to give accurate pixel intensity.

For the live cell imaging of endosome tubulation induced by CI-976, HeLa cells stably expressing galactosyltransferase-GFP (HeLa-GalT-GFP) (Storrie et al., 1998Go) cells were used. Cells were allowed to endocytose Alexa568-Tf for 45 minutes, washed to remove unbound transferrin, and then placed in media (MEM, 2.6 mM sodium bicarbonate, 5 mM HEPES) containing 25 µM CI-976 and imaged immediately on the spinning disc confocal microscope.


    Results
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Response of Golgi subcompartments to CI-976
In our initial studies, CI-976 was found to stimulate tubulation from medial and trans Golgi cisternae, as assayed by immunofluorescence of mannosidase II (ManII) and fluorescence microscopy of galactosyltransferase-GFP (GTase-GFP) (Drecktrah et al., 2003Go). To determine if other regions of the Golgi complex responded in a similar manner to CI-976, we examined a number of compartment-specific markers. The assumption is that wherever the LPAT acts in regulating tubulation, inhibition of that activity by CI-976 will stimulate that region of the Golgi complex to form tubules.

We first examined the cis Golgi (and CGN) using well-characterized antibodies or GFP constructs of markers found primarily in this region: GPP130 (Puri et al., 2002Go), GS28 (Subramaniam et al., 1996Go), a mutant form of the KDEL-R coupled to GFP (Sciaky et al., 1997Go), and the 10E6 antigen (Wood et al., 1991Go). With the exception of 10E6, all were found in tubules extending from the juxtanuclear Golgi region after short incubations (10-15 minutes) in CI-976 (Fig. 1B,E). By 20-30 minutes, all of the cis Golgi markers, including 10E6, started to become diffuse in the cytoplasm in an ER-like pattern. After extended periods of time (>50 minutes), a large proportion of the cells had this diffuse staining pattern (Fig. 1C,F). The medial marker, ManII, behaved similar to the cis markers (Fig. 1G-I). Two markers of the trans Golgi network (TGN), TGN38 and the mannose 6-phosphate receptor (M6PR), were also found in tubules following short-term treatment (10-15 minutes) with CI-976; however, these markers were not found diffuse in the cytoplasm after extended periods of time (>50 minutes) (Fig. 1J-O). Instead, they assumed a juxtanuclear distribution, similar but not identical to control cells in that stained elements were more tightly clustered following long-term CI-976 treatment (Fig. 1L,O). This redistribution of TGN was different than cis, medial or the trans markers, all of which assumed a diffuse ER-like pattern after an extended period of time in CI-976. To determine which subcompartment was most responsive to CI-976, we counted the number of cells with tubules as revealed by each maker antibody. These studies were complicated by the fact that some antibodies produce brighter signals than do others. However, the use of multiple antibodies against cis or trans compartments provided a measure of confidence in the results that suggested the following order of responsiveness to CI-976: medial ≥trans>cis. In addition, the ability of CI-976 also to induce tubule formation in HeLa cells (data not shown) indicates that this effect is not cell-type specific.



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Fig. 2. Tubules can form independently from Golgi subcompartments following CI-976 treatment. Clone 9 rat hepatocytes were treated with solvent control (A,B) or 20 µM CI-976 (C-H) for various amounts of time as indicated. Cells were then fixed and processed for double-immunofluorescence staining of Golgi marker proteins, ManII (A,C,E,G) and TGN38 (B,D,F,H).

 



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Fig. 3. TGN and early endosomes do not fuse. Clone 9 cells were treated with solvent control (A) or 20 µM CI-976 (B) for 50 minutes. Cells were then labeled with 1 mg ml–1 Alexa568-dextran for 5 minutes prior to fixing and staining for immunofluorescence localization of M6PR. The sets of three images (A1-A3 and B1-B3) are from consecutive slices in the Z plane taken using a spinning disc confocal microscope (Perkin-Elmer).

 
To determine if these subcompartment-derived tubules reflected an extensive mixing of membranes via cisternal fusion as occurs following BFA treatment (Lippincott-Schwartz et al., 1990Go; Lippincott-Schwartz et al., 1989Go), cells were treated with CI-976 and then processed for double-immunofluorescence staining with cisterna-specific antibodies. The results showed that within a single cell, multiple tubules could form, each of which contained a single cisternal marker (Fig. 2). Occasionally, single tubules could also be seen to contain two cisternal markers (Fig. 2G,H). These results suggest that CI-976 does not cause wholesale fusion of Golgi cisternae and that cis, medial and trans regions are all sensitive to CI-976.

