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J. Biol. Chem., Vol. 281, Issue 8, 5094-5105, February 24, 2006
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1
From the
Cambridge Institute for Medical Research, Departments of Clinical Biochemistry and Medicine, University of Cambridge, Wellcome Trust/MRC Building, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2XY, United Kingdom
Received for publication, August 5, 2005 , and in revised form, November 28, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Over the last few years, significant progress has been made in identifying the signals and machinery involved in protein sorting into the luminal vesicles of MVBs. Much of this information has come from studies in the budding yeast Saccharomyces cerevisiae. The majority of yeast proteins studied so far use ubiquitin attached to their cytosolic domain as an MVB sorting signal, although there is also a ubiquitin-independent pathway (5-7). As well as identification of a sorting signal, yeast studies have provided the first clues to the molecular machinery of MVB protein sorting. Genetic screens in yeast identified 18 genes, the "class E" vacuolar protein sorting (VPS) genes, which encode proteins involved in MVB protein sorting (8-12, for review, see Ref. 13). Deletion of any class E VPS gene results in the secretion of the vacuolar hydrolase carboxypeptidase Y and the formation of a large, aberrant endosomal compartment in which proteins en route to the vacuole (the yeast lysosome) accumulate. 17 of the class E VPS genes encode soluble or peripherally membrane-associated proteins, 12 of which form 4 endosomal sorting complexes required for transport (ESCRT complexes 0, I, II, and III, see Table 1), and the remaining 5 are directly or indirectly associated with ESCRTIII (5, 13-16). The sodium/proton exchanger Nhx1p is the only transmembrane class E Vps protein, and its role in MVB protein sorting remains unclear (17).
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Mammalian homologues of all the soluble yeast class E Vps proteins have now been identified (see Table 1 for homologues of the ESCRT proteins) and have in some cases been shown to perform similar functions to their yeast counterparts. For example, components of ESCRTI (TSG101, hVps28p, and hVps37Ap) are involved in mammalian MVB protein sorting (28-30). However, nothing is currently known about the role of the putative mammalian ESCRTII complex in endosomal sorting. The yeast ESCRTII complex is made up of two copies of Vps25p, and one each of Vps22p and Vps36p, and the structure of the core (i.e. without the N-terminal 395 amino acids of Vps36p) has been solved by x-ray crystallography at a resolution of 3.6 Å (31, 32). Yeast ESCRTII is a Y-shaped complex with a core consisting of eight winged-helix domains. By amino acid sequence alignments the mammalian homologues of the yeast ESCRTII proteins are the ELL-associated proteins, EAP20 (hVps25p), EAP30 (hVps22p), and EAP45 (hVps36p) (33, 34). ELL is an RNA polymerase II transcription factor that can increase the rate of transcription elongation by the polymerase from both promoter dependent and-independent templates (35). ELL can also negatively regulate polymerase activity in promoter-specific transcription initiation in vitro (36). hVps25p, hVps22p, and hVps36p were found in a complex with ELL, and hVps22p can derepress the inhibitory activity of ELL in vitro (33, 34). Yeast two-hybrid analysis suggests that hVps22p and hVps36p interact with TSG101 (an ESCRTI component) (37). In addition, two independent studies show yeast two-hybrid interactions between hVps22p, hVps25p, hVps36p, and hVps20p (also called CHMP6, an ESCRTIII component) (37, 38). hVps25p and hVps20p have also been shown to associate in immunoprecipitation experiments, and this interaction was shown to be direct by pull-down assays using purified hVps25p and hVps20p (39). Evidence that human ESCRTII is found on endosomes comes from the finding that hVps25p colocalizes with overexpressed hVps20p on punctate endosomal structures, and hVps36p coexpressed with hVps25p in cells stimulated with epidermal growth factor (EGF) colocalizes with endosomal markers (39, 40).
We have investigated the role of the putative mammalian ESCRTII complex in MVB protein sorting in mammalian cells. hVps25p, hVps22p, and hVps36p interact with one another and with their yeast homologues. In addition, hVps25p can associate with endosomes in the presence of a dominant-negative mutant of hVps4Bp. Surprisingly, we find no evidence that mammalian ESCRTII plays a role in MVB protein sorting.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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For protein purification, full-length hVPS25 was subcloned into pMW172-GST such that the C terminus of hVPS25 was in-frame with a thrombin-cleavable GST tag. A fragment corresponding to residues 232-end of hVPS36 (the CB fragment), and a second fragment corresponding to residues 82-end (the C1 fragment) of hVps22p, each preceded by a ribosome-binding site were sequentially subcloned into pMW172-hVPS25-GST. hVps22p had an N-terminal His6 tag. This construct allowed co-expression of the three ESCRTII subunits from one plasmid under the control of a single T7 promoter. The dsRed-VPS4B E235Q construct was a kind gift from Prof. Wes Sundquist (Salt Lake City, UT) (37).
