Article |
Address correspondence to Margaret S. Robinson, University of Cambridge, CIMR, Wellcome Trust/MRC Building, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2XY, UK. Tel.: (44) 1223-330163. Fax: (44) 1223-762640. E-mail: msr12{at}mole.bio.cam.ac.uk
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Abstract |
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Key Words: coated vesicles; adaptors; clathrin; mocha; pearl
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Introduction |
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Although the AP-3 complex was identified relatively recently, it has been much more amenable to genetic analysis than the AP-1 or AP-2 complexes. When the subunit of the complex was first cloned, it was found to be the mammalian homologue of the protein encoded in Drosophila by the garnet gene, one of the classical eye color genes (Ooi et al., 1997; Simpson et al., 1997). garnet mutants have defective pigment granules, and because of similarities between pigment granules and lysosomes, this suggested a role for the AP-3 complex in the trafficking of proteins destined for lysosomes and related organelles. Subsequent studies in yeast showed that deleting any of the four AP-3 subunit genes causes missorting of a subset of proteins normally destined for the vacuole, the yeast equivalent of the lysosome (Cowles et al., 1997; Stepp et al., 1997). Two naturally occurring mouse mutants have also been identified with mutations in AP-3 subunits. The mocha (mh) mouse has a null mutation in the
subunit of the complex (Kantheti et al., 1998), whereas the pearl (pe) mouse has effectively a null mutation in the ß3A subunit (small amounts of mRNA can be detected which encode a trunctated protein, missing the last 125 amino acids, but the protein has not been detected on Western blots) (Feng et al., 1999; Zhen et al., 1999). The mouse mutants have a similar phenotype to the human genetic disorder Hermansky Pudlak syndrome (HPS), and indeed a subset of HPS patients has been identified with mutations in ß3A (Dell'Angelica et al., 1999). In both the mice and the humans, AP-3 deficiency results in hypopigmentation because of abnormalities in melanosomes, prolonged bleeding because of deficiencies in platelet dense granules, and reduced secretion of lysosomal enzymes into the urine. AP-3deficient fibroblasts appear relatively normal, but several studies have revealed that lysosomal membrane proteins in these cells show increased trafficking via the plasma membrane (Dell'Angelica et al., 1999, 2000; Le Borgne et al., 1998). The mh mouse also has neurological defects, although the pe mouse and the ß3A-deficient humans are neurologically normal. This is presumably because there is a second ß3 isoform, ß3B, which is specifically expressed in neurons and neuroendocrine cells (Kantheti et al., 1998). Together, these findings indicate that the AP-3 complex facilitates the trafficking of certain types of cargo to lysosomes and lysosome-related organelles.
Whether or not AP-3 is associated with clathrin is still somewhat controversial. Unlike AP-1 and AP-2, AP-3 is not enriched in purified clathrin-coated vesicles (Newman et al., 1995; Simpson et al., 1996). However, a clathrin-binding consensus sequence, L[L,I][D,E,N][L,F][D,E], has been identified in the hinge domains of ß1 and ß2, and similar sequences are found in ß3A and ß3B which can interact with clathrin in vitro (Dell'Angelica et al., 1998; ter Haar et al., 2000). AP-3 is also able to coassemble with clathrin in vitro and to support clathrin recruitment onto liposomes (Dell'Angelica et al., 1998; Drake et al., 2000). On the other hand, an in vitro system for the budding of synaptic-like microvesicles from endosomes has been shown to require AP-3 but not clathrin (Shi et al., 1998), indicating that the two can function independently. In addition, gene deletion studies in yeast indicate that AP-3 and clathrin function on different pathways; however, yeast ß3 does not contain a clathrin binding motif. Immunolocalization studies, at both the light and the electron microscope level, have yielded conflicting results (Simpson et al., 1996, 1997; Dell'Angelica et al., 1998). Thus, the question of whether or not there is an obligatory coupling between AP-3 and clathrin in mammalian cells has not yet been resolved.
Here we have taken advantage of the mouse mutants to address two questions. First, what happens to the other AP-3 subunits if one of them is missing? And second, to what extent can we rescue the phenotype of mocha and pearl cells by transfecting them with either a wild-type copy of the missing subunit, or alternatively with one that has been altered in some manner? In particular, are ß3A subunits that no longer have the clathrin binding motif still functional? These studies provide insights into the assembly and function not only of the AP-3 complex, but of the other AP complexes as well.