TGN and early endosomes do not fuse
As noted above, the two TGN markers, TGN38 and M6PR, were found in tubules following short-term treatment with CI-976, but after extended periods of time they were more tightly clustered. This response is similar to that seen in BFA-treated cells, in which the TGN fuses with the early endosomes (Lippincott-Schwartz et al., 1991Go; Wood et al., 1991Go). To determine whether the TGN was behaving similarly in the presence of CI-976, cells were treated with 20 µM CI-976 for 50 minutes and then labeled with Alexa568-dextran for 5 minutes, while still in CI-976, to allow endocytic delivery to early endosomes. M6PRs served as a marker for the TGN and its fusion with early endosomes as demonstrated in previous studies with BFA (Wood and Brown, 1992Go; Wood et al., 1991Go). The results of confocal imaging showed that in control (Fig. 3A1-A3) and CI-976-treated cells (Fig. 3B1-B3), Alexa568-dextran internalized for 5 minutes did not co-localize with M6PRs to any significant extent. We conclude that the TGN and early endosomes do not fuse in the presence of CI-976.



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Fig. 4. Endosome tubule formation is stimulated by CI-976. HeLa cells were incubated with Alexa568-Tf for 45 minutes to label endocytic compartments, CI-976 (25 µM) was added, and live cells were imaged immediately by spinning disk confocal microscopy. The time interval between each panel is 6.4 seconds. The arrows point to tubules that formed during the course of these observations. The micrographs are inverted images from the originals.

 
Endosome tubule formation induced by CI-976
We observed that, similar to the Golgi complex, endosomes appeared to tubulate after incubation in CI-976. To investigate this effect more closely, cells were imaged live following labeling with Alexa568-Tf. In control cells, endosome tubule formation was not appreciable (data not shown). However, in 25 µM CI-976 it was clear that some endosomes began to form tubules almost immediately. This tubulation appeared at times to be transient, which is to say that tubules were seen moving throughout the cytoplasm, but at other times tubules appeared to be stable among structures in the cell (Fig. 4).

CI-976 does not inhibit initial uptake of Tf
Because CI-976 did not cause the TGN to fuse with endosomes but did cause endosomes to form tubules, we wondered if CI-976 might also be affecting endocytic compartments and trafficking. To investigate this issue, the well-characterized intracellular trafficking of TfR and its ligand Tf were followed (Maxfield and McGraw, 2004Go). First we assessed whether CI-976 affected initial uptake of Tf. TRITC-Tf was bound to Clone 9 cells for 2 hours at 4°C, and the unbound TRITC-Tf was removed by extensive washing. Cells were then treated for 15 minutes with 20 µM CI-976 (or DMSO as a solvent control) at 4°C prior to shifting to 37°C for 5 minutes to allow TRITC-Tf uptake. Treatment with CI-976 did not prevent uptake of TRITC-Tf from the cell surface (Fig. 5).



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Fig. 5. CI-976 does not inhibit initial uptake of Tf. TRITC-Tf was pre-bound to the surface of Clone 9 cells at 4°C. Following 15 minutes treatment with either (A) DMSO as a solvent control or (B) 20 µM CI-976 at 4°C, the cells were shifted to 37°C for 5 minutes to allow Tf uptake.

 



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Fig. 6. Tf accumulates in a juxtanuclear cluster in cells treated with CI-976. Clone 9 cells were treated for 10 minutes with (A) DMSO as a solvent control, (B) CI-976 (20 µM), (C) DuP-128 (5 µM) or (D) PKF-058-035 (10 µM). TRITC-Tf was then added (in the continuous presence of compounds indicated above) for 45 minutes at 37°C.

 
Tf accumulates in a juxtanuclear cluster in cells treated with CI-976
Although CI-976 had little effect on a brief pulse of Tf uptake, the drug could affect later steps in the endocytic pathway. To investigate this question, Clone 9 cells were pretreated with 20 µM CI-976 for 10 minutes and then in its continued presence, the cells were incubated with TRITC-Tf for 45 minutes to label all endocytic compartments. In control cells, TRITC-Tf was found in numerous punctate structures located throughout the cytoplasm indicative of its uptake and delivery to multiple endocytic compartments, including early sorting and the central endocytic recycling compartment (Fig. 6A). In CI-976-treated cells, however, TRITC-Tf was found in a more compact, juxtanuclear staining pattern (Fig. 6B). To determine whether this effect was due to inhibition of an ACAT activity, we used the more specific and potent ACAT inhibitors, DuP-128 and PKF-058-035 that have no effect on the Golgi-associated LPCAT (Drecktrah et al., 2003Go). Even at concentrations higher than the IC50s for ACAT inhibition (Harte et al., 1995Go; Patankar and Jurs, 2000Go), these antagonists had no effect (Fig. 6C,D). Therefore, the effect of CI-976 on Tf trafficking is not due to ACAT inhibition.

