Article |
Address correspondence to Harald Stenmark, Dept. of Biochemistry, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway. Tel.: 47-22934951. Fax: 47-22508692. email: stenmark{at}ulrik.uio.no
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Abstract |
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Key Words: endocytosis; lysosome; membrane traffic; Tsg101; protein sorting
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Introduction |
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The core constituents of the machinery responsible for endosomal sorting and MVB formation have been identified by yeast genetics through the isolation of vacuolar protein sorting (vps) mutants (Rothman and Stevens, 1986; Odorizzi et al., 1998). Recent work has revealed that 10 out of the 17 known Vps class E proteins participate in three endosomal sorting complexes required for transport (ESCRT), ESCRT-I, -II, and -III, and genetic evidence suggests that these complexes function sequentially in protein sorting and MVB formation (Katzmann et al., 2001; Babst et al., 2002a, b). ESCRT-I consists of three proteins and is involved in the sorting of ubiquitinated proteins into MVBs. One of the subunits of ESCRT-I is Vps23p, which recognizes ubiquitin via its NH2-terminal ubiquitin E2 variant domain (Katzmann et al., 2001). The ESCRT complexes are conserved through evolution, and the mammalian homologue of Vps23p, Tsg101, has been shown to mediate the sorting of ubiquitinated proteins into the degradative pathway, much like its yeast counterpart (Babst et al., 2000; Bishop et al., 2002). Remarkably, Tsg101 is also required for the budding of several enveloped RNA viruses such as HIV and Ebola from the plasma membrane (Garrus et al., 2001; Martin-Serrano et al., 2001). The ubiquitin E2 variant domain of Tsg101 binds to a P(S/T)AP sequence in viral proteins required for budding, and the interaction may be strengthened by the simultaneous binding to a nearby ubiquitin moiety (Pornillos et al., 2002a,b). It is thought that the binding of Tsg101 to viral proteins recruits the Vps class E machinery to the plasma membrane and engages it in a budding process topologically similar to that of MVB formation (Carter, 2002; Pornillos et al., 2002c).
One of the Vps class E proteins that has not been assigned to any of the ESCRT complexes is Vps27p and its mammalian homologue Hrs. Like Vps23p and Tsg101, Vps27p and Hrs bind ubiquitinated proteins and mediate their sorting into MVBs (Bilodeau et al., 2002; Raiborg et al., 2002; Shih et al., 2002). This raises the question of how Vps23p/Tsg101 and Vps27p/Hrs recruitment is coordinated in time and space. Here, we have tried to address this question.
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Results |
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Hrs is required for the localization of ESCRT-I to membranes
Because Hrs binds to the endosome-specific lipid phosphatidylinositol 3-phosphate (Gaullier et al., 1998; Gillooly et al., 2000), this protein is likely to interact directly with the endosome membrane. The finding that Hrs interacts with Tsg101, therefore, raised the possibility that Hrs may function to recruit ESCRT-I to endosome membranes. Should this be the case, then a reduction in the cellular level of Hrs might lead to a decrease in membrane-associated ESCRT-I subunits. To investigate this possibility, we used a specific small interfering RNA (siRNA) molecule for the knockdown of Hrs expression (Elbashir et al., 2002; Bache et al., 2003). As shown in Fig. 2 (A and B), HeLa cells incubated with the siRNA duplex showed a strong decrease in Hrs expression (as assessed with Western blotting with antibodies specific for Hrs) when compared with cells that had been incubated in the presence of a control RNA. Importantly, Western blotting with anti-Tsg101 antibodies showed that there was a significant reduction of membrane-associated Tsg101 levels in cells treated with siRNA to Hrs. We also investigated whether Hrs is required for membrane localization of the other known human ESCRT-I subunit, hVps28, which does not bind directly to Hrs. Like Tsg101, the amount of hVps28 in the membrane fraction was reduced by 50% in cells treated with siRNA against Hrs (Fig. 2, B and C). In the case of both Tsg101 and hVps28, the decrease in membrane-associated proteins was less than that of Hrs in Hrs-depleted cells. We do not know the reason for this, but one explanation could be that ESCRT-I is also associated with endosomes via other molecules than Hrs (see Discussion). In any case, these results indicate that Hrs is required for efficient membrane targeting of ESCRT-I.
