Article |
Address correspondence to Michael S. Marks, Dept. of Pathology and Laboratory Medicine, School of Medicine, University of Pennsylvania, 230 John Morgan Bldg./6082, Philadelphia, PA 19104-6082. Tel.: (215) 898-3204. Fax: (215) 573-4345. E-mail: marksm{at}mail.med.upenn.edu
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Abstract |
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Key Words: furin; endosome; proteolysis; fibril; lysosome-related organelle
* Abbreviations used in this paper:
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Introduction |
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Melanosomes serve as an excellent model to define biogenetic mechanisms among lysosome-related organelles. Melanosomes are specialized organelles within melanocytes and retinal pigment epithelial cells in which melanin pigments are generated and stored (Marks and Seabra, 2001; Raposo and Marks, 2002). As they are synthesized within maturing (stage III and IV) melanosomes, melanins are deposited along characteristic intralumenal fibers that appear before pigment deposition in stage II premelanosomes. Like the dense cores of secretory granules, the fibers serve to sequester and concentrate cargoin this case melanins; they also likely function to detoxify oxidative melanin intermediates (Seiji et al., 1963; King et al., 1995). The fibers are thought to be proteinaceous because they lack the ultrastructural hallmarks of a lipid bilayer (Moyer, 1966; Maul, 1969; Hearing et al., 1973). Thus, they are similar to intralumenal fibrils that form in certain pathologies associated with amyloid deposition (Badman et al., 1996; Kim et al., 1999; Chen et al., 2001). The formation of fibrils represents a key step in melanosome biogenesis, but their composition and the molecular mechanisms underlying their formation are unknown.
Pmel17 (also known as gp100, ME20, and Silver) is a type I integral membrane protein, uniquely expressed by melanocytes and retinal pigment epithelial cells, that localizes to the premelanosome matrix (Kwon et al., 1987; Vennegoor et al., 1988; Adema et al., 1994; Kobayashi et al., 1994; Zhou et al., 1994; Lee et al., 1996; Raposo et al., 2001). A role for Pmel17 in melanosome function is supported by the pigmentation defect in silver mice (Dunn and Thigpen, 1930), in which the Pmel17 protein is truncated (Martínez-Esparza et al., 1999). Pmel17 immunolocalizes to the fibrils within stage II premelanosomes (Berson et al., 2001; Raposo et al., 2001), and Pmel17 expression is sufficient to induce the formation of premelanosome-like fibrils within multivesicular bodies (MVBs)* of nonpigment cells (Berson et al., 2001). Nevertheless, Pmel17 in nondenaturing detergent extracts of melanocytes or transfected HeLa cells remains covalently tethered to its transmembrane domain, even after the precursor polypeptide is cleaved into two fragments (Berson et al., 2001). This implies a continued association with membranes, belying the inferred nonmembranous nature of the striations. To probe the mechanism by which Pmel17 initiates fibril formation, we asked (1) what is the function of the intralumenal cleavage; and (2) what is the biochemical nature of Pmel17 on the intralumenal striations? Our results highlight a role for proprotein convertases (PCs) in organelle biogenesis, and reveal a striking parallel between the formation of the morphological hallmark of premelanosomes and the pathological fibril formation in amyloid disease.
