![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 19, Issue 6, 2650-2660, June 2008
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||
Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan
Submitted October 23, 2007;
Revised March 19, 2008;
Accepted April 3, 2008
Monitoring Editor: Akihiko Nakano
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). ADP-ribosylation factors (ARFs) are a family of small GTPases that trigger budding of coated carrier vesicles by recruiting coat protein complexes onto donor membranes. Protein complexes that are recruited by ARFs include the COPI complex onto the cis-Golgi, the AP-1 clathrin adaptor complex onto the trans-Golgi network (TGN) and endosomes, the AP-3 complex onto endosomes, and the monomeric GGA proteins onto the TGN.
ARFs cycle between an inactive GDP-bound state and an active GTP-bound state during which they interact with coat proteins and other effectors. Exchange of bound GDP for GTP on ARFs is stimulated by a family of guanine nucleotide exchange factors (GEFs) that contain a Sec7 catalytic domain, whereas intrinsic GTPase activity of ARFs is enhanced by a family of GTPase-activating proteins (Donaldson and Jackson, 2000; Jackson and Casanova, 2000; Shin and Nakayama, 2004; Nie and Randazzo, 2006). The ARF-GEFs are classified into several groups including the high-molecular-weight GEFs of the Gea/GBF and Sec7/BIG groups. These are involved in the regulation of membrane traffic and are sensitive to brefeldin A (BFA), which inhibits various trafficking processes and causes deformation of the Golgi apparatus and recycling endosomes (Jackson, 2000; Jackson and Casanova, 2000; Shin and Nakayama, 2004).
GBF1 is a sole member of the mammalian Gea/GBF group and functions primarily in the trafficking between the cis-Golgi and the ER-Golgi intermediate compartment (Claude et al., 1999; Kawamoto et al., 2002; Zhao et al., 2002; García-Mata et al., 2003; Zhao et al., 2006). On the other hand, BIG2 and BIG1 of the Sec7/BIG group (Morinaga et al., 1996, 1997; Mansour et al., 1999; Togawa et al., 1999) show considerable similarity to each other in their primary sequence and domain organization (Mouratou et al., 2005). Although early studies showed that both BIG2 and BIG1 are associated mainly with the TGN (Mansour et al., 1999; Yamaji et al., 2000; Shinotsuka et al., 2002a,b; Zhao et al., 2002), we and others later showed that BIG2 is also associated with recycling endosomes, where it recruits the AP-1 complex through activating ARFs and regulates trafficking of cargo proteins through these compartments (Shin et al., 2004; Shen et al., 2006). However, it is currently not clear if BIG2 and BIG1 play distinct roles or share some roles. In the present study, we exploit an RNA interference (RNAi) approach and reveal that knockdown of BIG2 alone affects localization of proteins associated with recycling endosomes; knockdown of BIG1 alone shows no obvious effects on organelle markers; and knockdown of both BIG2 and BIG1 causes delocalization of various proteins that reside in the TGN and recycling endosomes and blocks retrograde trafficking of furin from late endosomes to the TGN.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Monoclonal mouse antibodies against CD4, EEA1, GM130, golgin-245, GGA3, and 
-adaptin (Clone 88) were purchased from BD Biosciences (San Jose, CA); polyclonal rabbit antibodies against β-COP and furin from Affinity Bioreagents (Golden, CO); monoclonal mouse antibodies against -adaptin (Clone 100.3) and FLAG (M2) from Sigma (St. Louis, MO); monoclonal mouse anti-transferrin receptor (TfnR) from Zymed Laboratories (South San Francisco, CA); monoclonal rat anti-CD4 antibody and polyclonal sheep anti-TGN46 antibody from Serotec (Raleigh, NC); AlexaFluor-conjugated secondary antibodies and AlexaFluor488-conjugated EGF from Molecular Probes (Eugene, OR); and Cy3-conjugated and horseradish peroxidase (HRP)-conjugated secondary antibodies from Jackson ImmunoResearch Laboratories (West Grove, PA). Polyclonal rabbit anti-EEA1 antibody, monoclonal mouse anti-lysobisphosphatidic acid (LBPA) and Cy3-cojugated Shiga toxin 1 were kind gifts from Marino Zerial (MPI-CBG, Germany), Toshihide Kobayashi (RIKEN, Japan), and Naoko Morinaga (Chiba University, Japan). Construction of an expression vector for a CD4-furin fusion protein with an exoplasmic domain of CD4 and transmembrane and cytoplasmic domains of furin (see Figure 6A) was described previously (Takahashi et al., 1995). Expression vectors for fluorescent protein-tagged Rab proteins were described previously (Shin et al., 2004). An expression vector for FLAG-tagged TGN38 was a kind gift from Nobuhiro Nakamura (Kanazawa University, Japan).
Cell Culture, RNAi Suppression, Antibody Uptake Experiments, and Immunofluorescence Analysis
HeLa cells were cultured in minimal essential medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. An HeLa cell line stably expressing CD4-furin or FLAG-TGN38 was established by transfection of a pcDNA3-based expression vector followed by selection in the presence of 800 µg/ml G418 sulfate. Knockdown of BIG2 (nucleotide residues 600-1366) or BIG1 (residues 1-988) alone or of both ARF-GEFs was performed as described previously (Ishizaki et al., 2006). Knockdown of AP-1 was performed by incubating cells with a pool of small interfering RNAs (siRNAs) directed for an mRNA region of µ1A covering nucleotide residues 95-1120 (when the A residue of the initiation Met codon is assigned as residue 1, prepared using a BLOCK-iT RNAi TOPO Transcription kit and a BLOCK-iT Dicer RNAi kit (Invitrogen, Carlsbad. CA). Indirect immunofluorescence analysis of cells cultured on coverslips was performed as described previously (Shin et al., 1997, 2004; Shinotsuka et al., 2002b). For staining with anti-LBPA antibody, the cells fixed with 3% paraformaldehyde in phosphate-buffered saline (PBS) were incubated with the antibody in 0.05% saponin/PBS at room temperature for 30 min.
