1 Department of Biochemistry, Institute for Cancer Research, the Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway
2 Institute of Pathology, the National Hospital, N-0027 Oslo, Norway
*Author for correspondence (e-mail: stenmark{at}ulrik.uio.no)
Accepted March 18, 2001
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SUMMARY |
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Key words: Endocytosis, FYVE domain, Membrane traffic, Phosphoinositide, Rab5
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INTRODUCTION |
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We have previously characterised the subcellular targeting of the early endosomal autoantigen EEA1. This large protein, which regulates endocytic membrane fusion (Simonsen et al., 1998b; Mills et al., 1998), is found exclusively on early endosomes (Mu et al., 1995; Wilson et al., 2000). Targeting of EEA1 to early endosomes has been found to rely on a cooperative lipid and protein interaction. The FYVE zinc finger domain of EEA1 binds to the phosphatidylinositol 3-kinase (PI 3-kinase) product, phosphatidylinositol 3-phosphate (PtdIns(3)P), whereas an adjacent domain binds to the endosomal GTPase, Rab5 (Simonsen et al., 1998b). Even if the two individual interactions are of low affinity, the dual interaction is sufficiently strong to efficiently recruit EEA1 to early endosomal membranes. Early endosome membranes are presumably the only cellular membranes that contain the combination of PtdIns(3)P and Rab5 (Gillooly et al., 2000), and this may explain the highly specific localisation of EEA1.
Like with EEA1, the FYVE domain of Hrs binds to PtdIns(3)P (Gaullier et al., 1998; Gillooly et al., 2000), and the membrane association of Hrs is regulated by PI 3-kinase (Komada and Soriano, 1999; Urbé et al., 2000). This raises the question whether Hrs has the same mechanism of membrane targeting as EEA1. In addition to the FYVE domain, Hrs also contains an N-terminal VHS-domain (Lohi and Lehto, 1998), a proline-rich domain, two coiled-coil domains (CC1 and CC2, respectively), and a C-terminal proline- and glutamine-rich domain (Komada and Kitamura, 1995) (see Fig. 1). Based on the recently solved X-ray structure of the N terminus of Hrs, a model has been proposed for its membrane association (Mao et al., 2000). According to this model, a dimeric FYVE domain of Hrs binds to two molecules of PtdIns(3)P in the membrane, and the membrane interaction is further stabilized by the interaction of the VHS domains with the cytoplasmic face of the membrane. To date, this model has not been tested experimentally. Here we have investigated the roles of the various Hrs domains in its subcellular targeting. We find that targeting of Hrs to early endosomes is Rab5 independent and is executed by the FYVE domain in cooperation with the SNAP-25-binding CC2 domain.
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MATERIALS AND METHODS |
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Plasmid constructs
Hrs or the deletion constructs indicated were generated by PCR with mouse Hrs (Komada and Kitamura, 1995) as a template. Synthetic oligonucleotides were from MedProbe (Oslo, Norway). The FYVE and CC2 domain constructs used consisted of residues 147-223 and 420-573 of Hrs, respectively. The HrsC215S mutation has been described before (Gaullier et al., 1998). The HrsR183A mutant was prepared by PCR mutagenesis. PCR errors were excluded by sequencing. For expression in mammalian cells with the T7 RNA polymerase vaccinica virus system, constructs were cloned behind the myc-epitope of pGEM-myc4 (Simonsen et al., 1998a). For expression in mammalian cells with Fugene(R) (Roche) transfection, we constructed a myc-eptiope-tagged variant (pcDNA3-myc) of pcDNA3 (Invitrogen). Constructs were cloned in-frame behind the myc-epitope of this plasmid. For use in the two-hybrid system, constructs were cloned into pLexA/pBTM116 (Vojtek et al., 1993) as bait and pGAD GH (Clontech) as prey. For expression as GST fusions in E. coli BL-21(DE 3) cells, the FYVE domains (residues 147-223) of Hrs, HrsR183A and HrsC215S were cloned into pGEX-6P-3 (Pharmacia Amersham). Expression and purification were performed as described (Gaullier et al., 2000).
Transient expression in BHK cells
In order to minimise possible artefacts associated with transient protein expression, we used two different transient expression systems. The pGEM constructs were expressed in cells using the modified Ankara T7 RNA polymerase recombinant vaccinia virus system and lipofection as described (Stenmark et al., 1995a; Sutter et al., 1995), and the cells were analyzed 6 hours after transfection. The pcDNA3 constructs were expressed in cells using Fugene(R) (Roche) according to the manufacturers instructions. In these cases, cells were analysed 24 hours after transfection. SDS-PAGE of transfected cells followed by immunoblotting with anti-myc antibodies indicated that the various constructs were expressed at comparable levels.