Antibodies—The rabbit polyclonal anti-calreticulin (PA3-900) antibody was from Affinity BioReagents, the mouse monoclonal anti-TSG101 (4A10) antibody was from Abcam, the mouse monoclonal anti-transferrin receptor antibody (H68.4) was from Zymed Laboratories Inc., the mouse monoclonal anti-LAMP1 antibody (H4A3) was from the Developmental Studies Hybridoma Bank, University of Iowa, the mouse monoclonal anti-clathrin (X22) was from Abcam, and the mouse monoclonal anti-EEA1 antibody was from BD Biosciences. The rabbit polyclonal antibodies against AMSH (antibody 850), Alix, and UBPY were generously provided by Sylvie Urbé (42), Harald Stenmark (43), and Pier Paolo Di Fiore (44), respectively. The anti-MHC class I mouse monoclonal W6/32 and rabbit anti-mannose 6-phospate (MPR) antibodies have been described previously (45, 46).
To generate antibodies against hVps25p, a GST-hVps25p fusion protein was purified from bacteria as previously described (16) and used to immunize rabbits (Harlan Sera-Lab Ltd). Anti-hVps25p antibodies were affinity-purified using purified His6-hVps25p covalently bound to a CNBr-Sepharose column (Amersham Biosciences) and the method described in Mullock et al. (47).
Yeast Two-hybrid Analysis—Yeast were grown and transformed using standard genetic techniques and grown in rich medium (YEPD: 1% yeast extract, 2% peptone and 2% glucose (48)) or synthetic dextrose minimal medium with appropriate amino acid supplements (SD: 0.67% yeast nitrogen base without amino acids and 2% glucose (48)). Amino acid drop-out mixes were from Formedium Ltd. The yeast strains and two-hybrid plasmids have been described previously (41). Yeast strain pJ69-4
was transformed with GAL4-DNA binding domain plasmids (pGBDU) and plated onto SD without uracil. Yeast strain pJ69-4a was transformed with GAL4-DNA activation domain plasmids (pGAD) and plated onto SD without leucine. The and a strains were mated on YEPD for 8 h at 30°C, and then diploids were selected on SD without uracil and leucine. After replating onto SD without uracil and leucine, diploids were replica-plated onto SD without adenine. Interaction of the binding- and activation-domain fusion proteins was scored by growth of the yeast on SD without adenine over 8 days.
Protein Purification—pMW172 with hVPS25-GST, hVPS36 (CB), and His6-hVPS22 (C1) was used to transform BL21(DE3)pLysS Escherichia coli (Novagen). Cells were grown at 37 °C until an A600 of 0.8 was reached, induced with 0.1 mM isopropyl 1-thio-
-D-galactopyranoside, and grown overnight at 22 °C. Cell pellets were resuspended in 10 mM Hepes, pH 7.4, 200 mM NaCl with protease inhibitors and DNase, and lysed using a cell disruptor. Proteins were purified either on glutathione-Sepharose (Amersham Biosciences) or Ni-NTA (Qiagen) columns. Columns were washed extensively and eluted with 20 mM glutathione or 300 mM imidazole, respectively.
siRNA Knockdowns—The siRNA oligonucleotides were purchased from Dharmacon, Inc. The TSG101, Alix, and AMSH siRNA duplexes were as described (42, 49, 50). The hVps25p and UBPY depletions were performed using Dharmacon SMARTpools consisting of four oligonucleotides each. The target sequences of hVPS25 were CAGAACAACTCCGTCTTTA, GCACAAGGCCGAGATCATC, GGGAAACTCATCTATCAGT, and GTCGATCCAGATTGTATTA, and the target sequences of UBPY were GAAGATCAATGACCAGTTT, TGAAATACGTGACTGTTTA, GGACAGGACAGTATAGATA, and AAATAAAGCTCAACGAGAA. The oligonucleotides all had 3'-UU overhangs and were synthesized using Dharmacon Option A4. siRNA oligonucleotides against hVPS22 and hVPS36 were Dharmacon SMARTpools with 3'-UU overhangs.
4 x 105HeLa cells were seeded into 6-cm dishes and transfected the following day with siRNA oligonucleotides using the procedure described in Motley et al. (51). This is a double transfection procedure, with transfections on days 0 and 2. Following the first transfection, on day 1 the cells were transferred to 9-cm dishes. For TSG101 siRNA transfections, cells were transfected on days 1 and 2 to avoid the extensive cell death observed with the longer protocol (see Ref. 52).