Uptake experiments of anti-CD4 or anti-FLAG antibody by cells stably expressing CD4-furin or FLAG-TGN38 were performed as described previously (Takahashi et al., 1995; Shin et al., 2004, 2005) with some modifications. Briefly, HeLa cells stably expressing CD4-furin or FLAG-TGN38 were mock-treated (a pool of siRNAs for LacZ) or treated with a pool of siRNAs for BIG1 and/or BIG2 or µ1A for 80 h and before antibody uptake were incubated with 15 mM sodium butyrate for 16 h. The cells were then incubated with monoclonal anti-CD4 (Leu3a) or anti-FLAG (M2) antibody in combination with AlexaFluor488-conjugated EGF at 19°C for 60 min, subjected to an acid wash (0.5% acetic acid, pH 3.0, 50 mM NaCl) in the case of the anti-FLAG uptake, and incubated at 37°C for the indicated periods of time. When indicated, the number of fluorescent structures of internalized anti-CD4 antibody present within the EGF-positive compartments were calculated using IPLab 4.0 software (Solution Systems, Funabashi, Japan). Briefly, punctate structures containing fluorescent EGF were set as regions of interest (ROIs) in more than fifty cells, and the number of CD4 signals in the EGF ROIs was estimated.
Cell surface levels of CD4-furin and FLAG-TGN38 were estimated as follows. HeLa cells stably expressing CD4-furin or FLAG-TGN38 in a well of a 24-well plate were incubated with 15 mM sodium butyrate for 16 h to induce expression of the recombinant protein. The cells were then incubated with biotin-conjugated anti-CD4 or anti-FLAG antibody, respectively, at 4°C for 60 min, washed three times with PBS, and lysed in biotin cell lysis buffer (20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1% Triton X-100). After centrifugation of the lysate at 13,000 rpm in a microcentrifuge, the biotin-conjugated antibody in the supernatant was recovered with immobilized streptavidin beads (Pierce, Rockford, IL) according to the manufacturer's instructions. The beads were washed with biotin wash buffer (50 mM Tris-HCl, pH 7.4, 500 mM NaCl, 0.1% Triton X-100) and with TBS-T (Tris-buffered saline containing 0.1% Tween 20), and incubated with HRP-conjugated anti-mouse IgG at room temperature for 30 min. The beads were then washed four times with TBS-T and developed with HRP substrate reagents (R&D Systems, Minneapolis, MN).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
); when HA-tagged BIG1 or BIG2 was expressed in HeLa cells, HA-BIG2 but not HA-BIG1 was significantly localized on peripheral punctate structures containing TfnR (a marker for early and recycling endosomes) and lacking EEA1 (an early endosomal maker; see Supplementary Figure S1). To explore roles specific for BIG2 or BIG1 and their redundant roles, we depleted HeLa cells of BIG2 or BIG1 alone or both ARF-GEFs by RNAi; as established in our previous study (Ishizaki et al., 2006), BIG1, BIG2, or both proteins were almost completely depleted by transfecting pools of siRNAs into HeLa cells, as confirmed by immunoblotting (Supplementary Figure S2A) and immunofluorescence analysis (Figure S2B).
Knocking down BIG2 alone resulted in tubular extensions from peripheral punctate structures positive for TfnR and the AP-1 subunit (Figure 1, c and h), but did not affect perinuclear TGN-like structures for AP-1 (Figure 1h). The tubulation of the TfnR-containing compartment we observed in BIG2 knockdown cells is consistent with the tubulation observed in our previous study when dominant negative BIG2 was expressed and with that of Shen et al. by RNAi-mediated knockdown of BIG2 (Shin et al., 2004; Shen et al., 2006). BIG2 knockdown did not significantly alter the TGN localization of CD4-furin (a chimeric protein with the exoplasmic domain of CD4 and the transmembrane and cytoplasmic domains of furin; see Figure 6A), TGN46, golgin-245, or GGA3 (Figure 1, m and r, and Supplementary Figure S3).
|
; Zerial and McBride, 2001), we concluded that BIG2 knockdown selectively induces tubulation of the Rab11-positive subdomains of recycling endosomes. This conclusion is apparently incompatible with our previous observations that overexpression of dominant-negative BIG2, BIG2(E738K), caused tubulation of Rab4-positive as well as Rab11-positive structures (Shin et al., 2004). Although the exact reason for the differential effects of the BIG2 knockdown and the dominant-negative BIG2 overexpression is not clear, it is possible that the effects of BIG2(E738K) overexpression on the endosomal integrity is much more drastic than that of BIG2 depletion and thereby the BIG2(E738K) overexpression impairs the entire recycling endosomes, whereas the BIG2 depletion impairs only the Rab11-positive subdomains. In support of this possibility, the endosomal tubules induced by the BIG2(E738K) overexpression (Shin et al., 2004) were much more prominent that those induced by the BIG2 depletion (Figure 2c). The more drastic effects of the BIG2(E738K) overexpression compared with the BIG2 depletion might result from sequestering not only ARFs through the mutated Sec7 catalytic domain but also unknown binding partners through regions outside of the Sec7 domain on the membrane.
|
30% of the double knockdown cells (Figure 1s, cells indicated by asterisks). Although we do not know the exact reason for the uneven redistribution of TGN46, we suspect this redistribution may require complete depletion of BIG2 and BIG1. Knocking down both BIG2 and BIG1 also alters the localization of CD4-furin. Specifically, its staining decayed and became more peripheral than that in control cells and often showed tubular appearance (Figure 1n). The disappearance of the TGN staining for AP-1 and TGN46 appeared to be specific effects of the double knockdown on these proteins but did not result from gross changes in Golgi morphology, because localization of other TGN markers (GGA3 and golgin-245) as well as cis-Golgi markers (GM130 and β-COP) was unaffected (Supplementary Figure S3).