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Results |
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Fig. 2 (left) shows that under these conditions both ß3 and µ3 can be detected in the mh cells, and furthermore that they assemble into a heterodimer since both subunits come down with anti-ß3. In contrast, the 3 in these cells appears to exist as a monomer, since it does not coimmunoprecipitate with any of the other subunits. In the pe cells (Fig. 2, middle),
and
3 form heterodimers which come down with antibodies against both subunits, and in addition, a small amount of µ3 can be detected which coprecipitates with the
/
3 dimers. This suggests either that the µ3 may be assembling with the
and
3 subunits into a heterotrimer, or alternatively, since the pe mutation is not a true null, that there are small amounts of truncated ß3A expressed in these cells which can form heterotetramers. To distinguish between these two possibilities, we immunoprecipitated extracts from pe spleen under nondenaturing conditions with a ß3A-specific antibody (the reason for using spleen for this experiment is that it is possible to get a more highly concentrated extract from tissues than from cultured cells, and AP-3 is known to be expressed at particularly high levels in spleen). Fig. 2 (right) demonstrates that there are indeed small amounts of truncated ß3A made in pe cells which can coassemble with the other three subunits to form a heterotetramer, although we cannot rule out the possibility that some of the µ3 may also be assembled into a heterotrimer. Together, these studies indicate that ß3 and µ3 can interact with each other, forming heterodimers in mh cells, and that
and
3 also interact with each other, forming heterodimers in pe cells. The interactions that we can detect in the mutant cells are shown diagrammatically in the bottom part of Fig. 2.
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To determine whether the complexes could be recruited onto membranes, cells expressing each of the six constructs were labeled for immunofluorescence with anti-. In nontransfected pe cells,
has a diffuse cytoplasmic distribution (Fig. 1 d); however, in cells expressing each of the five constructs, the
labeling was punctate, indicating that it was associated with membranes (Fig. 6). We also compared the distribution of AP-3 in cells expressing the various constructs with that of clathrin, using a confocal microscope (Fig. 7; AP-3 is green and clathrin is red). In cells expressing wild-type ß3A, AP-3 and clathrin were found to have distinct distributions: (a) they are often in the same general vicinity, and in some cases there may be overlap (yellow), but much of the AP-3 labeling is negative for clathrin. In contrast, when cells are double labeled for the AP-1 adaptor complex and clathrin (b), essentially all of the structures that are positive for AP-1 (green) are also positive for clathrin (red; note that there is extensive yellow labeling and very little green). In cells expressing the various constructs, the extent of overlap between AP-3 and clathrin was very similar to that in cells expressing wild-type ß3A. This is in spite of the fact that the ß3Aß2 chimera (c) has more potential clathrin binding sites than wild-type ß3A (not only the clathrin-binding consensus sequence LLNLD in its hinge domain, but also three copies of another motif implicated in clathrin binding, DLL [Morgan et al., 2000], as well as clathrin binding activity in its ear domain [Owen et al., 2000]). Constructs lacking a clathrin binding domain, like ß3A817AAA (d), did not look appreciably different from the other constructs: again, there were some yellow structures, as well as red and green. However, at the light microscope level, it is impossible to resolve two structures that are less than
0.2 µm apart from each other, so to quantify the true extent of overlap between AP-3 and clathrin in cells expressing the various constructs, it will be necessary to go to the electron microscope level.