Confocal fluorescence microscopy
BHK cells grown on coverslips were transiently transfected, fixed with 3% paraformaldehyde and stained for fluorescence microscopy as described (Simonsen et al., 1998a). In some experiments, cells were permeabilised with 0.05% saponin (Simonsen et al., 1998a) prior to fixation. When indicated, cells were incubated with Alexa488-transferrin (25 µg/ml) prior to fixation. Toto-3 (Molecular Probes) was used for the labelling of nuclei. Coverslips were examined using a Leica TCS NT confocal microscope equipped with a Kr/Ar laser and a PL Fluotar 100x/1.30 oil immersion objective. Appropriate emission filter settings and controls were included in order to exclude bleed-through effects.
Electron microscopy
To identify early endosomal compartments cells were incubated with 3-7 nm BSA-coated colloidal gold (Slot and Geuze, 1985) in the medium at 37°C for 10 minutes. At the end of the incubation with BSA-gold the cells were washed with PBS and immediately fixed with 0.1% glutaraldehyde/4% paraformaldehyde in Soerensen phosphate buffer. Following fixation the cells were scraped from the culture dish, pelleted, infused with 2.3 M sucrose, mounted, frozen and stored in liquid nitrogen. Immunocytochemical labelling was performed on thawed cryosections as described (Griffiths et al., 1984), using mouse anti-myc antibodies followed by rabbit-anti mouse IgG antibodies and 15-nm protein A-gold (purchased from G. Posthuma and J. Slot, Utrecht, The Netherlands). The labelled cryosections were viewed in a Phillips CM120 electron microscope.
Circular dichroism (CD) spectroscopy
CD spectra of wild-type and mutant GST-FYVE fusion proteins were recorded using a Jasco J-810 spectropolarimeter calibrated with ammonium d-camphor-10-sulfonate. Measurements were performed at 20°C using quartz cuvettes with a path length of 0.1 cm. All the measurements were performed with a protein concentration of 0.15 mg/ml in 10 mM sodium phosphate, pH 7.0. Samples were scanned 5 times at 50 nm/minute over the wavelength range 200-240 nm. The data were averaged and the spectrum of a protein-free control sample was subtracted. The resultant spectra were then smoothed with the binominal method. All measurements were conducted at least twice.
Surface plasmon resonance
Surface plasmon resonance was recorded at 25°C on a BiaCore X (BiaCore, Sweden). The liposomes used contained 63% phosphatidyl-choline, 20% phosphatidylserine, 15% phosphatidylethanolamine and 2% PtdIns(3)P (Echelon) (Gaullier et al., 2000). Liposomes (0.35 mg/ml) were loaded onto a Biacore L1 chip by three successive injections of 80 µl liposomes at a flow rate of 5 µl/minute. The reference cell was loaded with similar liposomes lacking PtdIns(3)P. Sensorgrams were recorded upon the injections of 0.1-2 µg protein at a flow rate of 20 µl/minute. The lipid surface was regenerated using 10 mM NaOH.
Two-hybrid methods
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 duplicate transformants were determined as previously described (Guarente, 1983).
Subcellular fractionation
Cells grown in 10-cm plastic dishes were collected with a rubber policeman, and post-nuclear supernatants, membrane and cytosol fractions were prepared as described (Stenmark et al., 1994). Equal samples of the post-nuclear supernatant, cytosol and membrane fractions were analysed by SDS-PAGE and immunoblotting with anti-myc antibodies. For detection, we used horseradish peroxidase-conjugated goat anti-mouse IgG antibodies and the SuperSignal chemoluminescence system (Pierce), according to the instructions from the manufacturer.
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RESULTS |
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A functional FYVE domain is important for the subcellular targeting of Hrs
Even though incubation of cells with wortmannin causes the dissociation of endogenous Hrs from endosomes (Komada and Soriano, 1999; Urbé et al., 2000), conflicting results have been obtained regarding the importance of the FYVE domain (Hayakawa and Kitamura, 2000; Urbé et al., 2000). Of two different studies employing the PtdIns(3)P-binding deficient FYVE mutant HrsC215S, one concluded that this mutation abolishes the endosomal targeting of Hrs (Urbé et al., 2000), whereas the other one found no effect (Hayakawa and Kitamura, 2000). When we expressed HrsC215S and analysed the cells by electron microscopy, we noticed that this protein, when expressed at high level, accumulates in proteinaceous aggregates devoid of membranes (Fig. 5B), which were never observed with wild-type Hrs (Fig. 5A). These aggregates were often observed close to endocytic profiles (see arrow in Fig. 5B), and since they resemble endosomes when examined by fluorescence microscopy, they may complicate the interpretation of the intracellular localisation of HrsC215S. The C215S mutation affects zinc binding of the FYVE domain, and the corresponding mutation in EEA1 has been shown to cause a distortion of the FYVE structure (Stenmark et al., 1996; Gaullier et al., 2000). We therefore sought to introduce a mutation in the PtdIns(3)P binding basic pocket of Hrs, R183A, which is predicted to abolish PtdIns(3)P binding without causing any structural rearrangements (Gaullier et al., 2000; Misra and Hurley, 1999). Indeed, when the wild-type and mutant FYVE domains were analysed by circular dichroism (CD) spectroscopy, the spectrum of the R183A mutant was similar (albeit not identical) to that of the wild-type protein, whereas the spectrum of the C215S mutant indicated a much less ordered structure (Fig. 6A). The structural distortion caused by the C215S mutation presumably explains the propensity of HrsC215S to aggregate when expressed in cells. To study if the R183A mutation causes a reduced affinity for PtdIns(3)P as predicted, we analysed the wild-type and R183A mutant FYVE domains by surface plasmon resonance (Fig. 6B). While we could detect the binding of the GST-tagged wild-type Hrs FYVE domain to PtdIns(3)P by surface plasmon resonance even at low concentrations, we were unable to detect any binding of GST-FYVER183A even at high protein concentrations. Thus, the R183A mutation causes a >100-fold loss of affinity for PtdIns(3)P.