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Discussion |
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What is the exact role of Hrs in MVB formation? Previous work has shown that the UIMs of Hrs and Vps27p are essential for sorting of ubiquitinated membrane proteins into the degradative pathway (Bilodeau et al., 2002; Raiborg et al., 2002; Shih et al., 2002). Interestingly, mutations in the UIMs of Vps27p specifically inhibit sorting of ubiquitinated proteins without affecting MVB formation (Bilodeau et al., 2002), suggesting that ubiquitin recognition may be uncoupled from the formation of intraluminal vesicles. Our finding that Hrs binds to Tsg101 and is required for membrane recruitment of both Tsg101 and hVps28 indicates that Hrs acts in the recruitment of ESCRT-I to endosomes. This provides the following plausible explanation for the lack of MVB formation in the absence of Hrs: because the ESCRT complexes function coordinately in the formation of inward endosomal invaginations (by mechanisms that are still not understood), a failure to recruit ESCRT-I will effectively inhibit this process.
Our finding that siRNA-mediated depletion of Hrs inhibits MVB formation agrees with previous studies of Hrs homologues in yeast and Drosophila (Odorizzi et al., 1998; Lloyd et al., 2002). Even though enlarged early endosomes have been observed in Hrs -/- mouse embryos (Komada and Soriano, 1999) and in Hrs-depleted HeLa cells (Bache et al., 2003), it was more surprising to observe that lysosomes increased greatly in size in HeLa cells treated with siRNA against Hrs. This indicates that Hrs not only controls MVB formation but also controls the morphology of endosomes and lysosomes.
The UIM has been shown to function as a signal for mono-ubiquitination of Hrs (Polo et al., 2002). Because Tsg101 binds with increased avidity to HIV Gag when this protein is ubiquitinated (Pornillos et al., 2002b), it is possible that ubiquitination of Hrs may strengthen its interaction with Tsg101. Ubiquitination of Hrs cannot be absolutely required, though, because Hrs287573, which interacted strongly with Tsg101 in vitro and in the two-hybrid system, does not contain the UIM. Even though HrsP351A had a reduced ability to bind Tsg101, overexpression of this mutant caused a similar accumulation of Tsg101 on early endosomes as wild-type Hrs (unpublished data). This can probably be explained by its residual ability to bind Tsg101 (Fig. 1 B), and the fact that we are unable to saturate Hrs binding sites on endosomes (Raiborg et al., 2001b). However, it is interesting to note that membrane recruitment of yeast Vps23p by Vps27p requires a region that contains the PSAP-like sequence PTVP, and which is also required for sorting of a ubiquitinated protein into the vacuole lumen (see Katzmann et al., 2003, in this issue). This indicates that membrane recruitment of Vps23p (Tsg101) by Vps27p (Hrs) is required for normal MVB sorting.
Previous work has shown that Hrs partially colocalizes with another FYVE domain containing protein, EEA1 (Urbé et al., 2000; Raiborg et al., 2001b). However, whereas EEA1 is strictly confined to sorting early endosomes (Wilson et al., 2000), Hrs is also present on the limiting membrane of MVBs (Tsujimoto et al., 1999; Sachse et al., 2002). Given that Hrs partially colocalizes with EEA1, we were initially surprised to find so little colocalization between Tsg101 and EEA1 (Fig. 7, AC). However, this lack of colocalization can be explained if EEA1 localizes to sorting endosomes and Tsg101 to MVBs/late endosomes. The finding that Tsg101, but not Hrs, is present on late (LAMP-1 positive) endosomes indicates that these proteins are not permanently associated. We speculate that Hrs is involved in the initial recruitment of Tsg101 to form MVBs, and that Tsg101 remains membrane associated through interactions with other molecules. Accordingly, when Hrs is overexpressed, it accumulates on early endosomes and causes a strong redistribution of Tsg101 to these structures and an altered appearance of late endosomes. The observation that Tsg101 is associated with LBPA-positive MVBs and late endosomes, which by definition contain intraluminal vesicles (Gruenberg, 2001; Katzmann et al., 2002), raises the possibility that ESCRT proteins may remain associated with the limiting membrane of the MVB even after formation of intraluminal vesicles has taken place.