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For quantitative studies, each stably transfected cell line was incubated for 6 h either with the antiLAMP-1 antibody or with an isotype control antibody (anti-KLH), which also had been conjugated to Alexa Fluor 488. The cells were then trypsinized, fixed, and analyzed by flow cytometry. Fig. 8 b shows the results obtained with one of the cell lines expressing wild-type ß3A. The peak marked "control" shows the fluorescence intensity of the ß3A-expressing cells incubated with the control antibody, which is likely to be taken up by fluid phase endocytosis only. The peaks marked "ß3A" and "empty vector" show the levels of fluorescence intensity observed when the ß3A-expressing line and one of the cell lines transfected with empty vector were incubated with the antiLAMP-1 antibody. In both cases, the fluorescence intensity is higher than that observed with the control antibody, indicating that the antiLAMP-1 has been taken up by receptor-mediated endocytosis as well as fluid-phase endocytosis. However, the fluorescence intensity of the ß3A-expressing line is 4.41 times that of the control. In contrast, the fluorescence intensity of the cell line transfected with empty vector is 16.61 times that of its own control (unpublished data, but similar results were obtained with all of the cell lines incubated with the control antibody). Thus, the amount of specific uptake of antiLAMP-1 in the ß3A-expressing line is 27% of that in the empty vectortransfected line. This is comparable to results obtained by others who have investigated antiLAMP-1 uptake in normal and AP-3-deficient primary fibroblasts from both mice and humans: wild-type cells were reported to internalize between 20 and 45% as much antibody as mutant cells (Dell'Angelica et al., 1999, 2000).
Fig. 8 c shows the extent of functional rescue obtained with all six constructs. In each case, three different cell lines stably expressing each of the six constructs were analyzed, as well as three cell lines transfected with empty vector. The experiments were repeated on different days and the results for each cell line were averaged. ß3A can be seen to give the best rescue, with antiLAMP-1 uptake 4.89 ± 0.237 (SD), as compared with 17.25 ± 0.821 in cells transfected with empty vector. The ß3B construct also rescued well, with antiLAMP-1 uptake 6.58 ± 0.421. Whether the difference between the ß3A- and ß3B-expressing cells reflects functional differences between the two ß3 isoforms, or whether it reflects other differences between the various cell lines (since wild-type cell lines can vary at least this much in their anti-LAMP-1 uptake), is not at present clear. The ß3Aß2 chimera and the ß3A807831 deletion mutant both partially rescued the missorting phenotype, with antiLAMP-1 uptake 10.30 ± 2.620 and 10.48 ± 1.635 respectively. However, there was no significant difference in antibody uptake between cells expressing the truncation mutant, ß3A807stop (15.73 ± 3.512), and cells transfected with empty vector. Thus, the distal hinge and/or ear domain of the ß3 subunit appears to be required for AP-3 function, even though the construct lacking these domains is still able to assemble into AP-3 complexes and be recruited onto membranes.
There are at least two possible explanations for the intermediate level of functional rescue that we observed with the ß3A807831 mutant. One possibility is that the clathrin-binding consensus sequence, which is missing from this construct, is required for full functional rescue. Alternatively, the construct might be defective in some other way: for instance, the ear might not be correctly folded. The point mutant ß3A817AAA enables us to distinguish between these two possibilities. Instead of a 25-amino acid deletion, this construct has only 3 amino acid substitutions, but (at least in vitro) it is unable to bind clathrin (Dell'Angelica et al., 1998). Fig. 8 c shows that when this construct is expressed in living cells, antiLAMP-1 uptake is 7.49 ± 1.128, which is only slightly more than in ß3A-expressing cells and not significantly different from ß3B-expressing cells. Thus, although the hinge and/or ear domains of ß3A are needed for protein function, the clathrin-binding consensus sequence appears to be dispensable.
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Discussion |
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We first investigated whether partial AP-3 complexes were able to assemble in the absence of a particular subunit. We found that mh cells, missing the subunit, assembled heterodimers from the ß3 and µ3 subunits, whereas the
3 subunit remained monomeric. The pe cells, which express very low levels of truncated ß3A, mainly assembled heterodimers consisting of
and
3 subunits, as well as a very small amount of complete heterotetramer. All of the subunits were destabilized to some extent by the absence of either
or ß3, but this was particularly striking in the case of the µ3 subunit, which seemed to exist only as a dimer with the ß3 subunit, even when the ß3 subunit was barely detectable.