Cell Surface Accumulation of CD4-Furin in Cells Depleted of Both BIG2 and BIG1
We next examined whether the morphological changes of recycling endosomes in cells depleted of BIG2 alone or in combination with BIG1 affect trafficking through these compartments. To this end, control or BIG-knockdown cells were incubated with AlexaFluor488-conjugated Tfn at 37°C for 60 min to allow its accumulation at early/recycling endosomes. After stripping surface-bound Tfn, the cells were then incubated at 37°C for appropriate time periods to follow its recycling through early/recycling endosomes. As shown in Supplementary Figure S4, we did not detect any significant difference in the recycling of fluorescent Tfn between the control and any of the BIG-knockdown cells. This is in line with previous observations that BFA, a specific inhibitor of ARF-GEFs of the Sec7/BIG and Gea/GBF groups (Jackson, 2000), causes gross tubulation of recycling endosomes but has marginal effects on Tfn recycling (Lippincott-Schwartz et al., 1991). We also examined retrograde transport of Shiga toxin through recycling endosomes to the Golgi, but again did not observe any significant difference between the control and BIG-knockdown cells (data not shown). These results are comparable with our previous observations that expression of a dominant-negative mutant of BIG2, BIG2(E738K), did induce tubulation of recycling endosomes but did not significantly affect transport of Tfn or Shiga toxin through these compartments (Shin et al., 2004). On the other hand, Shen et al. (2006) reported that release of accumulated Tfn from cells treated with BIG2 or BIG2+BIG1 siRNAs was slightly, but significantly, slower than from control cells. We do not know the reason for the difference between our data and that Shen et al., because our protocols to determine the Tfn recycling are essentially the same as those of Shen et al. (2006). In any way, the effects of BIG2 depletion on the Tfn recycling may be marginal in spite of the considerable effects on the recycling endosome architecture. However, it is also possible that, in the BIG2-depleted cells, because of the impairment of these compartments, a predominant fraction of internalized Tfn is recycled to the cell surface by bypassing recycling endosomes (i.e., directly from early endosomes).
We then set out to explore the reason why the levels of CD4-furin and TGN46 in the TGN region were reduced in the double knockdown cells. We speculated that the reduced levels might result from mislocalization and/or degradation of these transmembrane proteins. We first compared steady-state levels of CD4-furin in the control and knockdown cells by immunoblot analysis of whole cell lysates but failed to detect any significant difference in the CD4-furin levels between the control and double knockdown cells (Supplementary Figure S5A). These results exclude the possibility that the furin construct was missorted to the degradation pathway in cells depleted of BIG2 and BIG1. We then estimated the level of CD4-furin that accumulated on the cell surface by incubating the cells with anti-CD4 antibody at 4°C before fixation. As shown in Figure 3A, the cell surface level of CD4-furin approximately doubled in the double knockdown cells compared with that in the control cells or those depleted of BIG1 or BIG2 alone.
|
Block in Retrograde Transport of CD4-Furin at Late Endosomes and Its Missorting in Cells Depleted of Both BIG2 and BIG1
The high level of CD4-furin cell surface expression suggests a decrease in endocytosis or an increase in recycling of CD4-furin by knockdown of BIG2 and/or BIG1. To address these possibilities, we performed antibody uptake experiments in which retrograde transport of CD4-furin was monitored by following extracellularly applied anti-CD4 antibody. Internalization of CD4-furin from the cell surface to EEA1-positive early endosomes (antibody uptake at 19°C for 60 min) was not affected by the knockdown of either BIG2 or BIG1 or both (Figure 4A, top panels). However, further retrograde transport from early endosomes to the TGN was considerably delayed in cells depleted of both BIG2 and BIG1. In the control cells and those depleted of BIG2 or BIG1 alone, CD4-furin that accumulated in early endosomes was transported almost completely to perinuclear Golgi-like structures after temperature shift to 37°C for 60 min (Figure 4A, e–g, green). In marked contrast, a considerable fraction of CD4-furin remained in punctate endosome-like structures even after the temperature shift to 37°C in the double knockdown cells (Figure 4Ah; also see Figure 7Bc). However, the punctate CD4-furin labeling (green) was not significantly superimposed on the EEA1 staining (red), suggesting that the CD4-furin molecules that transit through early endosomes accumulate in distinct endosomal compartments.
