1 Institut Curie, CNRS UMR144, 26 rue d'Ulm, 75248, Paris, Cedex 05, France
2 E363 INSERM, Faculté de Médecine, Necker, 156 Rue de Vaugirard, 75015, Paris, France
3 Laboratoire des milieux désordonnés et hétérogènes, Universite Pierre et Marie Curie, Boite 86, 4 Place Jussieu, 75252 Paris Cedex 05, France
4 Department of Pathology and Laboratory Medicine, University of Pennsylvania, 3451 Walnut Street, Philadelphia, PA 19104, USA
Author for correspondence (e-mail: coudrier{at}curie.fr)
Accepted 2 August 2005
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Summary |
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Key words: Actin cytoskeleton, Biogenesis of melanosomes, Membrane traffic, Endocytosis
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Introduction |
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The acto-myosin generated forces could help to cluster the cargos prior to their transport. For example, the class VI myosin, which binds adaptors proteins containing PDZ domains, such as Dab2 or GIPC, has been proposed to cluster receptors with PDZ-binding motifs in clathrin-coated regions (Hasson, 2003). Acto-myosin tension might also drive morphological changes that could contribute to the formation of micro-domains involved in the sorting process. In this context, in Dictyostelium discoidum the knockout of two genes encoding class I myosins, myoA and myoB, leads to a disordered morphology of sorting recycling endosomes (Neuhaus and Soldati, 2000
).
We have previously reported that another myosin from class I, myosin Ib (Myo1b, also named myosin I alpha or Myr1), participates in the cytoplasmic distribution of endosomes and lysosomes and in the delivery of internalised molecules to lysosomes in mammalian cells (Cordonnier et al., 2001; Raposo et al., 1999
). Despite the involvement of this myosin in the traffic along the endocytic pathway, Myo1b is unable to move endocytic compartments on actin filaments in vivo (Cordonnier et al., 2001
). However, Myo1b, together with actin, might drive morphological changes of the endocytic compartments, similarly to myoA and myoB in Dictyostelium discoidum. It might also act with actin as a dynamic scaffold and be involved in the sorting of specific cargos.
To investigate further the mechanism by which Myo1b contributes to the traffic of internalized molecules towards the lysosomes, we used the pigmented human melanoma cell line MNT-1, which displays a well defined endocytic system. In these cells, endosomes that were traced with gold-labelled bovine serum albumin 15 minutes post uptake and displayed bilayered clathrin coats and internal vesicles, are intermediates for proteins transported to lysosomes and for proteins involved in the biogenesis of melanosomes (Raposo and Marks, 2002; Raposo et al., 2001
). These endosomes, which had a defined structure and were involved in targeting of proteins to two different organelles, present the hallmark of sorting endosomes (Raiborg et al., 2003
; Raposo and Marks, 2002
; Raposo et al., 2001
; Sachse et al., 2002
). We will refer to these compartments as early sorting MVEs in the present study. We report here that overexpression of Myo1b or the inhibition of actin dynamic by drugs affect the morphology of these early sorting MVEs.
One peculiarity of the MNT-1 cellular model is the expression of Pmel17, a melanosome-specific protein (and the product of murine silver locus, also named GP100) that transits in these early sorting MVEs. After its biosynthesis Pmel17 is delivered to early sorting MVEs where, concomitantly or after its delivery, it is cleaved by a furin-like proprotein convertase (Berson et al., 2001; Berson et al., 2003
). This cleavage leads to the formation of intra-lumenal fibrils that characterizes stage-II premelanosomes. Using this unique feature of the MNT-1 model, we investigated the contribution of Myo1b in the trafficking and the processing of Pmel17 in the early sorting MVEs. We report here that, the overexpression of Myo1b delays the processing of Pmel17 and thereby impairs the formation of the intra-lumenal fibrils in stage-II premelanosomes, and that Myo1b associated with endosomes co-immunoprecipitates with Pmel17. All together, these observations indicate that Myo1b and actin are involved in the morphological organisation of the early sorting MVE and in the transfer of cargo proteins such as Pmel17 towards internal vesicles of these endosomes.