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DISCUSSION |
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According to the crystal structure of the N terminus of Hrs, the VHS domain is likely to interact with the membrane surface (Mao et al., 2000). However, since deletion of the VHS domain did not interfere with the ability of Hrs to localise to early endosomes, the putative membrane interaction of the VHS domain may have a regulatory function rather than a role in the subcellular targeting of Hrs. In contrast, since the endosomal localisation of Hrs requires PI 3-kinase activity (Komada and Soriano, 1999; Urbé et al., 2000) as well as a functional FYVE domain, the interaction of the FYVE domain with PtdIns(3)P appears to be crucial for the intracellular targeting of Hrs. In addition, the CC2 domain is required, and the most straightforward interpretation of our results is that the binding of the CC2 domain to an endosomal membrane molecule contributes to the targeting of Hrs to endosomes, in cooperation with the FYVE-PtdIns(3)P interaction. On the other hand, at present we cannot rule out the possibility that one of these domains confers an allosteric regulation to the other, rather than binding directly to the endosome membrane.
What is the identity of the CC2-interacting molecule that contributes to the endosomal targeting of Hrs? From our previous studies of EEA1, we had expected to find an interaction between Hrs and Rab5, but we were unable to detect any interaction with Rab5 and other endosomal Rab GTPases, suggesting that the mechanism of targeting of Hrs to early endosomes is principally different to that of EEA1. It is interesting to note that the CC2 domain of Hrs binds to the the SNARE molecule SNAP-25, which regulates membrane fusion (Kwong et al., 2000). SNAP-25 is present mainly at the plasma membrane, but related molecules have been found on endosomes (Chen and Whiteheart, 1999). Thus, SNAP-25-related SNARE molecules may be regarded as possible candidates for targeting Hrs to early endosomes.
Conflicting results have been obtained regarding the importance of the FYVE domain for the subcellular targeting of Hrs (Hayakawa and Kitamura, 2000; Urbé et al., 2000). In order to clarify this issue we used two early endosome markers (EEA1 and endocytosed transferrin) and tested four different cell lines. Furthermore, we performed a quantitative analysis of confocal micrographs and employed a novel FYVE construct with a mutation in the PtdIns(3)P-binding pocket. All our results indicate that the FYVE domain is essential for the targeting of EEA1 to early endosomes. This is perhaps best illustrated by the quantitation in Fig. 10, which shows that a minimal construct consisting of the FYVE and CC2 domain is targeted to early endosomes, whereas the individual FYVE and CC2 domains are not.
Even though the PtdIns(3)P-binding-defective HrsR183A mutant was mainly cytosolic, a fraction of this protein did localise to early endosomes. If PtdIns(3)P-binding by the FYVE domain is involved in the subcellular targeting of Hrs, how can this result be explained? One possibility is that overexpressed Hrs constructs may dimerise with endogenous Hrs on the membrane. This interpretation is supported by the fact that the N terminus of Hrs crystallises as a dimer (Mao et al., 2000), and that coexpression of wild-type Hrs increases the amount of HrsR183A found on endosome membranes (C. Raiborg and H. Stenmark, unpublished results).
The ability of overexpressed Hrs to cause the clustering of endosomal structures is intriguing and may give a clue about the function of Hrs. We see two possible explanations for this clustering effect. First, Hrs overexpression may cause an increased docking between endosomes, without increasing their fusion. Second, it may affect the motility of endosomes. Further work is needed in order to distinguish between these possibilities, but it is interesting to note that even the construct consisting only of the FYVE and CC2 domains, HrsFYVE+CC2, caused the endosomal clustering effect. One of these domains is therefore likely to interact with the endosomal docking or motility apparatus.
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ACKNOWLEDGMENTS |
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