Western Blotting—Cells were scraped into 5 ml of ice-cold PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) and pelleted at 1000 x g for 5 min at 4 °C. Cells resuspended in ice-cold lysis buffer (0.5% Nonidet P-40, 150 mM NaCl, 2 mM EDTA, 20 mM Tris, pH 8, with an EDTA-free protease inhibitor mixture (Roche Diagnostics Ltd.)). Following a 15-min incubation on ice, the lysates were spun for 20 min at 16,000 x g, and the postnuclear supernatant was removed to a fresh tube. Protein assays were carried out on the samples (using the BCA assay, Perbio Science UK Ltd.), and equal amounts of protein were loaded per lane on SDS-polyacrylamide gels. For ECL detection (Amersham Biosciences), the proteins were transferred to nitrocellulose membranes, and the membranes probed using primary antibodies followed by goat anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies (Sigma-Aldrich Co Ltd.).
Visualization of GFP and dsRed-tagged Proteins—For permeabilization before fixation: cells expressing GFP-hVPS25 and dsRed-VPS4B E235Q were washed once in cytosol extraction buffer (25 mM Hepes-KOH, pH 7.4, 25 mM KCl, 2.5 mM magnesium acetate, 5 mM EGTA, 150 mML-glutamic acid) and then incubated in cytosol extraction buffer with 0.05% saponin for 30 s. Cells were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature and permeabilized in PBS with 0.05% saponin. All antibody incubations and washes were carried out in PBS with 0.2% BSA and 0.05% saponin. Cells were stained with anti-MPR antibodies followed by an anti-rabbit secondary antibody conjugated to Alexa Fluor 647 (Invitrogen). Cells were visualized with a 63x Plan Apochromat objective (numerical aperture, 1.4) on a Zeiss Axiovert 200M inverted microscope with an LSM 510 confocal laser scanning attachment.
Immunofluorescence—24 h after the second siRNA transfection, HeLa cells were seeded onto glass coverslips and grown for a further 24 h. Cells were either fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 for 4 min (for EEA1, TfR, and clathrin antibody staining) or fixed and permeabilized by incubation with methanol for 5 min at -20 °C (for MPR and LAMP1 antibody staining). All antibody incubations were carried out in PBS with 0.2% BSA. The secondary antibody reagent was either goat anti-mouse or anti-rabbit Alexa Fluor 488 (Invitrogen). Coverslips were mounted on slides using Mowiol 4-88 (Merck Biosciences Ltd.) with 2.5% (w/v) 1,4-diazobicyclo[2,2,2]octane. Stained cells were viewed with a 63x Apochromat objective (numerical aperture, 1.4) on a Zeiss Axioplan microscope and photographed with a digital camera (Princeton Instruments Inc.). For MHC I cell surface staining, cells were fixed with paraformaldehyde as above and stained intact. The secondary antibody reagent was anti-mouse Alexa Fluor 568 (Invitrogen), and coverslips were mounted as above. Stained cells were visualized as for GFP-tagged proteins.
Uptake of Fluorescently Labeled EGF—Cells on glass coverslips were incubated in serum-free medium with 21 µM leupeptin (Sigma-Aldrich) for 3 h at 37°C. Cells were washed 3x in BM (RPMI (Sigma-Aldrich) with 0.1% BSA, 10 mM HEPES, pH 7.4) and incubated in BM with 21 µM leupeptin, 1 µg/ml EGF-Alexa Fluor 488 (biotinylated EGF complexed to Alexa Fluor 488 streptavidin, Invitrogen) for 1 h at 20 °C. After a further incubation at 37 °C for 45 min, the cells were fixed and stained for LAMP1 as described (53). The secondary antibody reagent was anti-mouse Alexa Fluor 568 (Invitrogen). Stained cells were visualized as for GFP-tagged proteins.