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Materials and Methods |
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Cell culture and isolation of stable transformants
Cells of the human melanoma MNT-1 cell line were grown in MNT-1 culture medium, consisting of Dulbecco's modifies Eagle's medium (DMEM, Gibco-BRL Life Technology, Paisley, UK) supplemented with 10% AIMV medium (Gibco-BRL Life Technology), 20% foetal calf serum (Gibco-BRL Life Technology), penicillin (10 units/ml) and streptomycin (10 mg/ml) (Gibco-BRL Life Technology). Transfection with the recombinant DNA constructs encoding EGFP, EGFP-Myo1b and EGFP-Myo1b-Tail (Raposo et al., 1999), and isolation of stable transformants were performed as described by Durrbach et al. (Durrbach et al., 2000
). Three cellular clones producing EGFP (EGFP cells), three cellular clones producing EGFP-Myo1b (EGFP-Myo1b cells), and four cellular clones producing EGFP-Myo1b-Tail (EGFP-Myo1b-Tail cells) were selected and permanently grown in the MNT-1 culture medium in which penicillin-streptomycin was replaced by 0.7 mg/ml Geneticin (Gibco-BRL Life Technology). These cellular clones were treated overnight with 10 mM sodium butyrate (Sigma-Aldrich, France) before experiments.
HeLa cells were maintained in DMEM supplemented with antibiotics. Stable transfectants were generated with pCI-Pmel17 (Berson et al., 2001; Berson et al., 2003
) after transfection using FuGene6 (Roche Biochemicals, Indianapolis, IN) as previously described (Marks et al., 1995
).
Cell homogenate and membrane fractionation Membrane fractionation
Wild-type-expressing, EGFP-expressing and EGFP-Myo1b-expressing cells were grown 4 days in a 150 cm2 flask, washed with 5 ml of homogenisation buffer (HB) containing 50 mM imidazole pH 7.4, 0.25 M sucrose, 1 mM EDTA, 0.5 mM EGTA, 5 mM MgSO4, 1 mM DTT, 150 µg/ml casein from bovine milk and protease inhibitors (Sigma Aldrich, France). They were then scraped off into 5 ml HB and homogenized by passing them twice through a cell cracker. Post-nuclear supernatant (PNS) collected after a 7-minute centrifugation at 600 g was then centrifuged for 5 minutes at 2500 g to separate the membrane-rich fraction and the cytosol (S1) from melanosomes. A membrane-rich fraction (P2) was collected after centrifugation of the supernatant (S1) for 20 minutes at 100,000 g.
Magnetic endosomes
To isolate magnetic endosomes, MNT-1 cells that had been grown 4 days in a 300 cm2 flask, were incubated with 1 mM of Fe nanoparticles (CoFe2O4) coated with negatively charged citrate ligand for 1 hour at 4°C. They were then chased for 30 minutes at 37°C as described by Wilhelm et al. (Wilhelm et al., 2002). Cells were homogenized as described above, and PNS was loaded onto a magnetic column (5 ml/high magnetic field gradient). The non-magnetic fraction (NMF) was eluted first. The magnetic fraction (MF) was collected in the absence of magnet, and washed and magnetized twice more in 5 ml HB before its final collection in 200 µl HB.
Metabolic labelling, immunoprecipitation, and immunoblotting Metabolic labelling
Cells were harvested, starved of methionine and cysteine, pulse-labelled for 10 minutes with [35S]methionine-cysteine labelling mix (200 µCi), chased with excess methionine and cysteine for indicated periods of time and washed with PBS. After chase incubation, culture medium was collected and clarified by centrifugation.