This compact juxtanuclear staining pattern suggests that CI-976 was inhibiting the recycling of Tf. To examine this further, and to establish that the CI-976 effect is not cell-type specific, a series of dose-response experiments on Tf recycling were quantified using HeLa cells. As shown in Fig. 7, it is clear that CI-976 inhibits recycling at >2 µM and that the effect becomes more pronounced up to 50 µM. These experiments could only be done up to 50 µM CI-976 because cells became unhealthy at higher concentrations. These results suggest that CI-976 inhibited the exit of Tf from the central endocytic recycling compartment.



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Fig. 7. CI-976 inhibits recycling of Tf. (A-C) Fluorescence micrographs of HeLa cells labeled with TRITC-Tf. (A) Cells incubated with TRITC-Tf for 45 minutes and then fixed. (B) Cells pulse-labeled by TRITC-Tf uptake for 45 minutes followed by a chase without Tf for 25 minutes. (C) Same treatments as B except the chase was in the presence of CI-976 (25 µM). (D) Quantitation of inhibition of Tf recycling in the presence of different concentrations of CI-976; experiments performed as in A-C. Results are expressed as the mean with the error bars representing 1 s.d.

 

To determine if TfRs also accumulate in this juxtanuclear cluster, HeLa cells were treated as described immediately above and then stained for immunofluorescence localization of TfR. In control cells, Tf and TfR were co-localized in both peripheral sorting and central recycling endosomes (Fig. 8A,B). In the presence of CI-976, Tf and TfR were found to co-localize in a compact, juxtanuclear staining pattern (Fig. 8C,D), suggesting that recycling of TfRs in these cells is similarly inhibited by CI-976.



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Fig. 8. Tf and TfR accumulate in a juxtanuclear cluster of tubulo-vesicular organelles in HeLa cells treated with CI-976. Cells were treated for 10 minutes with DMSO as a solvent control (A,B) or 20 µM CI-976 (C,D). TRITC-Tf (A,C) was then added for 45 minutes at 37°C. Cells were then fixed and stained for immunofluorescence localization of TfR (B,D).

 
Treatment with CI-976 prevents recycling of TfR
To determine if CI-976 prevents recycling of TfRs back to the cell surface, Clone 9 cells were treated with 20 µM CI-976 for a longer period of time (50 minutes) prior to pulse labeling for 5 minutes with Alexa568-Tf. In control cells, a significant amount of Alexa568-Tf was endocytosed and delivered to peripheral early endosomes (Fig. 9A). By contrast, in CI-976 treated cells, very little Alexa568-Tf was endocytosed (Fig. 9D). The simplest interpretation of these results, and consistent with the above studies, is that CI-976 inhibits recycling of TfRs from internal endosome compartments, thereby preventing subsequent uptake of Tf from the cell surface. We note that when this experiment was repeated with HeLa cells, some Tf did seem to enter the cells, although TfRs still accumulated in a juxtanuclear cluster. The difference in the ability of Clone 9 and HeLa cells to endocytose Tf after 50 minutes of CI-976 treatment may be due to a difference in the amounts of TfRs present in these cell types.



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Fig. 9. Long-term treatment (50 minutes) with CI-976 prevents subsequent uptake of Tf from the cell surface. Clone 9 cells were treated for 50 minutes at 37°C with DMSO as a solvent control (A-C) or 20 µM CI-976 (D-F). Alexa568-Tf (A,D) was then added for 5 minutes at 37°C. Cells were fixed and stained for immunofluorescence localization of M6PRs (B,E). These results are consistent with the conclusion that CI-976 inhibits recycling of TfRs back to the cell surface.

 

To see if other endocytic trafficking pathways were disrupted by CI-976, we examined the uptake and delivery of LDL via the endocytic degradation pathway to lysosomes. In these experiments, fluorescently labeled DiI-LDL and Alexa488-EGF were followed in pulse-chase experiments, and we found that CI-976 did not detectably inhibit the transport of LDL or EGF from peripheral early sorting endosomes to larger, centrally located late endosomes or lysosomes (Fig. 10). These results show that CI-976 had a selective effect on trafficking in the endocytic recycling pathway.



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Fig. 10. CI-976 does not qualitatively affect the uptake and delivery of DiI-LDL or Alexa488-EGF to centrally located late endosomal compartments. Clone 9 cells were pre-treated in the presence or absence of 25 µM CI-976 for 15 minutes, pulse labeled with DiI-LDL (5 µg ml–1) and Alexa488-EGF (2 µg ml–1) for 10 minutes, and then chased for either 15 minutes or 1 hour, as indicated on the figure. In treated cells, CI-976 was present throughout the pulse and chase periods.