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Results |
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To determine whether PCs are required for the cleavage, Pmel17 or the cs variant were expressed in LoVo, a human adenocarcinoma cell line lacking furin (Takahashi et al., 1995) and several other PCs (Miranda et al., 1996). Cleavage in transfected LoVo or control HeLa cells was assessed by Western blotting of whole-cell lysates with
PEP13h, an antibody to the cytoplasmic domain. Lysates from HeLa cells expressing wild-type Pmel17, like melanocytic cells, contain two immunoreactive bands with Mr 28,000 and
100,000, representing Mß and the full-length, endoglycosidase H (endoH)-sensitive P1 precursor, respectively (Fig. 1 D; see Fig. 5 A). By contrast, in both HeLa cells expressing Pmel17
cs (Fig. 1 D) and LoVo cells expressing wild-type Pmel17 (Fig. 1 E), only the
100-kD P1 band and not Mß was observed, indicating that cleavage did not occur (a faint residual band that co-migrates with Mß in LoVo cells expressing Pmel17
cs may result from cleavage by NARC-1, a related protease that is active within the ER and for which a sequence similar to the consensus recognition site is generated by the KR468469
QQ mutation; Seidah et al., 2003). Mß was restored in LoVo cells by coexpression of both Pmel17 and furin, but not Pmel17
cs and furin (Fig. 1 E). Thus, furin or a similar PC can cleave Pmel17 at the dibasic recognition site. Importantly, cleavage at the only other dibasic sites in Pmel17, KR147, or RR192, would not generate fragments of the appropriate size for M
and Mß.
To determine whether a furin-like PC cleaves Pmel17 in melanocytic cells, we inhibited PCs in the pigmented human melanoma, MNT-1, by expressing 1-antitrypsin Portland (
1-PDX), a PC-specific variant of
1-antitrypsin inhibitor (
1-AT; Anderson et al., 1993; Jean et al., 1998, 2000). FLAG epitopetagged
1-PDX or
1-AT were expressed in MNT-1 cells by infection with recombinant adenoviruses. Transgene expression and Pmel17 processing were assessed by metabolic pulse/chase, immunoprecipitation, SDS-PAGE, and phosphorimaging analysis. Both transgenes were efficiently expressed, although a lower multiplicity of infection (moi) with the
1-PDX virus was required to obtain the same level of expression as
1-AT (Fig. 2 A; likely due to contaminating nonrecombinants in the
1-AT preparation). As described previously, Pmel17 in uninfected cells first appears as endoH-sensitive P1 and Pmel17-s forms (Berson et al., 2001). P1 is the full-length core glycosylated precursor, and Pmel17-s is the product of an alternatively spliced mRNA that is generated in melanocytic cells (Nichols et al., 2003), but not transfected HeLa cells (Berson et al., 2001); Pmel17-s is distinguished from M
based on its direct reactivity with
Pep13h, its appearance in the pulse-labeled samples, and its sensitivity to EndoH (see Fig. 5). P1 and Pmel17-s are subsequently modified in the Golgi to endoH-resistant P2, and P2 is then cleaved to M
and Mß (Fig. 2 B, lanes 15). All fragments disappeared from detergent cell lysates with a half-life of 2.5 h. In cells expressing
1-AT, the pattern and kinetics of Pmel17 processing were virtually unchanged (Fig. 2 B, lanes 610). However, in cells expressing
1-PDX, as in HeLa cells expressing Pmel17
cs, P2 accumulated, and generation of M
/Mß was inhibited by at least 90% (Fig. 2 B, lanes 1120; Fig. 2 C). Cleavage was equally inhibited in cells expressing two different levels of
1-PDX (Fig. 2, B and C), likely reflecting the secretion of
1-PDX by infected cells and uptake by uninfected cells. By contrast,
1-PDX did not affect the maturation and half-life of another melanosome protein, Tyrp1 (unpublished data). These data confirm that furin or a furin-like PC mediates Pmel17 cleavage in melanocytic cells.
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Association of the lumenal domain fragment with TX-insoluble complexes
The dependence of striation formation on PC cleavage of Pmel17 would support a model in which M fragments form intralumenal proteinaceous fibrils upon their release from membranes. By analogy to amyloid, such fibrils might be insoluble in nondenaturing detergents such as TX. Indeed, M
was observed in TX-insoluble fractions of MNT-1 cells. By Western blotting, an endoH-resistant, N-glycanase Fsensitive M
band was detected in whole MNT-1 cell lysates with
Pmel-N (antibody to the lumenally exposed NH2 terminus of Pmel17), but not with
Pep13h (antibody to the cytoplasmically exposed COOH terminus; Fig. 5 A), distinguishing it from the endoH-sensitive,
Pep13h-reactive alternative splice product, Pmel17-s. Although most Mß, Pmel17-s, and P1 isoforms were soluble in TX, a significant fraction of M
was detected in the TX-insoluble pool (Fig. 5 B).