|
; Schapiro et al., 2004; see Figure 8A) and the EGF receptor is known to be transported through late endosomes to lysosomes for degradation in a ligand-dependent manner. When CD4-furin–expressing HeLa cells were incubated with anti-CD4 and fluorescently labeled EGF at 19°C for 60 min, both anti-CD4 (red) and EGF (green) accumulated on punctate early endosomal structures in the control and knockdown cells (Figure 4B, a–d). After temperature shift to 37°C for 60 min, the majority of anti-CD4 reached perinuclear Golgi region in control cells and those depleted of BIG2 alone (Figure 4B, e–g, red). In these cells, a considerable fraction of the EGF labeling disappeared because of its degradation in lysosomes, although a certain fraction was still found on punctate late endosomal structures (e–g, green). On the other hand, in the double knockdown cells (Figure 4Bh), the punctate structures containing CD4-furin (red) and late endosomes labeled by fluorescent EGF (green) showed a good codistribution. Note that transport of EGF through late endosomes to lysosomes for degradation was not apparently influenced by the double knockdown (Supplementary Figure S6). Quantitative estimation revealed that, after temperature shift to 37°C for 60 min, the amount of CD4-furin present within the EGF-positive endosomes in the double knockdown cells was approximately three times as much as that in the control cells; 65% of internalized CD4-furin colocalized with internalized EGF in the double knockdown cells, whereas only 20% colocalized in the control cells (Figure 4C). The quantitative estimation also revealed that, even in cells depleted of BIG1 alone and possibly those depleted of BIG2 alone, internalized CD4-furin tends to be trapped in EGF-positive endosomes to a lesser extent (Figure 4C), although the single knockdown appeared not to significantly affect the steady-state localization of CD4-furin (Figure 1, l and m). These observations suggest that BIG1 and BIG2 are redundantly involved the retrograde transport from endosomes to the TGN, and BIG1 may contribute preferentially to this transport pathway.
To further define the late endosomal compartment where retrograde transport of internalized CD4-furin was blocked, we stained the CD4-furin–internalized cells with antibodies to some late endosomal markers. Although the punctate staining for CD4-furin accumulated in the double knockdown cells did not overlap with the staining for Lamp-1 (a marker for late endosomes and lysosomes) or Hrs (a marker for early and late endosomes; data not shown), it partially but significantly overlapped with the staining for LBPA (Kobayashi et al., 1998, 1999), a marker for late endosomes/multivesicular bodies (Figure 4D). These observations indicate that knocking down both BIG2 and BIG1 blocks retrograde transport to the TGN of the furin construct at some population of late endosomes.
Taking into account the data that the cell surface level of CD4-fuirn is elevated (Figure 3A) whereas its total level is unchanged (Supplementary Figure S5A) in the double knockdown cells compared with control cells, it is likely that the block in retrograde transport of CD4-furin resulted in its recycling to the cell surface (see Figure 8B). In support of this speculation, a population of CD4-furin was found along TfnR-positive tubules derived from recycling endosomes in the double knockdown cells (Figure 5).
|
; Schäfer et al., 1995; Takahashi et al., 1995; Voorhees et al., 1995; reviewed in Thomas, 2002). In previous studies, we and others have shown that steady-state localization to the TGN and recycling from the cell surface of furin both involve a Tyr-based motif, YKGL, and an acidic cluster sequence, SDSEEDE, containing Ser residues that are phosphorylated by protein kinase CK2, within its cytoplasmic domain (Figure 6A). As shown in Figure 6B, a CD4-furin construct with a WT cytoplasmic domain underwent retrograde transport to the TGN (a), whereas transport of constructs with mutations at both the Tyr and Ser residues within the cytoplasmic domain, YA/SA, were blocked at late endosomes (b–d).
|
; Wan et al., 1998; Teuchert et al., 1999; Crump et al., 2001). Moreover, AP-1 is recruited to membranes in an ARF-dependent manner (Stamnes and Rothman, 1993; Zhu et al., 1998; Austin et al., 2000). We therefore decided to examine the effects of AP-1 (µ1A subunit) depletion on the localization and retrograde trafficking of CD4-furin as well as the localization of other proteins. Even though we could not confirm depletion of the µ1A subunit due to unavailability of an anti-µ1A antibody, the level of the subunit was specifically decreased in the µ1A-knockdown cells (Figures 1j and 7A), probably due to its instability from the lack of the µ1A subunit in the AP-1 complex. In the AP-1 knockdown cells, as in cells depleted of both BIG2 and BIG1, CD4-furin was distributed more peripherally than in control cells and was found on punctate, often tubular, endosome-like structures (Figure 1o), and its abundance at the cell surface was increased (Figure 3A). These observations are in line with those of the previous studies obtained using cells derived from AP-1 knockout mice and using AP-1 knockdown cells (Meyer et al., 2001; Hirst et al., 2005). Furthermore, TfnR and Rab11 redistributed into tubular structures in the AP-1 knockdown cells (Figures 1e and 2e) as in the double knockdown cells. However, the steady-state localization of other Golgi and TGN proteins examined was unaffected in the AP-1–depleted cells as in cells depleted of BIG2 and BIG1 (Supplementary Figure S3).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Yamaji et al., 2000). Cherfils and colleagues recently showed that N-terminal DCB and HUS domains of BIG1, BIG2, and GBF1 are important for intra- and intermolecular interactions (Mouratou et al., 2005; Ramaen et al., 2007), suggesting that BIG1 and BIG2 are capable of functioning as homomeric or heteromeric complexes. On the other hand, we previously revealed that BIG2 but not BIG1 is required for the integrity of recycling endosomes. These data led us to examine how the specific or redundant functions of BIG1 and BIG2 are regulated in a cellular context by examining relevant subcellular compartments after RNAi-mediated knockdown of these proteins. We failed to detect any obvious effect of BIG1 knockdown on the localization of TGN and endosomal proteins and on trafficking through these compartments. In contrast, BIG2 knockdown induces tubular extensions from recycling endosomes positive for TfnR, AP-1, and Rab11. Knocking down both BIG2 and BIG1 abolishes TGN-localization of AP-1 and TGN46 and causes redistribution, often into tubular endosomal structures, of CD4-furin and AP-1, in addition to inducing the phenotype observed in the cells knocked down of BIG2 alone. Moreover, we made essentially the same observations in AP-1 knockdown cells as for those (but for AP-1 localization) in the double knockdown cells. Our data allow us to formulate a model that: 1) BIG2 functions at recycling endosomes, and BIG2 and BIG1 play redundant roles at the TGN; and 2) these ARF-GEFs activate ARFs at these compartments, and the activated ARFs in turn regulate AP-1 functions.