 

Tf accumulates in Rab11-positive recycling endosomes
The central location of Tf/TfRs in CI-976-treated cells suggests that the endocytic recycling compartment is the site of export blockage (Maxfield and McGraw, 2004Go). To examine this further, Clone 9 cells were transiently transfected with a GFP-tagged Rab11 construct, a known marker of the central endocytic recycling compartment (Sonnichsen et al., 2000Go). The cells were then pretreated for 10 minutes with 20 µM CI-976 prior to labeling with Alexa568-Tf for 45 minutes in the continued presence of CI-976. The cells were visualized by confocal microscopy. Images shown are three consecutive slices 0.3 µm apart. In control cells in which Tf is delivered to all endocytic compartments, both individual and merged images show that Tf and Rab11 co-localize slightly (Fig. 11A1-A3). However, when cells were treated with CI-976, Tf and Rab11 had a high level of co-localization in a tight juxtanuclear staining pattern (Fig. 11B1-B3). This result shows that the Tf-TfR complexes accumulate in the central endocytic recycling compartment. Interestingly, in CI-976 treated cells, GFP-Rab11 also became more concentrated in a tight juxtanuclear position, suggesting that its cycling is also affected by CI-976. Although Tf-TfR accumulated in the compact endocytic recycling compartment following CI-976 treatment, electron microscopy revealed no obvious differences in the morphology of the compartment (data not shown).



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Fig. 11. Tf accumulates in Rab11-positive recycling endosomes. Clone 9 cells were transiently transfected with GFP-Rab11. After 24 h of transfection, the cells were then treated with (A) DMSO as a solvent control or (B) 20 µM CI-976 for 10 minutes at 37°C prior to labeling with Alexa568-Tf for 45 minutes at 37°C. Arrows indicate juxtanuclear cluster of vesicles that contain both GFP-Rab11 and Alexa568-Tf, as evidenced by the yellow merged images in B1-B3.

 

Tf recycling is irreversibly inhibited by CI-976
To determine if the effect of CI-976 on the Tf trafficking pathway was reversible, cells were pretreated with CI-976 for 10 minutes and then pulse-labeled with TRITC-Tf for 45 minutes to accumulate Tf in the endocytic recycling compartment. Cells were then extensively washed and chased in medium without CI-976. As above, in control cells with no chase, TRITC-Tf was found in peripheral and central endosomes (Fig. 12A), whereas in CI-976 treated cells TRITC-Tf was found in a tight juxtanuclear staining pattern (Fig. 12C). As expected in control cells following a 1 hour chase, TRITC-Tf staining was significantly reduced, consistent with its recycling and loss into the extracellular medium (Fig. 12B). However quite different results were obtained with CI-976 treated cells. Following a 1 hour chase in which CI-976 had been removed, TRITC-Tf was not lost from cells but instead remained in a tight juxtanuclear staining pattern (Fig. 12D). Interestingly, even when the chase was extended to 24 hours, TRITC-Tf remained in the juxtanuclear cluster (Fig. 12E). Similar experiments on HeLa cells showed that although Tf did not chase from the cells after 24 hours, Tf-positive vesicles were somewhat more diffuse throughout the cytoplasm, when compared to Clone 9 cells. Although recovery of TfR recycling was extremely slow, cell viability was not significantly affected and cells continued to grow indicating that recovery of recycling eventually occurs. These results suggest that the molecular target of CI-976 in the endocytic recycling pathway is very slowly reversible, and perhaps even irreversible. The slow recovery seen could be due to new protein synthesis.



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Fig. 12. Tf recycling appears to be irreversibly inhibited by CI-976. Clone 9 cells were treated for 10 minutes with DMSO as a solvent control (A,B) or 20 µM CI-976 (C-E). TRITC-Tf was then added for 45 minutes at 37°C. Cells were either fixed (A,C) or were quickly washed in MEM containing 10% Nu-Serum, and subsequently incubated in MEM with 10% Nu-Serum for 1 hour (B,D) or 24 hours (E) at 37°C prior to fixing.

 

    Discussion
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
These studies demonstrate that the unique LPAT and ACAT inhibitor CI-976 affects membrane trafficking in the secretory and endocytic pathways. Because other more specific ACAT inhibitors had no effect on the secretory and endocytic pathways, we conclude that CI-976 must be exerting its effects on LPAT enzymes. Although superficially similar to the effects of BFA on the Golgi complex and endosomes (Klausner et al., 1992Go), CI-976 almost certainly produces its effects by mechanisms that are completely different from those of BFA. Thus, CI-976 provides another versatile tool for dissecting steps in membrane trafficking pathways.