Formation of TX-insoluble M was kinetically linked to both cleavage and dissociation from Mß by a metabolic pulse/chase assay in which TX-insoluble material from cell lysates was extracted by heating in 8M urea. Pmel17 isoforms from this TX-insoluble pool (after dilution of the urea) and from the soluble fraction were sequentially immunoprecipitated with
PEP13h and then
Pmel-N, then analyzed by SDS-PAGE and phosphorimaging. From the soluble pool,
Pmel-N only immunoprecipitated residual P1, P2, Pmel17-s, M
, and Mß in similar proportions as in the initial
Pep13h immunoprecipitates (Fig. 5, C and D; compare lanes 14 with lanes 58). From the TX-insoluble pool, although
Pep13h immunoprecipitated only full-length P1 and Pmel17-s (Fig. 5 C, lanes 912), subsequent
Pmel-N immunoprecipitates additionally contained an
80-kD band (lanes 1316); its failure to be depleted by
Pep13h suggests that this band is M
. The intensity of this band increased over time from undetectable at the pulse to most intense by 4 h of chase (Fig. 5, C and E). Thus, at least a portion of M
was released from Mß during the chase and accumulated in TX-insoluble complexes.
To confirm the identity of this TX-insoluble band as M and to determine whether its accumulation required PC cleavage, MNT-1 cells expressing
1-PDX were similarly analyzed by pulse/chase and immunoprecipitation. As shown in Fig. 5 F, expression of
1-PDX (but not
1-AT) inhibited the formation of TX insoluble M
. Appearance of the fragment was both delayed and reduced in efficiency by expression of
1-PDX. Instead, these cells accumulated a larger fragment of Pmel17, lacking the COOH terminus and possibly comprising the entire lumenal domain (Fig. 5 F; band marked "?"). Appearance of this band correlates with the disordered aggregates observed by IEM (Fig. 4 E), and likely results from cleavage within the juxtamembrane region by lysosomal proteases. These data show that formation of intact fibrils correlates with appearance of TX-insoluble M
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The premelanosome fibrils contain TX-insoluble M
If the fibrils consist of M fragments, they should label with antibodies to the Pmel17 lumenal domain, but not to the cytoplasmic domain. IEM analyses confirm this prediction. In MNT-1 cells,
PEP13h labels the coated endosome precursor to stage II premelanosomes, including the ILVs within them, but not the striated stage II premelanosomes themselves (Fig. 6 A; also see supplemental materials in Raposo et al., 2001, available at http://www.jcb.org/cgi/content/full/152/4/809/DC1/3). Concordantly, in transfected HeLa cells expressing high levels of Pmel17,
PEP13h labeling is observed on the ILVs of MVBs (Fig. 6 B), but not on the striated regions that form within the same multivesicular structures (Fig. 6 C). By contrast, antibodies to the Pmel17 lumenal domain label both the ILVs and the striations (Fig. 6, B and C). These data confirm that the fibrils lack the cytoplasmic domain present on full-length Pmel17 and the Mß fragment.
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Discussion |
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Role of PCs in organelle morphogenesis
PC cleavage is required to activate numerous secreted and cell surface proproteins and viral fusion proteins (Thomas, 2002), but few intracellular substrates are known. Here, we used two approaches to show that cleavage of Pmel17 by furin or a related PC is required to generate premelanosome fibrils. The formation of both fibrils and the mature Pmel17-derived fragments, M and Mß, was blocked by (1) mutagenesis of the PC recognition site of Pmel17 in an exogenous expression system, and (2) inhibition of PC activity in a melanocytic cell line. Previous reports demonstrated defects in Weibel-Palade bodylike structures in an ectopic expression system upon mutagenesis of a PC site in von Willebrand factor (Journet et al., 1993), and in lamellar body formation in lung epithelial type II cells upon treatment with a cysteine protease inhibitor (Guttentag et al., 2003). However, this is the first time that both approaches have been used to conclusively demonstrate a role for PCs or other proteases in the activation of a single substrate protein to initiate morphogenesis of a lysosome-related organelle.