EGF Degradation Assays—24 h after the second transfection with siRNA, cells were transferred to 24-well plates at 
50% confluence. Four wells were seeded for each cell type for each time point. The next day, cells were washed once with PBS and serum-starved for 4 h in Dulbecco's modified Eagle's medium. The plates were then placed on ice for 10 min before washing three times in ice-cold BM. Cells were incubated in 0.3 ml of 125I-EGF (PerkinElmer Life Sciences) at 2.5 ng/ml for 1 h on ice. For experiments at 20 ng/ml EGF, 11 ng/ml 125I-EGF was supplemented with cold EGF to a final concentration of 20 ng/ml. Excess 125I-EGF was removed by washing three times in ice-cold BM. 1 ml of BM at 37 °C was added to each well, and the plates were incubated at 37 °C for 30, 60, or 120 min (the 0-time point had 1 ml of cold BM per well). After the appropriate time, the 1 ml of BM was collected from each well, and the cells were washed twice in ice-cold PBS. Half the wells for each cell type were acid-stripped with two 3-min washes in EM (0.2 M acetic acid, 0.5 M NaCl), and all wells were washed twice in 1 ml of cold PBS. Cells were harvested in 800 µl of 0.1 M NaOH, and the samples were counted in a 
-counter. Protein assays were done on each sample after counting. For the media collected from the warm-up, 300 µlwas transferred to a fresh tube and counted on the -counter. A further 500 µl was transferred to a fresh tube, and 20% trichloroacetic acid was added. Samples were incubated on ice for 1 h, spun at 16,000 x g for 5 min, and the supernatant was transferred to a new tube. Both trichloroacetic acid pellets and supernatants were counted in the -counter. The graphs in Fig. 5 represent counts in the trichloroacetic acid supernatants (i.e. degraded 125I-EGF) per microgram of protein in each sample as a percentage of total counts bound at time zero (also per microgram of protein).
Flow Cytometric Analysis—48 h after the second siRNA transfection, 2 x 105 cells were incubated in suspension with anti MHC I antibody for 1 h at 4 °C in the presence of 0.1% BSA in PBS. Unbound antibody was removed by two 1-ml washes with 0.1% BSA in PBS at 4 °C. Cells were incubated in goat anti-mouse IgG (highly cross-absorbed) conjugated to Alexa Fluor 647 (Invitrogen) for 1 h at 4°C, followed by a further two washes in 0.1% BSA in PBS at 4 °C. Cells were pelleted by centrifugation and re-suspended in 300 µl of 1% paraformaldehyde in PBS and analyzed using the FACSCalibur system (BD Biosciences).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, vps25, or vps36).3 Binding Domains of the ESCRTII Subunits—We tested the interactions with other ESCRTII proteins of different regions of hVps36p: the N-terminal domain (N), a C-terminal region containing the entire Vps36 domain (CA), or a fragment equivalent to the C-terminal portion present in the structure of the yeast ESCRTII complex (CB). As shown in Fig. 2, the CA region of hVps36p was sufficient to bind both hVps25p and hVps22p. However, although the shorter CB region of hVps36p interacted strongly with hVps25p, we were unable to detect an interaction between the CB region and hVps22p. No interactions of the N-terminal region of hVps36p were found with any of the constructs tested. These data are summarized in Fig. 2c and are surprising, because in the structure of the yeast ESCRTII complex the C terminus of Vps36p (equivalent to CB) shows a significant interaction with Vps22p (31, 32).
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To investigate the interactions of the human ESCRTII complex in more detail, we used proteins purified from bacteria. We co-expressed hVps25p-GST, hVps36p (CB), and His6-tagged hVps22p (C1) from a single plasmid. All three proteins were eluted from a glutathione-Sepharose column after thrombin cleavage. The thrombin cleaves the GST tag from hVps25p and allows untagged hVps25p and associated proteins to be recovered. On subsequently passing the eluted material over Ni-NTA beads a mixture of His6-hVps22p (C1) and hVps25p bound to the column (Fig. 3a, lanes 3 and 4) and could be eluted. However, a mixture of hVps25p and hVps36p (CB) failed to bind the Ni-NTA column (Fig. 3a, lanes 1 and 2). Only a very small amount of hVps36p (CB) was associated with the His6-hVps22p·hVps25p complex (Fig. 3a, lanes 3 and 4). This suggests that with co-expression of hVps25p, hVps22p (C1) and hVps36p (CB) we generate two stable complexes (hVps25p·hVps22p (C1) and hVps25p·hVps36p (CB)) and very small amounts of a heterotetramer (two copies of hVps25p, hVps22p (C1), and hVps36p (CB)). Identical results were obtained with the longer (CA) hVps36p4.