Immunoprecipitation
Cells that had been grown for 4 days and [35S]methionine-cysteine metabolically labelled or not, were scraped off and lysed on ice in 50 mM Tris pH 7.5, containing 300 mM NaCl, 1 mM EDTA (lysis buffer) and 1% Triton X-100. The cell lysate was cleared by centrifugation for 20 min at 20,000 g, and by absorption with 25 µl of protein G sepharose beads (Amersham Bioscience Europe GmbH). The immuno-complex formed overnight (by incubating the pre-cleared cell lysate with 5 µg of purified antibody at 4°C) was absorbed by 25 µl protein G sepharose, washed 3x with lysis buffer containing 1% Triton X-100, 3xwith lysis buffer containing 0.1% Triton X-100, analysed by SDS-PAGE and immunoblotting.
Immunoblotting
Proteins separated by SDS-PAGE were transferred to nitrocellulose membranes in the presence of 20 mM Tris, 150 mM glycine and 0.05% SDS. Detection of antibodies was performed using the chemiluminescence blotting substrate from Roche Applied Sciences (Meylan, France) or Super Signal West Pico Chemiluminescent substrate from Pierce Biotechnology (Rockford, IL, USA).
Immunofluorescence microscopy
Cells were grown 4 days on cover-slips, fixed in PBS containing 3% paraformaldehyde and 0.025% glutaraldehyde, and permeabilized for 10 minutes in PBS containing 0.05% saponin. Cells were then incubated 30 minutes with primary antibodies, followed by a 30-minute incubation with FITC-conjugated or Cy3-conjugated antibodies (Rockland immunochemicals for research, Gilbertsville, PA 19525). Phalloidin (0.5 µg/ml) conjugated with TRITC (Sigma-Aldrich) was used to label F-actin. The cells were viewed with a confocal laser-scanning microscope (Leica, Vienna, Austria).
Electron microscopy
For conventional electronic microscopy, EGFP-Myo1b cells grown 4 days on cover slips were fixed with 2.5% glutaraldehyde in 0.066M cacodylate buffer for 3 hours at room temperature. After several washes with cacodylate buffer, cells were post-fixed with 2% OsO4 for 45 minutes, dehydrated with increasing concentrations of ethanol and embedded in EponTM. Ultra-thin sections were counterstained with 4% uranyl acetate in methanol for 5 minutes.
Ultra-cryomicrotomy and immunogold labelling was performed as previously described (Raposo et al., 1997). Cells were fixed with a mixture of 2% PFA and 0.125% glutaraldehyde for 3 hours at room temperature. Cells were pelleted, washed with 50 mM PBS-glycine and embedded in 10% gelatine at 37°C. After solidification on ice, small 1-mm3 blocks were prepared, infused in 2.3 M sucrose and frozen in liquid nitrogen. Ultra-thin cryosections were retrieved with a mixture of 2% methyl-cellulose2.3 M sucrose (vol/vol), thawed and single or double immunogold labelling was performed. Antibodies were detected directly with protein-A gold-conjugates (purchased from Department of Cell Biology, Institute for Biomembranes, Utrecht, Netherlands). Sections were cut with UCT and FCS Leica microtomes, and viewed and photographed with a TEM Philips CM 120 (FEI company, Eindoven, The Netherlands). Negatives were scanned with a Rotatif scanner (Highscan-Eurocore).
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Results |
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EGFP-Myo1b accumulated at the plasma membrane of MNT-1 cells in dynamic membrane areas that were enriched with actin cytoskeleton, such as membrane protrusions (Fig. 2B-D see arrow). EGFP-Myo1b was rarely associated with punctate structures in the cytoplasm and co-distribution with endosomes was difficult to determine at light-microscopic level. However, EGFP-Myo1b was detected at EM-level close to EEA1-labelled endosomes and in the magnetic fraction enriched in endosomes (Figs 3C and 9A). Furthermore, expression of EGFP-Myo1b in MNT-1 cells appeared to modify the distribution of the endocytic compartments labelled with anti-EEA1 antibodies. Endosomes accumulated in the perinuclear region in 73% of the cells expressing EGFP-Myo1b and only in 11% of the EGFP cells (Fig. 2, compare F and G with I and J, respectively).