The relevant PC in melanocytic cells may be furin; furin is expressed in melanocytic cells (unpublished results), is capable of cleaving Pmel17 in reconstituted LoVo cells (Fig. 1 E), and is somewhat selectively inhibited by 1-PDX (Jean et al., 1998). On the other hand, the complement of PCs expressed by pigment cells is not known, and a different PC may function physiologically because
1-PDX can inhibit other PCs (Jean et al., 1998), and the Pmel17 cleavage site differs from the consensus furin recognition site (Fig. 1 B). We have shown that cleavage occurs within an acidic, post-Golgi, prelysosomal compartment that kinetically precedes stage II premelanosomes (Berson et al., 2001), consistent with the known localization of furin and other PCs to the TGN, endosomes, and secretory granules (Molloy et al., 1999; Thomas, 2002). The residual cleavage that occurs in the presence of brefeldin A and that results in a small pool of endoH-sensitive Mß (Berson et al., 2001), like the residual cleavage of Pmel17
cs in LoVo cells (Fig. 1 E), is likely mediated by NARC-1 or a related protease (Seidah et al., 2003). Of note, the accumulation of Golgi-processed Pmel17 observed upon inhibition of PC cleavage adds to the growing evidence that premelanosomes derive from post-Golgi compartments, and not from smooth ER (Kushimoto et al., 2001).
In MNT-1 cells expressing 1-PDX, striated structures were less abundant than in control cells, and many cells displayed numerous abnormal pigment-containing structures (Fig. 4). Because melanogenic enzymes such as tyrosinase and Tyrp1 are normally delivered to preformed stage II premelanosomes (Raposo et al., 2001), these unusual pigmented structures likely represent the products of malformed premelanosomes, depleted of fibrils, to which melanogenic enzymes were still delivered. The failure to completely deplete stage II premelanosomes in
1-PDXtreated cells was likely due to the short duration of expression after adenovirus infection; "normal" melanosomes of all stages present within
1-PDXexpressing cells likely represent later stage melanosomes that were in the process of forming, and/or partial fibril formation by a pool of properly cleaved Pmel17 that was present within abundant stage I premelanosomes, before a threshold of
1-PDX expression. Tonic exposure to
1-PDX for extended periods of time would likely further reduce the level of normal melanosomal structures in these cells, and may permit a timeline for melanosome maturation to be derived.
Composition and formation of striations and parallels with fibrillary diseases
The striated fibrils of stage II and III melanosomes have been thought to consist of protein with no underlying membrane based on their periodicity of electron lucence and density and the frequent appearance of a "zigzag" shape by EM (Birbeck et al., 1956; Moyer, 1966; Maul, 1969; Hearing et al., 1973). This interpretation seemed inconsistent with the ability of the integral membrane protein, Pmel17, to induce formation of and become incorporated into the striations (Berson et al., 2001; Raposo et al., 2001). We now reconcile these observations by showing that the PC cleavage product, M, is eventually released from membranes and accumulates in TX-insoluble complexes. The insolubility of these complexes explains our previous inability to observe M
released from Mß in the TX-soluble fraction (Berson et al., 2001) and the short half-life of TX-soluble Pmel17 (Kobayashi et al., 1994; Donatien and Orlow, 1995; Berson et al., 2001), and supports earlier evidence for Pmel17-derived fragments in detergent-insoluble fractions of partially purified melanosomes (Orlow et al., 1993; Zhou et al., 1994). The identity of M
is confirmed by (1) its immunoreactivity with
Pmel-N but not
Pep13h and its insensitivity to endoH; (2) the failure to generate M
and Mß upon mutagenesis of KR469, inhibition of PCs by
1-PDX, or expression in PC-deficient LoVo cells; (3) the lack of any other potential dibasic cleavage sites in Pmel17 that could generate the appropriate sized fragments; and (4) the co-migration of intracellular M
with secreted Pmel17 (Berson et al., 2001), for which the sequence is known to end at V467 (Maresh et al., 1994). The presence of TX-insoluble, Mß-dissociated M
on isolated fibrils and the dependence of striation formation on PC cleavage of Pmel17 indicates that (1) Pmel17 is not only sufficient, but also necessary for the generation of striations; and (2) the striations consist of insoluble complexes composed at least in part of M
without Mß and underlying membrane.