We also co-expressed hVps25p-GST, hVps36p (CB), and His6-tagged hVps22p (C1), as above, but bound the soluble material first to a Ni-NTA column. As shown in Fig. 3b, lane 1, we were able to elute His6-hVps22p (C1) and hVps25p-GST from this column. The material that failed to bind the Ni-NTA column was subsequently passed over a glutathione-Sepharose column, and from this we were able to elute hVps25p-GST and hVps36p (CB) with a very small amount of His6-hVps22p (C1) (Fig. 3b, lane 2). Again our results suggest that there is very little interaction of the hVps25p·hVps22p (C1) complex with the hVps25p·hVps36p (CB) complex. To confirm this, we tried a GST-pull-down experiment with the purified hVps25p·GST·hVps36p (CB) complex mixed with the purified His6-hVps22p (C1)·hVps25p. We were able to detect only a very weak interaction between the two purified complexes4. Thus, our yeast two-hybrid results (summarized in Fig. 2c) are supported by the data from studies with purified proteins and suggest that the N-terminal region of Vps22p is required for its stable interaction with Vps36p. Unfortunately, we were unable to confirm this directly, because attempts to purify full-length hVps22p were unsuccessful as the protein formed insoluble aggregates.4
Full-length hVps25p, hVps22p, and hVps36p Form a Complex in Vivo—Evidence that full-length hVps25p, hVps22p, and hVps36p form a complex in vivo comes from protein depletion experiments using small interfering RNAs (siRNAs). As shown in Fig. 4, depletion of hVps25p using siRNA results in significant loss of the protein as determined by Western blotting using antibodies to hVps25p. Transfection of siRNA against hVPS22 or hVPS36 also resulted in depletion of hVps25p but no depletion of a control protein, calreticulin. We have been unsuccessful to date in attempts to produce antibodies against hVps22p or hVps36p and so could not assess depletion of these proteins. However, these data suggest that loss of one human ESCRTII protein destabilizes the remaining ESCRTII proteins and is consistent with them forming a complex in vivo. Similar results were obtained in yeast: deletion of any one of the three ESCRTII-encoding genes destabilized the remaining ESCRTII proteins (14). It should also be noted that while depletion of one human ESCRTII protein destabilizes the complex, it does not affect the cellular levels of other ESCRT components such as TSG101 (ESCRTI) or Alix (an ESCRTIII-associated protein) (see Fig. 6d).
Human ESCRTII Can Be Recruited to Endosomes—The data presented above suggested that hVps25p, hVps22p, and hVps36p may be orthologues of the yeast ESCRTII components. If these proteins form human ESCRTII, we would expect them to associate with endosomes. Expression of an N-terminally tagged GFP-hVps25p in HeLa cells by transient transfection produced predominantly cytosolic and nuclear staining. However, expression of dominant-negative versions of Vps4p in yeast or mammalian cells has been shown to increase the association of ESCRT components with endosomal membranes (5, 14, 15, 26, 37, 54, 55). We co-transfected HeLa cells with GFP-hVps25p and dsRed-hVps4Bp E235Q. This transfection produced enlarged, punctate structures in the cells as seen previously following overexpression of dominant-negative hVps4 constructs (55, 56). If the cytosolic pool of GFP-hVps25p was removed before fixation, the GFP-hVps25p was seen colocalized with the dsRed-hVps4Bp E235Q on the swollen structures, as well as in the nucleus (Fig. 5). To confirm that the swollen, punctate structures were endosomes, we costained them with antibodies against endosomal markers. Both the predominantly late endosomal protein MPR (Fig. 5) and the early endosomal protein EEA13 partially colocalized with dsRed-hVps4Bp E235Q and GFP-hVps25p. These data suggest that hVps25p (and presumably the ESCRTII complex) can be recruited to endosomes in an hVps4p-dependent manner. Taken together, our data show that the human ESCRTII proteins associate with one another and are likely to form a complex in vivo that can be recruited to endosomes.
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One simple explanation of our results is that there is redundancy in the mammalian MVB sorting system not seen in yeast. Therefore while ESCRTII may be essential for MVB sorting in yeast, in mammalian cells this essential function may be bypassed. Interestingly, analysis of mammalian ESCRT protein interactions has identified a link between ESCRTs I and III that we were unable to detect in the yeast system (16, 37, 38, 57). Alix, the mammalian homologue of Bro1p/Vps31p was found to interact with both TSG101 (ESCRTI) and hVps32A, -B, and -C (CHMP4A, -B, and -C; ESCRTIII) (37, 38, 57-59). We reasoned therefore that Alix may provide an alternative link between ESCRTs I and III that may bypass the requirement for ESCRTII in some situations. Therefore, depletion of both Alix and ESCRTII may be required to inhibit MVB sorting and thus degradation of EGF. To investigate this hypothesis, we transfected cells with siRNA directed against Alix and against both Alix and hVps25p. As shown in Fig. 6c, co-transfection of siRNAs against both hVps25p and Alix depleted levels of both proteins. However, depletion of hVps25p and Alix did not inhibit EGF degradation (Fig. 6b). In fact, depletion of Alix slightly increased EGF degradation, whereas depletion of Alix and hVps25p together had little effect on EGF degradation. Knock down of Alix in the double depletion situation (Alix and hVps25p) was never as efficient as a single Alix depletion (Fig. 6d), which may explain the smaller effect on EGF degradation observed. There are two alternative explanations for the effect of Alix depletion on EGF degradation: Firstly, we may be increasing the number of intraluminal vesicles formed and thus increasing the amount of EGF sorted for degradation. In this case, Alix would be a negative regulator of MVB formation, and this idea is consistent with the idea that Alix regulates the inward budding of MVB membranes in vitro via its association with the lipid lysobisphosphatidic acid (50). Secondly, depletion of Alix may allow a greater concentration of EGF in the inward-budding vesicles, again resulting in increased degradation. In this case, Alix would be a negative regulator of ESCRT complex function.