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Expression of EGFP-Myo1b modifies the morphology of early sorting MVEs
We next analysed how expression of EGFP-Myo1b affected the endosomes of MNT-1 cells at the ultra-structural level. MNT-1 cells exhibit the different compartments of the endocytic pathway that consist of small tubulovesicular endosomes accessible for internalised molecules within 5 to 10 minutes, early sorting MVEs accessible for internalised molecules within 20 minutes, MVEs and also melanosomes at different stages of maturation (Raposo et al., 2001). As determined by conventional electron microscopy, the expression of EGFP-Myo1b in MNT-1 cells, resulted often in the accumulation in the perinuclear region of early sorting MVEs, with a characteristic electron-dense coat at their cytosolic side (Fig. 3A). Consistent with previous reports, immunogold-labelling on ultra-thin cryosections of EGFP-Myo1b cells revealed the presence of EEA1 in the non-coated regions of these early sorting MVEs (Fig. 3B, see arrows). Interestingly, small membrane extensions and vesicles in their vicinity appeared to be more abundant on endosomes from EGFP-Myo1b-expressing cells compared with those of wild-type cells (Fig. 3B versus 3E). EEA1-positive compartments that are redistributed upon EGFP-Myo1b overexpression (Fig. 2F) might correspond to clusters of early sorting MVEs, which display increased amounts of small protrusions (Fig. 3A,B).
We also analysed the distribution of EGFP-Myo1b at the EM level. Low amounts were detected under the above described experimental conditions. However, EGFP-Myo1b appeared restricted to small vesicle-like structures in the vicinity of the early sorting MVEs on ultra-thin cryosections (Fig. 3C). Some of these structures may correspond to cross sections of the small protrusions. To better evaluate the distribution of EGFP-Myo1b, we used whole-mount immunogold labelling on cells in which endosomes had been crosslinked with internalised HRP (Stoorvogel et al., 1996). This method has been used previously to detect Myo1b on endosomes of BWTG3 cells (Raposo et al., 1999
). EGFP-Myo1b was detected on the vacuolar domain but appeared enriched on the small tubular membrane extension of the early sorting MVEs (Fig. 3D).
If Myo1b is driving morphological changes of these endocytic compartments then it might do this together with actin. Interestingly, EEA1-labelled endosomes that were observed at the light microscopic level co-distributed partially with actin filaments (Fig. 4A-C see arrows) and a consistent pool of actin was found in the close vicinity of these endosomes when they were observed at the electron microscopic level (Fig. 3E see long arrows). Particularly, actin was detected at the opposite side of the clathrin coated areas, where the small membrane vesicles and small tubular extensions were observed (Fig. 3E see small arrows). Furthermore, the treatment of MNT-1 cells with cytochalasin D induced the re-distribution of EEA1-labelled endosomes in the perinuclear region in close vicinity of actin patches (Fig. 4D,E,F see arrows) and facilitated the visualisation of the tubular extensions at the electron microscopic level (Fig. 3F see arrows).