How does M become incorporated into detergent-insoluble complexes? M
must first be released from the integral membrane Mß fragment, to which it is disulfide bonded on ILVs of multivesicular endosomes (Berson et al., 2001; Raposo and Marks, 2002). By analogy to the "sorting by retention" model of secretory granule formation (Arvan and Castle, 1998), the MVB may provide a structural framework to sequester Pmel17 and other molecules required for striation formation from other endosome components. Release of M
from the ILV must be mediated by either reduction of the disulfide bonds or proteolytic digestion of Mß; the latter is more likely, given the oxidative nature of melanin intermediates, the presence of lysosomal hydrolases in melanosomes (Orlow, 1995), and evidence for proteolytic maturation of Pmel17 in stage II premelanosome-enriched subcellular fractions (Chiamenti et al., 1996; Kushimoto et al., 2001). We speculate that release of M
from Mß is accompanied by a conformational change that favors fibrillogenic activity, perhaps facilitated by the low pH of forming premelanosomes (Raposo et al., 2001). Lumenal segments within Mß may serve to inhibit fibrillogenesis because longer Pmel17 fragments generated upon inhibition of PCs fail to properly form fibrils.
This model for physiological fibril formation in premelanosome biogenesis is reminiscent of the pathological process of fibril formation in a number of amyloid diseases. A specific requirement for PC cleavage and/or acidification in the formation of fibrils is observed in several pathogenic fibrillogenic substrates, including gelsolin in familial amyloidoses of Finnish type (Chen et al., 2001), BRI-L in familial British dementia (Kim et al., 1999), and pro-islet amyloid polypeptide in type II diabetes (Badman et al., 1996). Proteolytic maturation, acidification, and/or association with detergent-insoluble lipid raftsthe lipid contents of which are similar to the ILVs of MVBs (Mobius et al., 2002; Wubbolts et al., 2003)are common features in fibril formation by the amyloidogenic peptide in Alzheimer's disease (Esler and Wolfe, 2001; Ehehalt et al., 2003) and by prions (Taraboulos et al., 1995; Mayer et al., 1996; Zou and Cashman, 2002). Defining the fibrillogenic determinant within the Pmel17 lumenal domain and characterizing its properties will likely provide insights into the mechanism by which fibril formation is favored by proteolytic maturation, not only in premelanosomes, but also in these clinically important substrates.
Implications for biogenesis of lysosome-related organelles
PCs are well known to regulate the formation of dense cores in conventional secretory granules by permitting ordered aggregation and dense packing of granule contents (Arvan and Castle, 1998). Our work, together with the presumed role of PCs or other proteases in the formation of Weibel-Palade bodies or lamellar bodies, respectively, demonstrates that proteolytic activation plays a general role in lysosome-related organelle biogenesis as well. Other fibrillogenic substrates may include Langerin, a C-type lectin that is involved in the formation of Birbeck granules in Langerhans cells (Valladeau et al., 2000). Future studies will likely confirm the generality of this biogenetic function of proteases, and may reveal whether fibrillogenic activation steps are disrupted by genetic diseases of organelle biogenesis, such as Hermansky-Pudlak Syndrome (Spritz, 1999; Swank et al., 2000).