The simultaneous depletion of BIG2 and BIG1 appears not to affect anterograde transport of vesicular stomatitis virus G protein (VSVG) from the TGN to the plasma membrane (data not shown). We and Togawa et al. (1999) previously showed that BIG1 and BIG2 have a GEF activity toward ARF1 and ARF3 (see also Shin et al., 2004), and Volpicelli-Daley et al. (2005) observed that simultaneous knockdown of ARF1 and ARF3 shows marginal effects on VSVG transport to the plasma membrane. These data support our observation that the double knockdown of BIG2 and BIG1 does not affect the anterograde transport of VSVG from the TGN. It is thus unlikely that ARF activation by BIG2 and BIG1 at the TGN is required for VSVG transport to the plasma membrane.
Volpicelli-Daley et al. (2005) also reported that simultaneous knockdown of ARF1 and ARF3 significantly retarded Tfn recycling. Our preliminary analysis using the same shRNA constructs as those used by Volpicelli-Daley et al. also revealed a tendency that internalized Tfn is accumulated in cells depleted of ARF1 and ARF3. Given that double knockdown of BIG1 and BIG2 does not significantly affect Tfn recycling (Figure S4) albeit leading to marked morphological changes of recycling endosomes (Figures 1, d and i, and 2d), whereas simultaneous knockdown of ARF1 and ARF3 causes both the recycling block and the morphological changes, it is possible that the BIG1+BIG2 knockdown affects recycling though recycling endosomes alone, whereas the ARF1+ARF3 knockdown affects recycling through not only recycling endosomes but also early endosomes. Taken together with a previous report showing that BFA has marginal effects on Tfn recycling albeit causing endosomal tubulation (Lippincott-Schwartz et al., 1991), a BFA-insensitive ARF-GEF(s) might be responsible for activation of ARFs that participate in recycling of Tfn through early endosomes.
The double knockdown of BIG2 and BIG1 results in redistribution of a subset of TGN-localizing proteins (Figure 1 and Supplementary Figure S3). To our surprise, however, the double knockdown does not affect the localization of GGA3, which associates with the TGN in an ARF-dependent manner (for reviews, see Nakayama and Wakatsuki, 2003; Bonifacino, 2004). A most recent report could provide an explanation for this observation: Lefrançois and McCormick (2007) showed that GBF1 interacts with GGAs and is required for their recruitment onto Golgi membranes. Therefore, ARFs activated by BIG2 and BIG1 at the TGN recruits specific coat proteins and regulates transport of specific cargo proteins.
The simultaneous knockdown of BIG2 and BIG1 does not apparently affect retrograde transport of Shiga toxin or recycling of Tfn via recycling endosomes, despite considerable morphological changes in these compartments. Intriguingly, the double knockdown blocks retrograde transport of CD4-furin to the TGN, and consequently increases its accumulation in peripheral endosomes (Figure 4) and its expression on the cell surface (Figure 3A). It is therefore likely that, through activating ARFs, BIG2 and BIG1 play a crucial role in retrograde transport from endosomes to the TGN. The block in retrograde transport of CD4-furin at late endosomes appears to cause its recycling to the cell surface rather than its missorting to the lysosomal degradation pathway (Figure 8B) for the following reasons. First, we do not observe colocalization of internalized CD4-furin and lysosomal markers, such as Lamp-1, at any time point (data not shown). Second, in the double knockdown cells, the total amount of CD4-furin is not significantly changed (Supplementary Figure S5), whereas its cell surface expression is increased (Figure 3A). Finally, the recycling pathway appears not to be affected in the double knockdown cells (Supplementary Figure S4).
|
, 2005). Using a Tac-furin construct, Maxfield and colleagues carefully examined the role of the cytoplasmic domain of furin in its trafficking from the plasma membrane to the TGN through endosomal compartments. Their data suggested that phosphorylation of Ser residues in the acidic cluster sequence within the cytoplasmic domain plays an important role in selective sorting of Tac-furin into late endosomes and in retrieval from late endosomes to the TGN (Mallet and Maxfield, 1999; Schapiro et al., 2004). The behavior of CD4-furin we observe in the double knockdown cells and in AP-1 knockdown cells closely resembles that of cytoplasmic domain mutants with substitutions of the phosphorylatable Ser residues to unphosphorylatable Ala. Taken together with our data, it is likely that ARFs activated by BIG2 and BIG1 regulate AP-1, after which AP-1 regulates retrograde transport of furin.