We have previously shown that CI-976 inhibits a novel Golgi-associated LPAT and that this compound stimulates tubule formation and retrograde trafficking to the ER (Drecktrah et al., 2003Go). Here we show that CI-976, similar to BFA, is capable of inducing tubule formation from all regions of the Golgi complex, including the TGN, and endosomes. However, whereas BFA induces tubule-mediated retrograde trafficking of Golgi membranes to the ER and fusion of the TGN with early endosomes (Lippincott-Schwartz et al., 1991Go; Wood et al., 1991Go), CI-976 induced only the former. In addition, CI-976 inhibited export of Tf/TfRs from a Rab11-positive recycling endosome compartment, whereas endocytic recycling is often stimulated by BFA (Lippincott-Schwartz et al., 1991Go; Wood et al., 1991Go). Interestingly, reassembly of the Golgi complex following recovery from CI-976 treatment was fairly rapid, whereas inhibition of export from recycling endosomes was only very slowly reversible. These results strongly suggest that the molecular targets of CI-976 are different in the Golgi complex and endosomes.

The idea that membrane curvature and function is controlled by enzyme-catalyzed PL/LPL conversion has recently been gaining support (Brown et al., 2003Go; Burger, 2000Go; Huijbregts et al., 2000Go; Huttner and Schmidt, 2002Go; Shemesh et al., 2003Go), although no exact enzymes have yet been identified. Our previous studies have shown that PLA2 antagonists are potent inhibitors of membrane tubule formation (de Figueiredo et al., 1998Go; de Figueiredo et al., 2000Go; de Figueiredo et al., 1999Go; Drecktrah and Brown, 1999Go; Polizotto et al., 1999Go). These results led us to propose that cytoplasmic PLA2 enzyme(s) may act to generate localized increases in inverted cone-shaped LPLs, thus contributing to an increase in outward membrane bending and tubule formation (Brown et al., 2003Go; de Figueiredo et al., 1998Go). By inhibiting the reacylation of these LPLs with CI-976, a similar increase in membrane LPL concentration results (Drecktrah et al., 2003Go), leading to the formation of tubules seen here.

In addition to increasing outward curvature and tubulation via the accumulation of LPC, CI-976 might also inhibit membrane trafficking by inhibiting the formation of inward curve inducing lipids, such as phosphatidic acid (PA) or diacylglycerol (DAG), that have been implicated in vesicle formation. For example, endophilins A and B and CtBP/BARS are LPAATs that catalyze the conversion of LPA to PA and have been implicated in membrane fission (Scales and Scheller, 1999Go; Schmidt et al., 1999Go; Weigert et al., 1999Go). In these cases, re-acylation of inverted cone-shaped LPA back to cylindrical or cone-shaped PA might contribute to inward bending that occurs at the neck of a budding vesicle (Burger, 2000Go; Huijbregts et al., 2000Go; Shemesh et al., 2003Go). PA could also be metabolized to DAG by phospholipase D, an enzyme implicated in coated vesicle formation including most recently, COPII vesicles (Bi et al., 1997Go; Pathre et al., 2003Go; Roth et al., 1999Go). Interestingly, one known target of BFA is CtBP/BARS (Spano et al., 1999Go). Since BFA and CI-976 have similar effects on the Golgi complex, CtBP/BARS may be a possible target of CI-976. However, CtBP/BARS is an LPA-specific LPAAT, whereas CI-976 was found to be selective for a Golgi-associated LPCAT (Chambers and Brown, 2004Go; Drecktrah et al., 2003Go). In addition, CtBP/BARS is capable of inducing fission of membrane tubules when its LPAAT activity is compromised (Carcedo et al., 2004Go). Thus, the Golgi target of CI-976 cannot be CtBP/BARS.

Although CI-976 does not appear to influence the activity of a Golgi LPAAT, it could affect unknown LPAATs involved in vesicle production from endosomes, thus accounting for its ability to inhibit export of Tf/TfRs from recycling endosomes. Many studies have provided evidence that coated vesicles may work in concert with membrane tubules to facilitate export from endosomes (Bonifacino and Glick, 2004Go; Bonifacino and Lippincott-Schwartz, 2003Go). In other words, CI-976 could inhibit different acyltransferases that influence membrane trafficking by two specific mechanisms. First, failure to reacylate LPLs generated by PLA2 activity on Golgi membranes would stimulate tubule formation leading to inappropriate retrograde trafficking to the ER. Second, CI-976 could inhibit an unknown LPAAT involved in membrane fission and vesicle production from recycling endosomes, thus accounting for the inhibition of Tf recycling. Inhibition of vesicle fission by CI-976 might also explain why relocated Golgi enzymes fail to exit the ER (Drecktrah et al., 2003Go). Thus, it is tempting to speculate that CI-976 might be a fission inhibitor specific for some (COPII?), but not all (e.g. AP-2 clathrin coated vesicles) vesiculation events.