Hrs and the ESCRT complexes are essential for endosomal protein sorting and MVB formation. Here, we have identified a functional interaction between Hrs and the ESCRT-I subunit Tsg101. Viral late domains target Tsg101 and ESCRT-I to the plasma membrane via a P(S/T)AP sequence in order to facilitate viral budding (Carter, 2002; Pornillos et al., 2002c). Hrs appears to act in a similar manner at the early endosome and is critical for the formation of endosomal invaginations and vesicles. Indeed, Pornillos et al. (2003)(in this issue) show in a companion paper that the PSAP-containing part of Hrs can functionally replace the PTAP-containing part of the HIV Gag protein in budding of viruslike particles from the plasma membrane, indicating that HIV Gag mimics the Tsg101-recruiting effect of Hrs. Together, with previously published results (Katzmann et al., 2001; Bilodeau et al., 2002; Bishop et al., 2002; Lloyd et al., 2002; Raiborg et al., 2002; Shih et al., 2002), these results argue that Hrs plays a dual role in endosomal protein sorting: First, it recruits ubiquitinated proteins into clathrin-coated microdomains on early endosomes. Second, it recruits the ESCRT-I complex that is essential for further sorting and MVB formation.
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Materials and methods |
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Antibodies
Affinity-purified rabbit antibodies against recombinant Hrs have been described previously (Raiborg et al., 2001a). Antibodies against a recombinant maltose binding protein, hVps28 fusion protein, were prepared in a similar manner. These antibodies recognized endogenous hVps28 by Western blotting but not by immunocytochemistry. Mouse mAbs against Tsg101 were obtained from GeneTex. Human anti-EEA1 antiserum (Mu et al., 1995) was a gift from B. Toh (Monash University, Melbourne, Australia). Mouse mAbs against human LAMP-1 were from the Developmental Studies Hybridoma Bank of the University of Iowa. Mouse mAbs against LAMP-2 were provided by G. Griffiths (University of Oxford, Oxford, UK). Mouse mAbs against LBPA (Kobayashi et al., 1998) were provided by J. Gruenberg (University of Geneva, Geneva, Switzerland). Cy2-, Cy3-, and Cy5-labeled secondary donkey antibodies were from Jackson ImmunoResearch Laboratories. The Zenon® mouse IgG labeling kit was from Molecular Probes. It was used to label anti-Tsg101 antibodies with Alexa-588conjugated Fab fragments according to the manufacturer's instructions.
Expression of GST fusion proteins in E. coli
GST fusion of Hrs287573 and the P351A mutation of the same Hrs fragment was produced in E. coli BL21 (DE3) cells transformed with the respective pGEX constructs as described previously (Raiborg et al., 2001a). The recombinant protein was purified on glutathione-Sepharose 4B (Amersham Biosciences) after lysis of the bacteria in B-PERTM reagent (Pierce Chemical Co.), according to the manufacturer's instructions.
In vitro transcription and translation
pGEM-Tsg101 was linearized with SalI, and in vitro transcribed with T7 RNA polymerase (Promega), according to the manufacturer's protocol. The resulting mRNA was translated in the presence of [35S]methionine (Amersham Biosciences) in rabbit reticulocyte lysate according to the manufacturer's instructions. The lysate was dialysed overnight against assay buffer (20 mM Hepes, pH 7.2, 140 mM NaCl, and 1 mM dithiothreitol).