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Materials and methods |
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Antibodies
mAb specific for Tyrp1 (TA99/Mel-5; Thomson et al., 1985) and affinity-purified rabbit antibody to the Pmel17 COOH terminus (PEP13h; Berson et al., 2001) were described previously. Purchased mAbs were HMB50 and HMB45 (anti-Pmel17; Lab Vision), M2 (anti-FLAG; Sigma-Aldrich), H4A3 (anti-LAMP1; Developmental Studies Hybridoma Bank, Iowa City, IA), anti-CD63 (Immunotech), and YL 1/2 (anti-tubulin; Accurate Chemical). Rabbit antiserum
Pmel-N was generated by Genemed Synthesis, Inc. to a synthetic peptide (CTKVPRNQDWLGVSRQLR-CO2H) corresponding to the human Pmel17 NH2 terminus (residues 2440), and affinity purified on peptide coupled to SulfoLink beads (Pierce Chemical Co.).
Pmel-N had no reactivity with untransfected HeLa cells. Protein A gold conjugates were purchased from Dr. J. Slot (Utrecht Medical School, Utrecht, Netherlands).
Plasmids
pCI-Pmel17 encoding the Pmel17 long form in pCI (Promega) has been described previously (Berson et al., 2001). Pmel17cs was generated by site-directed mutagenesis using two-step amplification (Higuchi et al., 1988), replacing the endogenous sequence using unique KasI and NsiI sites, and sequence verified by automated dideoxy sequencing. HA11 epitope-tagged furin in pSX (Bosshart et al., 1994) was a gift of Dr. J.S. Bonifacino (National Institutes of Health, Bethesda, MD).
Recombinant virus production and infection
Recombinant adenoviruses encoding the tetracycline-responsive transactivator (tTA) and tetracycline-responsive, FLAG-epitope tagged 1-PDX and
1-AT were gifts of Drs. G. Thomas and L. Thomas (Vollum Institute, Oregon Health Sciences University, Portland, OR; Jean et al., 2000). Viruses were amplified and titred using the Tissue Culture Infectious Dose 50 method according to the AdEasyTM vector system application manual (Qbiogene). Crude virus preparations in PBS were used directly for double infections of MNT-1 cells, according to the AdEasyTM manual, using tTA virus at moi of 20 and
1-PDX or
1-AT virus at moi of 5 or 20. Infections were not cytotoxic for MNT-1 cells as judged by trypan blue exclusion, and resulted in transgene expression in
50% of the cells by IFM. Cells were harvested 2 d after infection for analyses.
Metabolic labeling and immunoprecipitation
Cells were metabolically pulse labeled for 1530 min with [35S]methionine/cysteine and chased for indicated times as described previously (Marks et al., 1996). Fresh or frozen cell pellets were lysed in 1% (wt/vol) TX for 30 min as described previously (Berson et al., 2000), and lysates were clarified by centrifugation for 15 min at 20,000 g. Where noted, the resulting TX-insoluble pellets were resuspended in 50 µl TX lysis buffer containing 8M urea and heated at 60°C for 10 min. After cooling, samples were diluted 20x with TX lysis buffer for immunoprecipitation. Immunoprecipitations, SDS-PAGE on 10% polyacrylamide gels, and phosphorimaging analysis were performed as described previously (Berson et al., 2000). For sequential immunoprecipitations, unbound material from the first precipitation was clarified by centrifugation at 20,000 g for 5 min before incubation with the subsequent immobilized antibody.