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All our initial EGF degradation experiments were performed with a 2.5 ng/ml concentration of 125I-EGF. A recent report suggests that, at this low concentration of EGF, the EGF·EGFR complex is internalized via a clathrin-mediated route (63). However, upon stimulation with a higher concentration of EGF (20 ng/ml), the EGF·EGFR complex is internalized predominantly via a clathrin-independent caveolae pathway (63). To determine whether the route of EGF·EGFR internalization affects the downstream requirement for ESCRTII in MVB protein sorting, we performed EGF degradation experiments with 20 ng/ml concentrations of EGF. As shown in Fig. 6c, we again found no effect of hVps25p depletion on EGF degradation, even with this higher concentration of EGF. Transfection of the cells with siRNA against Alix slightly increased EGF degradation, as seen with the lower concentration of EGF. TSG101 depletion inhibited EGF degradation (Fig. 6c), demonstrating that EGF degradation is TSG101-dependent and ESCRTII-independent irrespective of its method of internalization.
To show that EGF was still targeted to lysosomes for degradation in the hVps25p-depleted cells, we internalized fluorescently labeled EGF. As shown in Fig. 7, EGF-Alexa Fluor 488 internalized for 1 h at 20 °C and 45 min at 37 °C in the presence of the protease inhibitor leupeptin in mock transfected cells showed partial colocalization with the lysosomal marker lysosome-associated membrane protein 1 (LAMP1). Similar colocalization was seen following EGF-Alexa Fluor 488 uptake in the hVps25p-depleted cells. Our results suggest that EGF is taken up and degraded in lysosomes in hVps25-depleted cells.
Depletion of Mammalian ESCRTII Does Not Affect KK3-mediated Down-regulation of MHC Class I Molecules—As described above, depletion of ESCRTII did not inhibit EGF degradation. Therefore, it is possible that ESCRTII may be cargo-specific, and we decided to study another cargo of the MVB sorting pathway. The Kaposi's sarcoma-associated herpesvirus (KSHV) protein K3 (KK3) is a ubiquitin ligase (52). This protein is involved in the evasion of the immune system by virally infected cells. Expressed alone in HeLa cells this protein polyubiquitinates and causes down-regulation from the cell surface of MHC I molecules (52, 64). The down-regulation of MHC I in KK3-expressing cells is dependent on TSG101 and hVps4Bp (37, 52). We assayed the down-regulation of MHC I in HeLa cells or in HeLa cells stably expressing KK3 (HeLa-KK3). >99% of HeLa cells had MHC I on the cell surface, as determined by cytofluorometric analysis (Fig. 8a). This was unchanged in HeLa cells transfected with siRNA against hVps25p or TSG101 and consistent with the cell-surface staining of MHC I seen in these cells by immunofluorescence (Fig. 8b). In HeLa-KK3 cells, few cells had cell-surface MHC I (Fig. 8a, and b, HeLa-KK3 mock). In contrast, in HeLa-KK3 cells transfected with siRNA against TSG101, MHC I now appeared on the cell surface, as seen by a shift in the trace in Fig. 8a to the right and cell-surface fluorescence in Fig. 8b. However, HeLa-KK3 cells transfected with siRNA against hVPS25 showed identical results to the mock transfected cells (Fig. 8a, and b) suggesting that depletion of ESCRTII did not affect K3-mediated MHC I down-regulation. The transfection of HeLa-KK3 cells with siRNA against hVPS22 or hVPS36 produced similar results to those obtained with siRNA against hVPS25.5
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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From the yeast ESCRTII structure, the interaction of Vps20p with the complex is via Vps25p (31). Our data are consistent with a similar interaction in human ESCRTII, because we find that hVps25p binds hVps20p while hVps22p and hVps36p do not. The yeast ESCRTII structures were lacking the N-terminal 395 amino acids of Vps36p, suggesting that the C-terminal domain is sufficient to interact with Vps25p and Vps22p (31, 32). Consistent with the yeast data, we have shown that the C-terminal domain of hVps36p is sufficient to bind both hVps22p and hVps25p. However, our analysis with the human proteins suggests that the C-terminal region of hVps36p required for binding to hVps22p is larger than that predicted by the yeast structure. As summarized in Fig. 2c, we find that a construct containing amino acids 126-386 (CA) of hVps36p is capable of binding both hVps25p and hVps22p, but a construct corresponding to the shorter part of yeast Vps36p in the structure (hVps36p amino acids 232-386, CB) binds only Vps25p. This implies that amino acids 126-231 of hVps36p may be required either to mediate or strengthen the interaction between hVps36p and hVps22p in the mammalian complex. Also unexpected from the yeast ESCRTII structure was the finding that the N-terminal region of hVps22p was required for the binding of hVps36p. (It should also be noted that yeast Vps36p is significantly larger than human Vps36p.) Our yeast two-hybrid and purified protein interaction data suggest that human ESCRTII may have a modified structure compared with yeast ESCRTII, which may account for its unexpected redundancy or cargo-specificity in MVB protein sorting (see below).