Our attempts to unequivocally define the compartments accumulating CD4-furin in cells depleted of both BIG2 and BIG1 or those depleted of AP-1 have not been successful. Given that CD4-furin colocalizes with LBPA and internalized EGF but not with EEA1, Lamp-1, or internalized Tfn (Figure 4 and our unpublished observations), the CD4-furin–accumulating compartments are very likely to be late endosomes and/or intermediates of early and late endosomes. It is possible that BIG2 and BIG1 play a redundant roles at late endosomal compartments, although we have so far failed to show localization of BIG2 or BIG1 at these compartments because of limitations in the sensitivity of our anti-BIG2 and anti-BIG1 antibodies (Ishizaki et al., 2006).
Similar to CD4-furin, the staining for TGN46 is reduced in the double knockdown cells (Figure 1). A simple explanation for this observation is that retrograde transport of TGN46 from endosomes to the TGN is inhibited by the depletion of BIG2 and BIG1. As expected, we found that in the double knockdown cells retrograde transport of FLAG-TGN38 (TGN38 is a rat ortholog of human TGN46) to the TGN is inhibited (Figure 3C), although compartments accumulating internalized FLAG-TGN38 are not significantly overlapped with those positive for internalized EGF (data not shown), and the cell surface level of FLAG-TGN38 was not significantly changed (Figure 3B). The behavioral difference between CD4-furin and FLAG-TGN38 may be explained by the different retrograde routes taken by them, as previously shown by Mallet and Maxfield (1999). One of candidate regulators that discriminate between the retrograde transport of furin and TGN38 is PACS-1 (phosphofurin acidic cluster sorting protein-1). Thomas and colleagues proposed that PACS-1 is a critical connector between AP-1 and the acidic cluster containing phospho-Ser residues within the furin cytoplasmic domain and regulates steady-state distribution and trafficking of furin (Wan et al., 1998; Crump et al., 2001; Thomas, 2002). A recent RNAi study of Robinson and colleagues, however, has indicated that, for the furin localization and trafficking, PACS-1 is dispensable even though the acidic cluster is indeed the essential sorting signal and AP-1 and clathrin do play critical roles (Lubben et al., 2007). Our attempts to show a role of PACS-1 in trafficking of CD4-furin have been also unsuccessful so far (data not shown). Thus, the function of PACS-1 is currently unclear, and although not essential, it might play some sort of a regulatory role in the furin localization and trafficking.
In the present study, we demonstrate the first evidence that BIG2 and BIG1 play redundant roles in recruitment of AP-1 and in the retrograde transport from endosomes to the TGN. Moreover, we have determined the specific function of BIG2 for the integrity of recycling endosomes. Our study leads to further questions, namely, whether BIG1 and BIG2 carry out their redundant and nonredundant functions in a spatially and temporally regulated manner and how the formation of homomeric and heteromeric complexes is linked to the regulation of the function and localization of BIG1 and BIG2.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
* These authors contributed equally to this work. 
Address correspondence to: Kazuhisa Nakayama (kazunaka{at}pharm.kyoto-u.ac.jp)
Abbreviations used: ARF, ADP-ribosylation factor; HRP, horseradish peroxidase; GEF, guanine nucleotide exchange factor; PACS, phosphofurin acidic cluster sorting protein; TGN, trans-Golgi network; TfnR, transferrin receptor; VSVG, vesicular stomatitis virus G protein.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bonifacino, J. S. (2004). The GGA proteins: adaptors on the move. Nat. Rev. Mol. Cell Biol 5, 23–32.[CrossRef][Medline]
Claude, A., Zhao, B.-P., Kuziemsky, C. E., Dahan, S., Berger, S. J., Yan, J.-P., Armold, A. D., Sullivan, E. M., and Melançon, P. (1999). GBF1, a novel Golgi-associated BFA-resistant guanine nucleotide exchange factor that displays specificity for ADP-ribosylation factor 5. J. Cell Biol 146, 71–84.
Crump, C. M., Xiang, Y., Thomas, L., Gu, F., Austin, C., Tooze, S. A., and Thomas, G. (2001). PACS-1 binding to adaptors is required for acidic cluster motif-mediated protein traffic. EMBO J 20, 2191–2201.[CrossRef][Medline]
Dittie, A. S., Thomas, L., Thomas, G., and Tooze, S. A. (1997). Interaction of furin in immature secretory granules from neuroendocrine cells with the AP-1 adaptor complex is modulated by casein kinase II phosphorylation. EMBO J 16, 4859–4870.[CrossRef][Medline]
Donaldson, J. G., and Jackson, C. L. (2000). Regulators and effectors of the ARF GTPases. Curr. Opin. Cell Biol 12, 475–482.[CrossRef][Medline]
García-Mata, R., Szul, T., Alvarez, C., and Sztul, E. (2003). ADP-ribosylation factor/COPI-dependent events at the endoplasmic reticulum-Golgi interface are regulated by the guanine nucleotide exchange factor GBF1. Mol. Biol. Cell 14, 2250–2261.
Hirst, J., Borner, G.H.H., Harbour, M., and Robinson, M. S. (2005). The Aftiphilin/p200/-Synergin complex. Mol. Biol. Cell 16, 2554–2565.
Hirst, J., Miller, S. E., Taylor, M. J., Fischer von Mollard, G., and Robinson, M. S. (2004). EpsinR is an adaptor for the SNARE protein Vti1b. Mol. Biol. Cell 15, 5593–5602.
Ishizaki, R., Shin, H.-W., Iguchi-Ariga, S.M.M., Ariga, H., and Nakayama, K. (2006). AMY-1 (associate of Myc-1) localization to the trans-Golgi network through interacting with BIG2, a guanine-nucleotide exchange factor for ADP-ribosylation factors. Genes Cells 11, 949–959.