If the effects of CI-976 are indeed due to the inhibition of an LPAT, then prior treatment with a PLA2 antagonist, to prevent the formation of LPLs, should abrogate or reverse the effects of CI-976. In support of this idea, pretreatment of cells with PLA2 antagonists prior to CI-976 inhibits Golgi membrane tubule formation and retrograde trafficking to the ER (Drecktrah et al., 2003Go). Thus, one might expect that PLA2 antagonists would reverse the inhibitory effects of CI-976 on Tf recycling. However, that experiment cannot be done in the context of endocytic recycling because we have previously shown that PLA2 antagonists on their own inhibit both endosome tubule formation and Tf recycling (de Figueiredo et al., 2001Go). Taken together, the data strongly suggest that recycling endosomes utilize a two-step process for efficient export and recycling: PLA2-facilitated tubule formation followed by LPAAT-facilitated membrane fission to generate vesicles. In this regard it is worth noting that CI-976 does not cause COPI proteins or ADP-ribosylation factor to dissociate from the Golgi complex (Drecktrah et al., 2003Go); therefore, its proposed effects on vesiculation are consistent with inhibition of processes that follow coat protein binding, i.e. fission.

In conclusion, like BFA, CI-976 has been shown to be a versatile tool for studying membrane trafficking events in both the secretory and endocytic pathways. Owing to differences in the effects of BFA and CI-976 (particularly on the endocytic pathway), the mechanisms of action on these two compounds are most certainly very different. Here we propose that CI-976 inhibits multiple acyltransferases involved in various steps of the secretory and endocytic pathways, which is consistent with recent proposals that acyltransferases influence membrane shape and function by regulating PL and LPL levels.


    Acknowledgments
 
We would like to thank Kelly Moremen, Wanjin Hong, Adam Linstedt, and Jennifer Lippincott-Schwartz and Marino Zerial for sharing antibodies and reagents. We also thank Brian Jackson of GlaxoSmithKline Pharmaceuticals for supplying the CI-976. This work was supported by NIH grants DK51596 and GM60373 (to W.J.B.).


    References
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

Bi, K., Roth, M. G. and Ktistakis, N. T. (1997). Phosphatidic acid formation by phospholipase D is required for transport from the endoplasmic reticulum to the Golgi complex. Curr. Biol. 7, 301-307.[CrossRef][Medline]

Bonifacino, J. S. and Lippincott-Schwartz, J. (2003). Coat proteins: shaping membrane transport. Nat. Rev. Mol. Cell Biol. 4, 409-414.[CrossRef][Medline]

Bonifacino, J. S. and Glick, B. S. (2004). The mechanisms of vesicle budding and fusion. Cell 116, 153-166.[CrossRef][Medline]

Brown, W. J., Chambers, K. and Doody, A. (2003). Phospholipase A2 (PLA2) enzymes in membrane trafficking: mediators of membrane shape and function. Traffic 4, 214-221.[Medline]

Burger, K. N. (2000). Greasing membrane fusion and fission machineries. Traffic 1, 605-613.[CrossRef][Medline]

Carcedo, C. H., Bonazzi, M., Spano, S., Turacchio, G., Colanzi, A., Luini, A. and Corda, D. (2004). Mitotic Golgi partitioning is driven by the membrane-fissioning protein CtBP3/BARS. Science 305, 93-96.[Abstract/Free Full Text]

Chambers, K. and Brown, W. J. (2004). Characterization of a novel CI-976-sensitive lysophospholipid acyltransferase that is associated with the Golgi complex. Biochem. Biophys. Res. Commun. 313, 681-686.[CrossRef][Medline]

Christiansson, A., Kuypers, F. A., Roelofsen, B., Op den Kamp, J. A. and van Deenen, L. L. (1985). Lipid molecular shape affects erythrocyte morphology: a study involving replacement of native phosphatidylcholine with different species followed by treatment of cells with sphingomyelinase C or phospholipase A2. J. Cell Biol. 101, 1455-1462.[Abstract]

Corda, D., Hidalgo Carcedo, C., Bonazzi, M., Luini, A. and Spano, S. (2002). Molecular aspects of membrane fission in the secretory pathway. Cell Mol. Life Sci. 59, 1819-1832.[Medline]

de Figueiredo, P., Drecktrah, D., Katzenellenbogen, J. A., Strang, M. and Brown, W. J. (1998). Evidence that phospholipase A2 activity is required for Golgi complex and trans Golgi network membrane tubulation. Proc. Natl. Acad. Sci. USA 95, 8642-8647.[Abstract/Free Full Text]

de Figueiredo, P., Polizotto, R. S., Drecktrah, D. and Brown, W. J. (1999). Membrane tubule-mediated reassembly and maintenance of the Golgi complex is disrupted by phospholipase A2 antagonists. Mol. Biol. Cell 10, 1763-1782.[Abstract/Free Full Text]