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Expression of EGFP-Myo1b perturbs the cellular distribution and the maturation of Pmel17
The delocalization and the morphological changes of the early sorting MVEs induced by the expression of EGFP-Myo1b may consequently perturb the transport of proteins that are passing through these endocytic compartments. Therefore, we analysed the cellular distribution and the maturation of Pmel17, which is processed in early sorting MVEs in their transition to stage-II melanosomes (Berson et al., 2001; Berson et al., 2003
; Raposo et al., 2001
). Intracellular compartments labelled with HMB50, an antibody that detects Pmel17, were dispersed from the cell periphery to the perinuclear region in all EGFP cells analysed, while they were mostly concentrated in the perinuclear region in 56% of EGFP-Myo1b cells (Fig. 5A versus 5B). However, consistent with a previous report (Raposo et al., 2001
), the distribution of Pmel17 and specific markers of endosomes did not overlap when cells were examined through a light microscope. In that report, the majority of Pmel17 in MNT-1 cells was associated with premelanosomes and mature melanosomes (Raposo et al., 2001
). Thus, the Pmel17-positive compartments, which were concentrated in the perinuclear region of EGFP-Myo1b cells, might correspond to premelanosomes and melanosomes. By contrast, it has been reported that the majority of Pmel17 is associated with a subset of late endosomes in HeLa cells transiently expressing Pmel17 (Berson et al., 2001
). We isolated a cellular clone, derived from HeLa cells and expressing Pmel17. After 20 minutes of internalisation, Pmel17 co-distributed with EGF (data not shown). Interestingly, the transient expression of EGFP-Myo1b in HeLa cells expressing Pmel17 induced the perinuclear re-distribution of Pmel17 (Fig. 5 compare C with D). Overexpression of EGFP-Myo1b alters, therefore, the distribution of Pmel17 associated with endosomes in HeLa cells, and with mature and immature melanosomes in MNT-1 cells. We hypothesised that the disrupted localisation of Pmel17 associated with mature and immature melanosomes in MNT-1 cells is a consequence of the altered trafficking of Pmel17 in endosomes.
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To test this hypothesis, we followed the processing of Pmel17 in MNT-1 cells. Pmel17 is an integral membrane protein. It is synthesised in the endoplasmic reticulum as precursor P1, O-linked-glycan- and N-linked-glycan-modified in the Golgi complex to result in the precursor P2, and cleaved in a post-Golgi pre-lysosome compartment into two disulfide-linked subunits. These subunits include a C-terminal 28 kDa Mß subunit that contains part of the lumenal domain, the entire transmembrane and cytoplasmic domains and an 80 kDa M subunit with the remaining N-terminal region of the lumenal domain (Fig. 6A) (Berson et al., 2001
). We compared the processed forms of Pmel17 in EGFP cells with that in EGFP-Myo1b cells. P1 and P2 precursors and the two disulfide-linked subunits M
and Mß were immunoprecipitated together with a monoclonal antibody directed against the N-terminus of Pmel17, HMB50. The amounts of M
and Mß immunoprecipitated from EGFP-Myo1b cells in these experimental conditions appeared to be lower than the amount immunoprecipitated from EGFP-expressing cells (Fig. 6B). Conversely, the amount of the precursor P2 was slightly increased in EGFP-Myo1b cells compared with that of EGFP cells (Fig. 6B). To confirm this first observation, which suggests that Myo1b can control the transport of Pmel17 leading to its processing, we compared the posttranslational processing of Pmel17 in EGFP-Myo1b cells with that in EGFP-expressing cells. In agreement with the previous report by Berson and colleagues (Berson et al., 2001
), the endoplasmic reticulum precursor P1 immunoprecipitated 10 minutes after pulse-labelling, whereas P2 immunoprecipitated as a broad protein pattern that overlapped with the P1 precursor after 10 minutes of pulse-labelling and 30 minutes of chase (Fig. 6C). Furthermore, M
, which predominantly immunoprecipitated in a diffuse protein pattern co-migrating with an alternatively spliced form of Pmel17 and Mß, was detected after 1 hour of chase (Fig. 6C). Consistent with the observations at the steady state, Mß levels were significantly lower and M
was hardly detectable in EGFP-Myo1b cells, while levels of the precursor P2 appeared to slightly increase after 1 and 2 hours of chase in the EGFP-Myo1b-expressing cells (Fig. 6C and D).
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We next examined whether the delayed cleavage of Pmel17 perturbed the formation of the intralumenal fibrils of stage-II premelanosomes in EGFP-Myo1b cells as previously reported, when cleavage of Pme17 was inhibited by expressing a variant of 1-antitrypsin inhibitor (Berson et al., 2003
). Indeed, abortive fibrils were frequently observed in the immature melanosomes of the EGFP-Myo1b cells (Fig. 7). All together these observations suggest that MYO1B controls the processing of Pmel17, leading to the formation of the intralumenal fibrils of stage-II premelanosomes.