Jackson, C. L. (2000). Brefeldin A: revealing the fundamental principles governing membrane dynamics and protein transport. Subcell. Biochem 34, 233–272.[Medline]
Jackson, C. L., and Casanova, J. E. (2000). Turning on ARF: the Sec7 family of guanine-nucleotide exchange factors. Trends Cell Biol 10, 60–67.[CrossRef][Medline]
Jones, B. G., Thomas, L., Molloy, S. S., Thulin, C. D., Fry, M. D., Walsh, K. A., and Thomas, G. (1995). Intracellular trafficking of furin is modulated by the phosphorylation state of a casein kinase II site in its cytoplasmic tail. EMBO J 14, 5869–5883.[Medline]
Kawamoto, K., Yoshida, Y., Tamaki, H., Torii, S., Shinotsuka, C., Yamashina, S., and Nakayama, K. (2002). GBF1, a guanine nucleotide exchange factor for ADP-ribosylation factors, is localized to the cis-Golgi and involved in membrane association of the COPI coat. Traffic 3, 483–495.[CrossRef][Medline]
Kirchhausen, T. (2000). Three ways to make a vesicle. Nat. Rev. Mol. Cell Biol 1, 187–198.[CrossRef][Medline]
Kobayashi, T., Beuchat, M.-H., Lindsay, M., Frias, S., Palmiter, R. D., Sakuraba, H., Parton, R. G., and Gruenberg, J. (1999). Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport. Nat. Cell Biol 1, 113–118.[CrossRef][Medline]
Kobayashi, T., Stang, E., Fang, K. S., de Moerloose, P., Parton, R. G., and Gruenberg, J. (1998). A lipid associated with the antiphospholipid syndrome regulates endosome structure and function. Nature 392, 193–197.[CrossRef][Medline]
Lefrançois, S., and McCormick, P. J. (2007). ArfGEF GBF1 is required for GGA recruitment to Golgi membrane. Traffic 8, 1440–1451.[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]
Lubben, N. B., Sahlender, D. A., Motley, A. M., Lehner, P. J., Benaroch, P., and Robinson, M. S. (2007). HIV-1 Nef-induced down regulation of MHC class I requires AP-1 and clathrin but not PACS-1, and is impeded by AP-2. Mol. Biol. Cell 18, 3351–3365.
Mallet, W. G., and Maxfield, F. R. (1999). Chimeric forms of furin and TGN38 are transported from the plasma membrane to the trans-Golgi network via distinct endosomal pathways. J. Cell Biol 146, 345–359.
Mansour, S. J., Skaug, J., Zhao, X.-H., Giordano, J., Scherer, S. W., and Melançon, P. (1999). p200 ARF-GEP1, a Golgi-localized guanine nucleotide exchange protein whose Sec7 domain is targeted by the drug brefeldin A. Proc. Natl. Acad. Sci. USA 96, 7968–7973.
Meyer, C., Eskelinen, E.-L., Guruprasad, M. R., von Figura, K., and Schu, P. (2001). µ1A deficiency induces a profound increase in MPR300/IGF-II receptor internalization. J. Cell Sci 114, 4469–4476.[Medline]
Morinaga, N., Moss, J., and Vaughan, M. (1997). Cloning and expression of a cDNA encoding a bovine brain brefeldin A-sensitive guanine nucleotide exchange protein for ADP-ribosylation factor. Proc. Natl. Acad. Sci. USA 94, 12926–12931.
Morinaga, N., Tsai, S.-C., Moss, J., and Vaughan, M. (1996). Isolation of a brefeldin A-inhibited guanine nucleotide-exchange protein for ADP-ribosylation factor (ARF) 1 and ARF3 that contains a Sec7-like domain. Proc. Natl. Acad. Sci. USA 93, 12856–12860.
Mouratou, B., Biou, V., Joubert, A., Cohen, J., Shields, D. J., Geldner, N., Jürgens, G., Melançon, P., and Cherfils, J. (2005). The domain architecture of large guanine nucleotide exchange factors for the small GTP-binding protein Arf. BMC Genomics 6, 20.[CrossRef][Medline]
Nakayama, K., and Wakatsuki, S. (2003). The structure and function of GGAs, the traffic controllers at the TGN sorting crossroads. Cell Struct. Funct 28, 431–442.[CrossRef][Medline]
Nie, Z., and Randazzo, P. A. (2006). Arf GAPs and membrane traffic. J. Cell Sci 119, 1203–1211.
Ramaen, O. et al. (2007). Interactions between conserved domains within homodimers in the BIG1, BIG2 and GBF1 ARF guanine nucleotide exchange factors. J. Biol. Chem 282, 28834–28842.
Schäfer, W., Stroh, A., Berghöfer, S., Seiler, J., Vey, M., Kruse, M.-L., Kern, H. F., Klenk, H.-D., and Garten, W. (1995). Two independent targeting signals in the cytoplasmic domain determine trans-Golgi network localization and endosomal trafficking of the proprotein convertase furin. EMBO J 14, 2424–2435.[Medline]
Schapiro, F. B., Soe, T. T., Mallet, W. G., and Maxfield, F. R. (2004). Role of cytoplasmic domain serines inn intracellular trafficking of furin. Mol. Biol. Cell 15, 2884–2894.
Shen, X., Xu, K.-F., Fan, Q., Pacheco-Rodriguez, G., Moss, J., and Vaughan, M. (2006). Association of brefeldin A-inhibited guanine nucleotide-exchange protein 2 (BIG2) with recycling endosomes during transferrin uptake. Proc. Natl. Acad. Sci. USA 103, 2635–2640.