Functional Consequences of ESCRTII Depletion—In our experiments, depletion of human ESCRTII had no effect on EGF degradation. This was surprising, because previous studies have shown that ESCRTI and hVps4p are essential for this process (28-30, 55). Yeast studies also imply that ESCRTII is essential for MVB protein sorting, and current models imply that yeast ESCRTII is required in stoichiometric amounts (14, 16). In addition, recent studies show that mutation or depletion of Drosophila Vps25p results in enlarged endosomal structures where the receptors Notch and Dpp accumulate (65, 66). A similar phenotype was seen in patches of Drosophila tissue expressing mutant forms of the erupted gene product, the orthologue of mammalian TSG101 (67). However, although vps25 or erupted mutant Drosophila cells show ectopic Notch activation, cells expressing mutant forms of Hrs did not. Cells from hrs mutant Drosophila failed to degrade EGF leading to enhanced signaling (68). In contrast, Drosophila cells expressing mutant vps28 showed little or no effect on the trafficking of the Sevenless receptor or its ligand Boss, on EGFR trafficking, or on the trafficking of Delta (a ligand of Notch) (69). The different functional effects of mutating ESCRT subunits in Drosophila therefore make it difficult to fit the fly data with the current yeast model of MVB sorting. Nevertheless, it is significant that Drosophila ESCRTII appears important in endosomal sorting. Also of note is the finding from siRNA analysis in the Caenorhabditis elegans system: depletion of the VPS class E homologues resulted in a variety of different phenotypes (70). Depletion of the Vps36p homologue was larval-lethal, whereas depletion of the Vps22p or Vps25p homologues resulted in no observable phenotype.
By Western blotting, we were typically able to knock down expression of hVps25p by >90%. We were also able to show that transfection of siRNA oligonucleotides against hVps22p or hVps36p significantly reduced the level of hVps25p (Fig. 4). Thus knock down of one ESCRTII component destabilized the complex, and our cells had very little ESCRTII. However, the <10% ESCRTII remaining after siRNA transfection using our double transfection procedure is likely to be the result of 90% transfection efficiency of the siRNA. Thus we produce a mixed population of cells, 10% of which have relatively normal levels of ESCRTII and 90% of which have almost no ESCRTII. There are several possible explanations for our lack of MVB sorting phenotype on depleting mammalian ESCRTII: (i) Human ESCRTII may be cargo-specific. To address this issue, we studied the down-regulation of MHC I in cells expressing the KSHV ubiquitin ligase K3. KK3-mediated MHC I down-regulation is dependent on TSG101 (ESCRTI) and hVps4Bp (37, 52). As with EGF degradation, we saw no effect of ESCRTII depletion on MHC I trafficking in KK3-expressing cells. It should be noted that the EGFR is mono-ubiquitinated at multiple sites, whereas MHC I is poly-ubiquitinated by the KK3 E3 ubiquitin ligase (52, 71). Thus for two differently ubiquitinated cargo proteins we saw no requirement for ESCRTII in MVB protein sorting. It should also be noted that overexpression of ESCRTII proteins had no effect on HIV budding, whereas overexpression of other ESCRT proteins had a dominant-negative effect (38). However, our results do not rule out ESCRTII being specific for a cargo we have not yet tested. Interestingly, a recent report has suggested the presence of more than one type of MVB, with only a subpopulation of MVBs capable of sorting EGF·EGFR (72). Therefore, ESCRTII may be required for protein sorting at the MVBs that do not sort EGF·EGFR (or virally ubiquitinated MHC I). Yet another possibility is that ESCRTII is essential for the MVB sorting of cargo transported directly from the late Golgi to endosomes, rather than cargo endocytosed from the cell surface to endosomes. (ii) The mammalian MVB sorting system is likely to be more complex than the yeast system and may show more redundancy. The complexity of the mammalian system is demonstrated by the presence of several isoforms of proteins that have only one form in yeast. For example, there are two isoforms of Vps2p (hVps2Ap and -B, also known as CHMP2A and -B); two isoforms of Vps4p (hVps4Ap and hVps4Bp, also known as SKD1); two isoforms of Vps46p (hVps46Ap and -B, also called CHMP1A and -B); three isoforms of Vps32p (hVps32pA-C, also known as CHMP4A-C); and four isoforms of Vps37p (hVps37pA-D). In