de Figueiredo, P., Drecktrah, D., Polizotto, R. S., Cole, N. B., Lippincott-Schwartz, J. and Brown, W. J. (2000). Phospholipase A2 antagonists inhibit constitutive retrograde membrane traffic to the endoplasmic reticulum. Traffic 1, 504-511.[CrossRef][Medline]

de Figueiredo, P., Doody, A., Polizotto, R. S., Drecktrah, D., Wood, S., Banta, M., Strang, M. and Brown, W. J. (2001). Inhibition of transferrin recycling and endosome tubulation by Phospholipase A2 antagonists. J. Biol. Chem. 276, 47361-47370.[Abstract/Free Full Text]

Drecktrah, D. and Brown, W. J. (1999). Phospholipase A2 antagonists inhibit nocodazole-induced Golgi ministack formation: evidence of an ER intermediate and constitutive cycling. Mol. Biol. Cell 10, 4021-4032.[Abstract/Free Full Text]

Drecktrah, D., Chambers, K., Racoosin, E. L., Cluett, E. B., Gucwa, A., Jackson, B. and Brown, W. J. (2003). Inhibition of a Golgi complex lysophospholipid acyltransferase induces membrane tubule formation and retrograde trafficking. Mol. Biol. Cell 14, 3459-3469.[Abstract/Free Full Text]

Farsad, K., Ringstad, N., Takei, K., Floyd, S. R., Rose, K. and De Camilli, P. (2001). Generation of high curvature membranes mediated by direct endophilin bilayer interactions. J. Cell Biol. 155, 193-200.[Abstract/Free Full Text]

Fujii, T. and Tamura, A. (1979). Asymmetric manipulation of the membrane lipid bilayer of intact human erythrocytes with phospholipase A, C, or D induces a change in cell shape. J. Biochem. 86, 1345-1352.[Abstract]

Harte, R. A., Yeaman, S. J., Jackson, B. and Suckling, K. E. (1995). Effect of membrane environment on inhibition of acyl-CoA:cholesterol acyltransferase by a range of synthetic inhibitors. Biochim. Biophys. Acta 1258, 241-250.[Medline]

Harter, C. and Reinhard, C. (2000). The secretory pathway from history to the state of the art. Subcell. Biochem. 34, 1-38.[Medline]

Huijbregts, R. P. H., Topalof, L. and Bankaitis, V. A. (2000). Lipid metabolism and regulation of membrane trafficking. Traffic 1, 195-202.[CrossRef][Medline]

Huttner, W. B. and Schmidt, A. A. (2002). Membrane curvature: a case of endofeelin'. Trends Cell Biol. 12, 155-158.[CrossRef][Medline]

Jackson, C. L. (2000). Brefeldin A revealing the fundamental principles governing membrane dynamics and protein transport. Subcell. Biochem. 34, 233-272.[Medline]

Klausner, R. D., Donaldson, J. G. and Lippincott-Schwartz, J. (1992). Brefeldin A: insights into the control of membrane traffic and organelle structure. J. Cell Biol. 116, 1071-1080.[CrossRef][Medline]

Lawrence, J. B., Moreau, P., Cassagne, C. and Morre, D. J. (1994). Acyl transfer reactions associated with cis Golgi apparatus of rat liver. Biochim. Biophys. Acta. 1210, 146-150.[Medline]

Lippincott-Schwartz, J., Yuan, L. C., Bonifacino, J. S. and Klausner, R. D. (1989). Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER. Cell 56, 801-813.[CrossRef][Medline]

Lippincott-Schwartz, J., Donaldson, J. G., Schweizer, A., Berger, E. G., Hauri, H. P., Yuan, L. C. and Klausner, R. D. (1990). Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway. Cell 60, 821-836.[CrossRef][Medline]

Lippincott-Schwartz, J., Yuan, L., Tipper, C., Amherdt, M., Orci, L. and Klausner, R. D. (1991). Brefeldin A's effects on endosomes, lysosomes, and the TGN suggest a general mechanism for regulating organelle structure and membrane traffic. Cell 67, 601-616.[CrossRef][Medline]

Lippincott-Schwartz, J., Roberts, T. H. and Hirschberg, K. (2000). Secretory protein trafficking and organelle dynamics in living cells. Annu. Rev. Cell Dev. Biol. 16, 557-589.[CrossRef][Medline]

Maxfield, F. R. and McGraw, T. E. (2004). Endocytic recycling. Nat. Rev. Mol. Cell Biol. 5, 121-132.[CrossRef][Medline]