In addition to the naturally occurring AP-3 mouse mutants, knockout mice have been generated which lack the AP-1 and µ1A subunits. Both mouse strains die during embryogenesis; however, a fibroblast line has been established from the µ1A knockout mice, and these cells were found to assemble a heterotrimer consisting of
, ß1, and
1 subunits. This complex appears to be fairly stable, although (like the
/
3 heterodimer) it is unable to be recruited onto membranes (Meyer et al., 2000). The
knockout mouse dies so early that it has not been possible to establish a
null cell line, but cells from the heterozygote have been examined and found to express the
subunit at
50% normal levels. Both ß1 and µ1 completely comigrate with
by gel filtration in these cells, indicating that any excess ß1 and µ1 are unstable and get degraded (Zizioli et al., 1999). Similarly, our own studies show that in the absence of the
subunit, the stability of ß3 and µ3 is greatly reduced. However, somewhat different findings have been reported in yeast. When the AP-3
, ß3, or
3 subunits are deleted, the stability of µ3 does not appear to be affected, although it fractionates differently by gel filtration, in particular in the ß3-disrupted strain, where it appears to be monomeric (Panek et al., 1997). In contrast, deleting the
-like subunit of the AP-2related complex in yeast does not affect the ß2 subunit, but it has a profound effect on the stability of
2 (Yeung et al., 1999). Thus, it is difficult to predict how a particular AP subunit will behave in a particular organism; however, all of these studies consistently show that there are strong interactions between the
/
/
/
subunits and the
subunits, and between the ß and µ subunits.
These same interactions can be detected using the yeast two-hybrid system. The interactions that we report between the and
3 subunits, and between the ß3 and µ3 subunits, are consistent with interactions that we and others have reported between the corresponding pairs of subunits in the AP-1, AP-2, and AP-4 complexes (Page and Robinson, 1995; Aguilar et al., 1997; Hirst et al., 1999; Takatsu et al., 2001). In addition, in a previous study using the yeast two-hybrid system, we were able to demonstrate interactions between the two large subunits of the AP-1 and AP-2 complexes,
/
and ß (Page and Robinson, 1995). However, interactions between the large subunits appear to be more difficult to reconstitute, and so far we have been unable to demonstrate any interactions between
and ß3, or between the
and ß4 subunits of the AP-4 complex (Hirst et al., 1999).
and ß4 have been shown recently to interact in the presence of
4 using the yeast three-hybrid system, however (Takatsu et al., 2001), so it is possible that
3 may promote the interaction between
and ß3. One observation that supports the notion that
and ß3 interact in vivo is the finding that mh cells have greatly reduced levels of the ß3 and µ3 subunits. This suggests that ß3 may be unstable in the absence of
, which in turn would destabilize µ3.
We also used the yeast two-hybrid system to investigate whether the ubiquitously expressed (A) and neuronal-specific (B) ß3 and µ3 subunits were capable of interacting with each other. Interactions were detected between ß3A and µ3A, between ß3A and µ3B, and between ß3B and µ3A, although surprisingly not between ß3B and µ3B. The ability of the A and B isoforms to interact under more physiological conditions was demonstrated in our transfection experiments. When pe cells were transfected with ß3B, they formed heterotetramers containing the ubiquitously expressed µ3A subunit. Heterotetramers were also assembled from the other five constructs, as expected since they all contained the ß3A NH2-terminal domain. Perhaps less predictable was the ability of all the constructs to be recruited equally well onto membranes. Although studies from our own lab and others indicate that the COOH-terminal hinge and ear domains of the adaptor large subunits are not absolutely essential for membrane association, AP complexes containing earless or
are compromised in their ability to bind to their respective membranes (Robinson, 1993); however, the earless ß3A construct, ß3A807stop, showed no increased cytosolic background. We were also interested in the extent to which the various complexes colocalized with clathrin, in particular complexes containing the ß3Aß2 chimera, since multiple clathrin binding sites have been identified and/or predicted in the ß2 COOH-terminal hinge and ear domains (Dell'Angelica et al., 1998; Morgan et al., 2000; Owen et al., 2000). However, this construct, like the others, was at least partially localized to structures that were negative for clathrin. In contrast, AP-1 and AP-2 both show essentially complete colocalization with clathrin (Fig. 7; Robinson, 1987), indicating that the presence of the ß2 hinge and ear domains is not enough for a stable association with clathrin, and that there must be additional interactions, possibly involving the
and
subunits (Goodman and Keen, 1995; Morgan et al., 2000; Doray and Kornfeld, 2001).