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If the direct or indirect interaction of MYO1B with Pmel17 is involved in the transport of Pmel17, this interaction should take place on endosomes, where sorting and maturation of Pmel17 occurs. Taking advantage of a new method to isolate magnetic endosomes we determined whether MYO1B and Pmel17 could co-immunoprecipitate from a fraction enriched in endocytic compartments. Magnetic endosomes were isolated after internalising super paramagnetic nano-particles for 30 minutes (Wilhelm et al., 2002). This magnetic fraction was enriched in specific markers for early endosomes, such as Rab5, and exhibited low amounts of specific markers of late endosomes, such as Rab7, confirming the efficiency of this purification method (Fig. 9A). Endogenous MYO1B was enriched in the magnetic fractions isolated from wild-type and EGFP-Myo1b cells and EGFP-Myo1b was associated to some extend with the fraction isolated from EGFP-Myo1b cells (Fig. 9A). Precursor and mature forms of Pmel17 were also detected in the two magnetic fractions isolated from wild-type or EGFP-Myo1b cells (Fig. 9A). From the magnetic endosomes isolated from EGFP-Myo1b cells, anti-GFP antibody immunoprecipitated two proteins: one with the apparent molecular mass similar to the Pmel17 precursor P2 detected in the post nuclear supernatant (Fig. 9B compare lanes PNS and IP GFP) and one with higher molecular mass than the P2 precursor. This second protein is probably a degradation product because its mobility on the gel was slower than that of the P1 precursor when using the same antibodies for detection in post nuclear supernatant fraction (see lane PNS versus lane IP GFP Fig. 9B). Together, these observations indicate that, MYO1B and the Pmel17 precursor P2 are associated with the endocytic compartments (which are enriched in Rab5 protein), and bind directly or indirectly to each other. This interaction might occur through the C-terminal tail-domain of MYO1B.
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Discussion |
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Myo1b and the morphology of early sorting MVEs
It is generally accepted that the cellular distribution of the endocytic compartments depends upon the balance between anterograde and retrograde microtubules-motors that bind to these compartments. However, we have previously reported that Myo1b contributes to the distribution of lysosomes and transferrin receptor labelled endosomes (Cordonnier et al., 2001; Raposo et al., 1999
). Furthermore, we have also reported that Myo1b and actin filaments cooperate with microtubules for the movement of lysosomes (Cordonnier et al., 2001
). Consistent with these observations we show here that Myo1b and actin filaments control the distribution of EEA1 labelled endosomes. Myo1b and the actin networks might act as part of the regulatory machinery that transiently retains the endocytic compartments on actin filaments during their travelling on microtubules.
EEA1 is associated with early endosomes and early sorting MVEs with a bilayered clathrin coat. Early sorting MVEs are important sorting stations for the proteins that are delivered to lysosomes or involved in the biogenesis of lysosomes related organelles such as melanosomes (Raposo and Marks, 2002; Raposo et al., 2001
; Sachse et al., 2002
). We report here that, overexpression of EGFP-Myo1b or treatment with drugs that perturbe the dynamics of the actin cytoskeleton highlighted the presence of numerous small membrane extensions at the surface of these endosomes. We therefore suggest that MYO1B and actin contribute to the morphology of these endocytic compartments. Several lines of evidence suggest that MYO1B binds to the membrane of the endosomes. Using electron microscopy, we have previously reported that Myo1b was associated with endosomes and lysosomes from an hepatoma cell line and report here that EGFP-Myo1b is found on EEA1 labelled early sorting MVEs. Myo1b was also detected in fractions enriched with endosomes isolated from the hepatoma cell line (Raposo et al., 1999
) and from MNT-1 cells (this study). Furthermore, we have previously shown that Myo1b was released from membrane fractions in the presence of the tail-domain of this molecular motor, suggesting that the interaction of Myo1b with membranes is mediated by this domain. In parallel, recent observations suggest that in solution, Myo1b regulates the organisation of actin filaments (Stafford et al., 2005
). At low concentrations of Myo1b actin filaments are straighter and at high concentrations they form bundles. Modification of the cellular organisation of the actin network was never observed in the presence of EGFP-Myo1b at light microscopic level. However, we cannot exclude that MYO1B regulates the organisation of actin filaments at the surface of the early sorting MVE. In interacting with the membrane of endosomes on one hand and actin filaments on the other, MYO1B might transiently retain endosomes on actin filaments and exert tension on the endosomal membrane to form the small membrane extensions observed at the EM level.