Patankar, S. J. and Jurs, P. C. (2000). Prediction of IC50 values for ACAT inhibitors from molecular structure. J. Chem. Inf. Comput. Sci. 40, 706-723.[CrossRef][Medline]

Pathre, P., Shome, K., Blumental-Perry, A., Bielli, A., Haney, C. J., Alber, S., Watkins, S. C., Romero, G. and Aridor, M. (2003). Activation of phospholipase D by the small GTPase Sar1p is required to support COPII assembly and ER export. EMBO J. 22, 4059-4069.[Abstract/Free Full Text]

Polizotto, R. S., de Figueiredo, P. and Brown, W. J. (1999). Stimulation of Golgi membrane tubulation and retrograde trafficking to the ER by phospholipase A2 activating protein (PLAP) peptide. J. Cell Biochem. 74, 670-683.[CrossRef][Medline]

Presley, J. F., Cole, N. B., Schroer, T. A., Hirschberg, K., Zaal, K. J. and Lippincott-Schwartz, J. (1997). ER-to-Golgi transport visualized in living cells. Nature 389, 81-85.[CrossRef][Medline]

Puri, S., Bachert, C., Fimmel, C. J. and Linstedt, A. D. (2002). Cycling of early Golgi proteins via the cell surface and endosomes upon lumenal pH disruption. Traffic 3, 641-653.[CrossRef][Medline]

Roth, M. G., Bi, K., Ktistakis, N. T. and Yu, S. (1999). Phospholipase D as an effector for ADP-ribosylation factor in the regulation of vesicular traffic. Chem. Phys. Lipids 98, 141-152.[CrossRef][Medline]

Scales, S. J. and Scheller, R. H. (1999). Lipid membranes shape up. Nature 401, 123-124.[CrossRef][Medline]

Schmidt, A., Wolde, M., Thiele, C., Fest, W., Kratzin, H., Podtelejnikov, A. V., Witke, W., Huttner, W. B. and Soling, H. D. (1999). Endophilin I mediates synaptic vesicle formation by transfer of arachidonate to lysophosphatidic acid. Nature 401, 133-141.[CrossRef][Medline]

Sciaky, N., Presley, J., Smith, C., Zaal, K. J., Cole, N., Moreira, J. E., Terasaki, M., Siggia, E. and Lippincott-Schwartz, J. (1997). Golgi tubule traffic and the effects of brefeldin A visualized in living cells. J. Cell Biol. 139, 1137-1155.[Abstract/Free Full Text]

Shemesh, T., Luini, A., Malhotra, V., Burger, K. N. and Kozlov, M. M. (2003). Prefission constriction of Golgi tubular carriers driven by local lipid metabolism: a theoretical model. Biophys. J. 85, 3813-3827.[Abstract/Free Full Text]

Sonnichsen, B., De Renzis, S., Nielsen, E., Rietdorf, J. and Zerial, M. (2000). Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of Rab4, Rab5, and Rab11. J. Cell Biol. 149, 901-914.[Abstract/Free Full Text]

Spano, S., Silletta, M. G., Colanzi, A., Alberti, S., Fiucci, G., Valente, C., Fusella, A., Salmona, M., Mironov, A., Luini, A. et al. (1999). Molecular cloning and functional characterization of brefeldin A-ADP-ribosylated substrate. A novel protein involved in the maintenance of the Golgi structure. J. Biol. Chem. 274, 17705-17710.[Abstract/Free Full Text]

Storrie, B., White, J., Rottger, S., Stelzer, E. H., Suganuma, T. and Nilsson, T. (1998). Recycling of Golgi-resident glycosyltransferases through the ER reveals a novel pathway and provides an explanation for nocodazole-induced Golgi scattering. J. Cell Biol. 143, 1505-1521.[Abstract/Free Full Text]

Subramaniam, V. N., Peter, F., Philp, R., Wong, S. H. and Hong, W. (1996). GS28, a 28-kilodalton Golgi SNARE that participates in ER-Golgi transport. Science 272, 1161-1163.[Abstract]

Weigert, R., Silletta, M. G., Spano, S., Turacchio, G., Cericola, C., Colanzi, A., Senatore, S., Mancini, R., Polishchuk, E. V., Salmona, M. et al. (1999). CtBP/BARS induces fission of Golgi membranes by acylating lysophosphatidic acid. Nature 402, 429-433.[CrossRef][Medline]

Wood, S. A. and Brown, W. J. (1992). The morphology but not the function of endosomes and lysosomes is altered by brefeldin-A. J. Cell Biol. 119, 273-285.[Abstract]

Wood, S. A., Park, J. E. and Brown, W. J. (1991). Brefeldin A causes a microtubule-mediated fusion of the trans-Golgi network and early endosomes. Cell 67, 591-600.[CrossRef][Medline]





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