Although all of the constructs assembled into complexes and were recruited onto membranes equally well, they varied widely in their ability to rescue the missorting phenotype of the pe cells. Using a quantitative assay for LAMP-1 mislocalization, we were able to show that ß3B gave nearly as good functional rescue as ß3A; indeed, the differences that we saw could simply be due to variability from one cell line to another (Dell'Angelica et al., 1999, 2000). However, although ß3B can functionally substitute for ß3A, the opposite may not be true. Synaptic-like microvesicle budding from endosomes has been shown to be dependent upon brain AP-3; liver AP-3 will not suffice (Faundez et al., 1998). This suggests that the neuronal-specific isoform of either ß3 or µ3, or both, is required for this activity. Brain AP-3 contains a mixture of A and B isoforms of both ß3 and µ3, while the stably transfected cells express only ß3B and µ3A; thus, it should be possible to test the relative importance of neuronal-specific ß3 and µ3 by determining whether AP-3 complexes from the transfected cells will substitute for brain AP-3 in the in vitro budding assay.
Two of our constructs gave partial rescue: the ß3Aß2 chimera and the deletion mutant ß3A807831. In contrast, the premature truncation mutant ß3A807stop was unable to rescue even partially. This finding indicates that the distal hinge and/or ear domains of ß3 are essential for function. In the ß3A
807831 mutant, part of the distal hinge is missing, and this fragment may either contribute directly to ß3 function, or alternatively may aid in the folding of the ear. Virtually nothing is known about the ear domain of ß3, although the ß2 ear has been shown to bind both clathrin and certain coated vesicle accessory proteins, including AP180, epsin, and Eps15 (Owen et al., 2000). We have shown that AP-3 complexes containing truncated ß3A are recruited onto the membrane, and presumably they are able to select cargo since they contain µ3A (Ohno et al., 1998). Thus, the lack of functional rescue suggests that they fail to produce coated vesicles, consistent with an inability to recruit structural or accessory proteins that might be essential for this event. The ability of the ß3Aß2 construct to give partial rescue suggests that the ß3 and ß2 ears may recruit some of the same proteins. However, gels of GST pulldowns using the ß3A ear showed no obvious candidate binding partners, and clathrin could not be detected on Western blots of the pulldowns (D. Owen, personal communication; unpublished data). Thus, the precise function of the ß3 ear remains elusive. However, its importance is highlighted by its evolutionary conservation: the ß3 ear domains of both Drosophila and C. elegans are significantly homologous to the ear domains of mammalian ß3A and ß3B, although both S. cerevisiae and S. pombe ß3 subunits appear to lack any ear domain.
Yeast ß3 subunits also lack a clathrin-binding consensus sequence, but both C. elegans and Drosophila have such sequences in their ß3 distal hinge domains (LIDVD and LLDLD, respectively). However, we show here that it is possible to mutate this sequence to one that can no longer bind clathrin in vitro (Dell'Angelica et al., 1998), and yet still get functional rescue. This result can now be added to the weight of evidence against an obligatory coupling between AP-3 and clathrin. At present we cannot rule out the possibility that there might be other clathrin binding sites in AP-3, even though there are no obvious candidates (we have tested the hinge and ear domains of the subunit, but are unable to detect any clathrin in GST pulldowns [unpublished data]). Double labeling for AP-3 and clathrin at the electron microscope level should help to determine whether there is any colocalization between the two in cells expressing ß3 with a mutated clathrin binding domain. However, much of the evidence in support of an association between AP-3 and clathrin comes from studies on the LLDLD sequence, and the present study demonstrates that this sequence is not essential for AP-3 function in vivo.
The large subunits of the AP complexes contain a number of other intriguing motifs and domains, including the "KRIGY" and "WIIGEY" sequences (Hirst et al., 1999). These motifs are found not only in every member of the /
/
/
and ß families, but also in the very distantly related ß-COP and
-COP subunits of the coatomer complex. So far nothing is known about their function, but we are currently mutating these motifs in both the
and ß3 subunits. By taking advantage of the mh and pe cell lines, it should be possible to assess the importance of these and other sequences not only in the assembly and localization of coat protein complexes, but also in coated vesicle function.