Immunoblotting
Western blotting using whole-cell lysates prepared with 1% SDS, and endoH/ N-glycanase F treatments were as described previously (Berson et al., 2000). For fractionation into TX-soluble and -insoluble fractions, 106 cells were resuspended in 200 µl lysis buffer containing 1% TX and protease inhibitors at 4°C for 2 h. After centrifugation at 20,000 g for 15 min, supernatants (TX soluble) were harvested and 6x SDS-PAGE sample buffer was added before analysis. Pellets (TX insoluble) were washed once with 1% TX lysis buffer and then resuspended in 1x SDS-PAGE sample buffer for analysis. 8% PAGE/5% methanol transfer buffer was used for Pmel-N and anti-tubulin blots, and 12% PAGE/15% methanol transfer buffer for
Pep13h blots. Immobilon-P membranes (Millipore) with transferred proteins were probed with indicated antibodies, and bands were detected with alkaline phosphataseconjugated goat antirabbit Ig, enhanced chemifluorescence, and phosphorimaging analysis using a fluorescence imaging system (Storm® 860; Molecular Dynamics, Inc.) and ImageQUANT software (Amersham Biosciences).
Subcellular fractionation
See Fig. 7 A for schematic. MNT-1 cells (108) were homogenized with a hand-held Dounce homogenizer in 4 volumes of buffer I (25 mM Hepes, 1 mM EDTA, 0.1 mM EGTA, and 0.02% sodium azide, pH 7.4) containing 0.25 M sucrose and a cocktail of protease inhibitors (Berson et al., 2000). Cell debris and nuclei were pelleted at 700 g for 10 min. Post-nuclear supernatants were layered over a cushion of 2.0 M sucrose in buffer I and centrifuged at 11,000 g for 10 min. The black interface at the cushion (dense membrane fraction) and the diffuse supernatant (light membrane fraction) were harvested, and each was subjected to 100,000 g for 60 min to recover a pellet (total membrane fraction). Pellets were resuspended in 600 µl 150 mM NaCl/0.1 M Tris-HCl, pH 8.0; after a sample was removed for EM analysis, TX was added to a final concentration of 1%, and samples were rotated at 4°C for 2 h. Samples were then spun at 20,000 g for 15 min, and supernatants (TX-soluble fraction) and pellets (TX insoluble fraction) were harvested. Approximately 3% of the material in each fraction was used for each lane for Western blotting analyses. An aliquot of each fraction was fixed for EM analysis as described below.
Electron microscopy
Conventional EM of MNT-1 or transfected HeLa cells was performed as described previously (Raposo et al., 2001). For IEM, cells were fixed with 2% PFA/0.2% glutaraldehyde and single- or double-immunogold labeling of ultrathin cryosections was performed as described previously (Raposo et al., 1997, 2001) using protein A conjugated to 10-nm or 15-nm gold particles (PAG-10 or -15). For transfected HeLa cells, only cells expressing comparable levels of wild-type Pmel17 and Pmel17cs were analyzed in parallel. Expression per cell was assessed as total expression level (judged by Western blotting) divided by percentage of transfected cells (judged by IFM). Relative quantitation as described in the text was performed directly under the electron microscope by counting the number of affected and unaffected relevant compartments among 50 cell profiles. For EM analysis of subcellular fractions, 20-µl drops of each fraction, previously fixed with 2% PFA in PHEM buffer, as described in Raposo et al. (2001), were placed on formvar-carboncoated EM grids for 30 min. The grids were then immunogold labeled, contrasted, and embedded in a mixture of uranyl acetate and methylcellulose as described for ultrathin cryosections (Raposo et al., 1997).
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Acknowledgments |
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This work was supported by National Institutes of Health grants R01 EY 12207 and R01 AR 48155 (to M.S. Marks) and by Association de Recherche pour le Cancer and Vaincre les Maladies Lysosomales (to G. Raposo). J.F. Berson was supported in part by training grant T32 CA 09140 from the National Cancer Institute and by American Cancer Society Fellowship PF-99-336-01-CIM.
Submitted: 12 February 2003
Revised: 18 March 2003
Accepted: 19 March 2003
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References |
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