GST pull-down assay
Purified GST or GST fusion protein (100 pmol) was bound to 20-µl aliquots of glutathione-Sepharose (Pharmacia Biosciences) at 4°C for 60 min. The beads were washed with assay buffer and 50 µl of in vitro-translated Tsg101 was added. After rotation at 4°C for 60 min, the beads were washed three times with assay buffer and analyzed by SDS-PAGE and fluorography. Films were scanned (model ScanJet 6100C; Hewlett Packard), and images were processed with Adobe Photoshop 7.0.
Cell culture and transfection
HeLa cell cultures were maintained as recommended by the American Type Culture Collection. For expression in mammalian cells, we used the FuGENE system according to the manufacturer's instructions (Roche Diagnostic Corporation). Constructs to be expressed were cloned behind the myc epitope of pcDNA3 (Invitrogen). Cells were analyzed 48 h after transfection. Transfection of HeLa cells with siRNA was performed as described previously (Elbashir et al., 2002). The cells were first transfected with siRNA for 3 d, then the cells were replated, and the transfection was repeated for another 3 d.
Two-hybrid methods
For use in the two-hybrid system, constructs were cloned into pLexA/pBTM116 (Vojtek et al., 1993) as bait and pGAD GH (CLONTECH Laboratories, Inc.) as prey. The yeast reporter strain L40 (Vojtek et al., 1993) was cotransformed (Schiestl and Gietz, 1989) with the indicated pLexA and pGAD plasmids, and ß-galactosidase activities of transformants were determined as described previously (Guarente, 1983).
Preparation of membrane and cytosolic fractions of HeLa cells treated with siRNA against Hrs
HeLa cells were transfected with siRNAs against Hrs or a scrambled RNA duplex as control (Bache et al., 2003). The cells were washed three times with ice-cold PBS, and homogenized in homogenization buffer (10 mM Hepes, 3 mM imidazole, pH 7.2, 250 mM sucrose, and mammalian protease inhibitor cocktail [used according to the manufacturer's instructions]; Sigma-Aldrich) by repeated passages through a 22-gauge needle at 4°C. Membrane particulate and cytosolic fractions were prepared from postnuclear supernatants by ultracentrifugation for 15 min at 65,000 rpm in a TLA-100 rotor using a table top ultracentrifuge (Beckman Coulter).
Confocal immunofluorescence microscopy
Transfected or nontransfected HeLa cells grown on coverslips were permeabilized with 0.05% saponin, fixed with 3% PFA, and stained for fluorescence microscopy as described previously (Simonsen et al., 1998). Coverslips were examined using a META microscope (model LSM 510; Carl Zeiss MicroImaging, Inc.) equipped with a Neo-Fluar 100x/1.45 oil immersion objective. Image processing was done with Adobe Photoshop version 7.0.
Electron microscopy
To label lysosomes, we incubated cells with BSA gold (final OD520 value of 23) for 3 h followed by an overnight chase. Cells were fixed in a mixture of 4% formaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, washed, and scraped in 1% gelatin/PBS. After embedding with 12% gelatin/PBS, tissue blocks were infused with 2.3 M sucrose overnight at 4°C, mounted, and frozen in liquid nitrogen (Peters et al., 1991). Ultrathin cryosections were cut at -110°C on a microtome (Ultracut; Leica) and collected with a 1:1 mixture of 2% methyl cellulose and 2.3 M sucrose. Sections were transferred to formvar/carbon-coated grids and labeled with primary antibodies followed by protein Agold conjugates essentially as described previously (Slot et al., 1991). For plastic embedding, the cells were fixed in 2% glutaraldehyde in cacodylate buffer, fixed after in 2% osmium tetroxide, and stained with uranylacetate. After dehydration and embedding, the cells were sectioned and examined in an electron microscope (model CM100; Philips).
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Acknowledgments |
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This work was supported by the Top Research Programme, the Research Council of Norway, the Norwegian Cancer Society, the Novo-Nordisk Foundation, and the Anders Jahre's Foundation for the Promotion of Science.
Submitted: 24 February 2003
Accepted: 11 June 2003
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