addition, ESCRTs I and III are connected in mammalian cells via ESCRTII and (unlike in yeast) via Alix. We found that depleting both ESCRTII and Alix did not inhibit EGF degradation, suggesting that ESCRTI-III interaction via Alix does not compensate for loss of ESCRTII in our system. However, we are unable to exclude that idea that lack of ESCRTII may be compensated by an as yet unknown factor or factors. (iii) The mammalian MVB sorting system may have evolved such that ESCRTII plays a different role from its yeast counterpart. As mentioned above, the mammalian MVB sorting is likely to be more complex than the yeast system. This is necessarily so, because cells of multicellular organisms can be specialized for a huge number of different functions. Because we find the interactions of the human ESCRTII proteins with each other and with ESCRTIII, the human ESCRTII proteins bind the yeast proteins, and human ESCRTII can be recruited to endosomes, we suggest that human ESCRTII is likely to interact with the other ESCRT complexes in a similar way to its yeast homologue. However, instead of the essential role played by yeast ESCRTII in linking ESCRTs I and III and binding ubiquitinated cargo proteins, mammalian ESCRTII may play a regulatory role, perhaps in coordinating MVB sorting with other endosomal sorting events. (iv) Our ESCRTII depletions by siRNA techniques may not be sufficient to knock out ESCRTII function. As discussed above, we were typically able to knock down expression of mammalian ESCRTII almost completely in 90% of cells in the population. Similar levels of protein depletion were achieved with siRNAs against TSG101, UBPY, or AMSH, and yet these depletions were sufficient to produce either significant inhibition or stimulation of EGF degradation. Nevertheless, we cannot rule out the possibility that ESCRTII is required for MVB sorting at extremely low (non-stoichiometric) or catalytic levels.
ESCRTII or EAP Complex?—Our data and those of others suggest that hVps25p, hVps22p, and hVps36p make up the mammalian ESCRTII complex. However, do the same proteins (in a complex with the transcription factor ELL) have another role in the nucleus as part of the EAP complex? Several other mammalian class E Vps protein homologues are thought to play additional roles in the cell nucleus. Mammalian Vps46p (CHMP1) plays a role in chromatin remodeling; TSG101 binds the glucocorticoid receptor and inhibits receptor transcriptional activation; and mammalian Vps24p (CHMP3) was localized to the nucleus in addition to endosomes (73-75). In addition, other components of the endocytic protein sorting machinery have been shown to link endocytic sorting to transcriptional regulation (reviewed in Ref. 76). For example, Eps15, epsin, and -arrestin 2 (components of clathrin-coated pits) undergo nucleocytoplasmic shuttling (77-80). In addition, the Rab5 effectors APPL1 and APPL2 dissociate from early endosomal membranes and translocate to the nucleus following prolonged treatment of cells with EGF (81). In the nucleus, the APPL proteins bind the nucleosome remodeling and histone deacetylase complex NuRD/MeCP1. Therefore, the ESCRT proteins may provide a further link between transcriptional regulation and endocytic protein sorting.
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1 To whom correspondence should be addressed. Tel.: 44-1-223-336-782; Fax: 44-1-223-762-640; E-mail: kb123{at}cam.ac.uk.
2 The abbreviations used are: MVB, multivesicular body; BSA, bovine serum albumin; EAP, ELL-associated protein; EEA1, early endosome autoantigen 1; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ESCRT, endosomal sorting complex required for transport; KK3, KSHV protein K3; KSHV, Kaposi's sarcoma-associated herpesvirus; LAMP1, lysosome-associated membrane protein 1; MHC I, major histocompatibility complex class I; MPR, cation-independent mannose-6-phosphate receptor; NZF, npl4 zinc finger; PBS, phosphate-buffered saline; TfR, transferrin receptor; VPS, vacuolar protein sorting; GST, glutathione S-transferase; GFP, green fluorescent protein; EGFP, enhanced GFP; AMSH, associated molecule with the SH3 domain of STAM (signal transducing adaptor molecule); UBPY, ubiquitin isopeptidase Y; Ni-NTA, nickel-nitrilotriacetic acid; siRNA, small interfering RNA.
3 K. Bowers and J. P. Luzio, unpublished results.
4 M. Edeling and D. J. Owen, unpublished results.
5 S. C. Piper, J. P. Luzio, and P. J. Lehner, unpublished results.
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