Shin, H.-W., Kobayashi, H., Kitamura, M., Waguri, S., Suganuma, T., Uchiyama, Y., and Nakayama, K. (2005). Roles of ARFRP1 (ADP-ribosylation factor-related protein 1) in post-Golgi membrane trafficking. J. Cell Sci 118, 4039–4048.
Shin, H.-W., Morinaga, N., Noda, M., and Nakayama, K. (2004). BIG2, a guanine nucleotide exchange factor for ADP-ribosylation factors: its localization to recycling endosomes and implication in the endosome integrity. Mol. Biol. Cell 15, 5283–5294.
Shin, H.-W., and Nakayama, K. (2004). Guanine nucleotide exchange factors for Arf GTPases: their diverse functions in membrane traffic. J. Biochem 136, 761–767.
Shin, H.-W., Shinotsuka, C., Torii, S., Murakami, K., and Nakayama, K. (1997). Identification and subcellular localization of a novel mammalian dynamin-related protein homologous to yeast Vps1p and Dnm1p. J. Biochem 122, 525–530.
Shinotsuka, C., Waguri, S., Wakasugi, M., Uchiyama, Y., and Nakayama, K. (2002a). Dominant-negative mutant of BIG2, an ARF-guanine nucleotide exchange factor, specifically affects membrane trafficking from the trans-Golgi network through inhibiting membrane association of AP-1 and GGA coat proteins. Biochem. Biophys. Res. Commun 294, 254–260.[CrossRef][Medline]
Shinotsuka, C., Yoshida, Y., Kawamoto, K., Takatsu, H., and Nakayama, K. (2002b). Overexpression of an ADP-ribosylation factor-guanine nucleotide exchange factor, BIG2, uncouples brefeldin A-induced adaptor protein-1 coat dissociation and membrane tubulation. J. Biol. Chem 277, 9468–9473.
Sönnichsen, 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, 901–913.
Stamnes, M. A., and Rothman, J. E. (1993). The binding of AP-1 clathrin adaptor particles to Golgi membranes requires ADP-ribosylation factor, a small GTP-binding protein. Cell 73, 999–1005.[CrossRef][Medline]
Takahashi, S., Nakagawa, T., Banno, T., Watanabe, T., Murakami, K., and Nakayama, K. (1995). Localization of furin to the trans-Golgi network and recycling from the cell surface involves Ser and Tyr residues within the cytoplasmic domain. J. Biol. Chem 270, 28397–28401.
Teuchert, M., Schäfer, W., Berghöfer, S., Hoflack, B., Klenk, H.-D., and Garten, W. (1999). Sorting of furin at the trans-Golgi network: interaction of the cytoplasmic tail sorting signals with AP-1 Golgi-specific assembly proteins. J. Biol. Chem 274, 8199–8207.
Thomas, G. (2002). Furin at the cutting edge: from protein traffic to embryogenesis and disease. Nat. Rev. Mol. Cell Biol 3, 753–766.[CrossRef][Medline]
Togawa, A., Morinaga, N., Ogasawara, M., Moss, J., and Vaughan, M. (1999). Purification and cloning of a brefeldin A-inhibitable guanine nucleotide-exchange protein for ADP-ribosylation factors. J. Biol. Chem 274, 12308–12315.
Volpicelli-Daley, L. A., Li, Y., Zhang, C.-J., and Kahn, R. A. (2005). Isoform selective effects of the depletion of Arfs1–5 on membrane traffic. Mol. Biol. Cell 16, 4495–4508.
Voorhees, P., Deignan, E., van Donselaar, E., Humphrey, J., Marks, M. S., Peters, P. J., and Bonifacino, J. S. (1995). An acidic sequence within the cytoplasmic domain of furin functions as a determinant of trans-Golgi network localization and internalization from the cell surface. EMBO J 14, 4961–4975.[Medline]
Wan, L., Molloy, S. S., Thomas, L., Liu, G., Xiang, Y., Rybak, S. L., and Thomas, G. (1998). PACS-1 defines a novel gene family of cytosolic sorting proteins required for trans-Golgi network localization. Cell 94, 205–216.[CrossRef][Medline]
Yamaji, R., Adamik, R., Takeda, K., Togawa, A., Pacheco-Rodriguez, G., Ferrans, V. J., Moss, J., and Vaughan, M. (2000). Identification and localization of two brefeldin A-inhibited guanine nucleotide-exchange proteins for ADP-ribosylation factors in a macromolecular complex. Proc. Natl. Acad. Sci. USA 97, 2567–2572.
Zerial, M., and McBride, H. (2001). Rab proteins as membrane organizers. Nat. Rev. Mol. Cell Biol 2, 107–117.[CrossRef][Medline]
Zhao, X., Claude, A., Chun, J., Shields, D. J., Presley, J. F., and Melançon, P. (2006). GBF1, a cis-Golgi and VTCs-localized ARF-GEF, is implicated in ER-to-Golgi protein traffic. J. Cell Sci 119, 3743–3753.
Zhao, X., Lasell, T.K.R., and Melançon, P. (2002). Localization of large ADP-ribosylation factor-guanine nucleotide exchange factors to different Golgi compartments: evidence for distinct functions in protein traffic. Mol. Biol. Cell 13, 119–133.
Zhu, Y., Traub, L. M., and Kornfeld, S. (1998). ADP-ribosylation factor 1 transiently activates high-affinity adaptor protein complex AP-1 binding sites on Golgi membranes. Mol. Biol. Cell 9, 1323–1337.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||