Myo1b and the traffic of proteins that pass through early sorting MVEs
By controlling the morphology of the sorting MVE, MYO1B might participate in the trafficking of those proteins that exist in these endocytic compartments. The peculiar processing of Pmel17 allowed us to study the impact of the expression of EGFP-Myo1b in the traffic of proteins that are passing through these endocytic compartments. We found that, the expression of EGFP-Myo1b impaired the cellular distribution of Pmel17, delayed its proteolytic cleavage and, thereby, the maturation of melanosomes (Berson et al., 2001; Berson et al., 2003
; Sachse et al., 2002
). Furthermore, MYO1B associated to endosomes that co-immunoprecipitated with the precursor P2 of Pmel17. Consistent with the previous observations, which suggest that the interaction of Myo1b with membranes is mediated by its tail, this domain co-imunoprecipitates also with Pmel17. MYO1B might bind directly to the cytoplasmic domain of Pmel17 through its tail-domain or, these two proteins might belong to the same protein complex at the surface of the early sorting MVE. The interaction with the precursor P2 of Pmel17 might allow MYO1B to transiently retain P2 on the external membrane of the early sorting MVE and thereby prevent it from proteolytic cleavage. The interaction with Pmel17 on one hand and with actin filaments on the other might allow MYO1B to act as a dynamic scaffold that clusters proteins to be transported towards lysosomes through internal vesicles.
Altogether, the experimental evidence reported here strongly suggests that, MYO1B and actin filaments at the surface of MVEs act as a mechanical device that contribute to the morphology of these endocytic compartments and to the sorting of proteins that are transported to the internal vesicles of MVEs.
The machinery involved in sorting of protein cargos towards internal vesicles of MVEs has been recently unravelled in the case of ubiquitinated proteins. Cargo proteins are recognized at the cytosolic side of endosomes by a protein complex, called ESCRT-0, which contains HRS (mammalian hepatocyte receptor tyrosine kinase substrate) STAM (signal transduction adaptor molecule) and Eps15 (Raiborg et al., 2003). In this model HRS recruits Tsg101, a component of the ESCRT-I complex that in turn recruits the protein complexes ESCRT-II and ESCRT-III. These protein-complexes act together to sequester ubiquitinated cargo proteins into the inward-budding vesicles of the MVEs (Bache et al., 2003
; Lu et al., 2003
). Some non-ubiquitinated proteins, however, are also delivered to the internal vesicles of MVEs (Reggiori and Pelham, 2001
). A recent report indicates that HRS, but not Tsg101, is required for the transport of one of these proteins towards lysosomes (Hislop et al., 2004
). Pmel17 might belong to this second class of proteins because we have not been able to detect an ubiquitin motif on this protein (data not shown). The acto-Myo1b-based mechanism might represent a part of the alternative machinery to sort non-ubiquitinated proteins in specific membrane domains at the cytosolic side of endosomes. Alternatively or additionally the retention of the proteins to be sorted in specific membrane domains at the surface of endosomes by an acto-Myo1b-based mechanism, might constitute an additional step in the trafficking events occurring in sorting MVEs.
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Acknowledgments |
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Footnotes |
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References |
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