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Materials and methods |
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An immortal pe cell line was donated by R. Swank (Roswell Park Cancer Institute, Buffalo, NY). Although this line was generated from skin using methods designed to select for melanocytes, we were unable to detect the melanocyte-specific proteins tyrosinase and TRP-1 in these cells, indicating that they are another type of skin cell, most likely fibroblasts. The mouse melanocyte line, melan-a, was a gift from D. Bennett (St. George's Hospital Medical School, London, UK).
Antibody-based methods
Immunoprecipitations were performed both on nondenatured proteins and in the presence of SDS, using antibodies against both AP-3 and AP-1 subunits, as described previously (Simpson et al., 1996, 1997). Western blotting and immunofluorescence were also performed using previously published methods (Robinson and Pearse, 1986; Robinson, 1987). The polyclonal rabbit antibodies against AP-3 subunits have already been described (Simpson et al., 1996, 1997); in addition, a mouse monoclonal antibody was raised against the hinge domain (amino acids 608800) of the AP-3 subunit. The construct was expressed as a GST fusion protein and used to immunize female BALB/c mice. Fusion of the spleen cells with myeloma cells and screening of the resulting antibodies were performed as described by Bock et al. (1997). Ascites fluid was prepared by Joseph Beirao (Josman Laboratories, Napa Valley, CA). The rabbit anticlathrin antibody has been described previously (Simpson et al., 1997). The monoclonal anti-
antibody, mAb 100/3, was purchased from Sigma-Aldrich. The antiLAMP-1 antibody (1D4B) was initially obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA.
Yeast two-hybrid analysis
Most molecular biology techniques were performed as described by Sambrook and Russell (2001). To construct the plasmids for yeast two-hybrid analysis, the AP-3 subunits , ß3A, ß3B, µ3A, µ3B,
3A, and
3B were all cloned into the yeast two-hybrid vectors pGBT9 and pGAD424 (CLONTECH Laboratories, Inc.), using PCR to amplify the coding sequences and to introduce appropriate restriction sites. All constructs that had been made using PCR were sequenced by John Lester (University of Cambridge, Cambridge, UK) to confirm that no errors had been introduced. Interactions were monitored by assaying for growth in the absence of histidine.
Expression in mammalian cells
All plasmids for expression in mammalian cells were constructed using the vector pMEP (Girotti and Banting, 1996). The ß3Aß2 construct consists of the NH2-terminal 686 amino acids of ß3A fused to the last 334 amino acids of ß2. The deletion constructs and the point mutant were made by PCR and sequenced as above to confirm that they were error-free. Cells were transfected using QIAGEN SuperFect transfection reagent and selected in DME plus 10% fetal calf serum with Hygromycin (0.2 mg/ml). At least three cell lines were selected for each construct.
Flow cytometry
A quantitative assay for AP-3 dependent sorting was developed using flow cytometry. Two rat monoclonal antibodies, antiLAMP-1 (1D4B) and a control anti-KLH antibody (both IgG2as) were supplied by BD PharMingen. The antibodies were directly conjugated to Alexa Fluor 488 using the Molecular Probes Alexa 488 protein-labeling kit according to the manufacturer's instructions. Cells were then incubated with either antiLAMP-1 or control antibody for 6 h at 37° in serum-free media. The cells were rinsed in PBS, trypsinized, fixed in 3% paraformaldehyde, and analyzed on a fluorescence-activated cell scanner (FACScalibur; Becton Dickinson). To ensure that differences in antiLAMP-1 uptake were not due to differences in LAMP-1 expression levels, Western blots of each cell line were probed with the LAMP-1 antibody. Although there was some variability from one line to another, there was no correlation between LAMP-1 expression levels and the amount of antiLAMP-1 uptake.
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Footnotes |
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Andrew A. Peden's present address is Howard Hughes Medical Institute, Department of Molecular and Cellular Physiology, Stanford University School of Medicine, CA 94305-5426.
* Abbreviations used in this paper: AP, adaptor proteins; HPS, Hermansky Pudlak syndrome.
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Acknowledgments |
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This work was supported by grants from the Wellcome Trust and the Medical Research Council.
Submitted: 31 July 2001
Revised: 4 December 2001
Accepted: 4 December 2001
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References |
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