Article |
Address correspondence to Juan S. Bonifacino, Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, Building 18T/Room 101, National Institutes of Health, Bethesda, MD 20892. Tel.: (301) 496-6368. Fax: (301) 402-0078. E-mail: juan{at}helix.nih.gov
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Abstract |
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Key Words: lysosome biogenesis; vacuolar protein sorting; vesicle tethering; vesicle docking; lysosome fusion
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Introduction |
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Fusion of mammalian lysosomes with other membrane-bound organelles is key to their ability to acquire both internalized and biosynthetic materials (Kornfeld and Mellman, 1989; Luzio et al., 2000). Lysosomes have been shown to fuse with late endosomes (Mullock et al., 1998; Pryor et al., 2000; Ward et al., 2000) and phagosomes (Zimmerli et al., 1996; Funato et al., 1997) (heterotypic fusion), as well as with themselves (Bakker et al., 1997; Ward et al., 1997, 2000) (homotypic fusion). Late endosomelysosome fusion, in particular, has been the subject of much interest in recent years. Late endosomes have been proposed to establish transient contacts with lysosomes (i.e., kiss-and-run; Storrie and Desjardins, 1996) or to undergo complete fusion into a hybrid organelle from which lysosomes are later recovered by a fission event (i.e., fusionfission; Mullock et al., 1998; Pryor et al., 2000). Although the occurrence of lysosome fusion has been extensively documented, the identification of factors involved in this process, particularly in the specific tethering and/or docking events, is still in its infancy. In vitro studies of late endosomelysosome fusion have revealed a requirement for ATP, NSF, SNAPs, Rabs, as well as the SNARE syntaxin 7 (Mullock et al., 1998, 2000; Pryor et al., 2000; Ward et al., 2000), whereas in vivo overexpression studies have suggested a role for Rab7 (Bucci et al., 2000). To date, no tethering/docking factors have been identified that play a role in mammalian lysosome fusion.
Potential candidates for lysosomal tethering/docking factors are mammalian homologues of Saccharomyces cerevisiae gene products involved in vacuole fusion (for review see Wickner and Haas, 2000). Among these are Vps11p, Vps18p, Vps16p, Vps33p, Vam2p/Vps41p, and Vam6p/Vps39p, which assemble on the vacuolar membrane into a complex referred to as homotypic fusion and vacuole protein sorting (HOPS)* (Eitzen et al., 2000; Seals et al., 2000; Ungermann et al., 2000) or C-Vps complex (Sato et al., 2000; Wurmser et al., 2000). This complex has been shown to cooperate with the yeast homologue of mammalian Rab7, Ypt7p, to enable vacuole fusion mediated by the SNAREs (Ungermann et al., 1999, 2000; Eitzen et al., 2000; Sato et al., 2000; Seals et al., 2000; Wurmser et al., 2000). In this study, we report the identification and characterization of a human homologue of S. cerevisiae Vam6p/Vps39p (Nakamura et al., 1997), which we refer to as hVam6p. We show that overexpression of this protein in human cells induces massive clustering and fusion of lysosomes, whereas early endosomes and other organelles of the endocytic and secretory pathways remain unaffected. hVam6p exerts these effects by associating with the cytoplasmic face of the lysosomal membrane. An NH2-terminal citron homology (CNH) domain and a central clathrin homology (CLH) repeat domain in hVam6p are required for lysosome clustering and fusion. These observations suggest that hVam6p may function as a tethering/docking factor specifically involved in lysosome fusion.
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Results |
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hVam6p and its homologues also contain a CLH motif (Fig. 1, A and B) similar to seven such motifs present in the leg domain of the clathrin heavy chain (Ybe et al., 1999). Similar motifs are also found in the S. cerevisiae vacuolar sorting proteins Vam2p/Vps41p (Fig. 1 B), Vps18p, Vps11, and Vps8p. These motifs have been proposed to mediate proteinprotein interactions leading to homo- or heterooligomerization of the proteins (Ybe et al., 1999; Darsow et al., 2001).
Northern blot analysis demonstrated that hVam6p mRNA is expressed in all human tissues examined (Fig. 1 C), indicating that the protein may be widely expressed.
Coalescence of lysosomes and late endosomes caused by overexpression of hVam6p
S. cerevisiae Vam6p has been implicated in tethering and/or docking events that precede homotypic vacuole fusion (Eitzen et al., 2000; Price et al., 2000a,b; Seals et al., 2000; Wurmser et al., 2000). To assess whether hVam6p could play a similar role in lysosome tethering/docking, we examined the effects of overexpressing Myc epitopetagged hVam6p (MychVam6p) by transient transfection into HeLa cells. Fixed-permeabilized cells were analyzed by indirect immunofluorescence microscopy after double labeling with antibodies to the Myc epitope and to the lysosomal integral membrane proteins, lamp-1 (Fig. 2
, AC), lamp-2 (Fig. 2, DF), and CD63 (Fig. 2, GI). Untransfected cells exhibited the characteristic distribution of lysosomes, which were more concentrated in the juxtanuclear area of the cytoplasm but also extended toward the cell periphery (Fig. 2, AI). In contrast, cells overexpressing hVam6p displayed a striking coalescence of lysosomes into a few large juxtanuclear structures (Fig. 2, AI, arrows) with concomitant loss of peripheral lysosomes. These large structures were observed in virtually all hVam6p-overexpressing cells and contained all three lysosomal membrane proteins tested, as well as the lysosomal luminal hydrolase, cathepsin D (Fig. 2, JL, arrows). The distributions of the early endosomal marker, transferrin receptor, in cells overexpressing hVam6p (Fig. 2, MO, arrowhead), as well as various other endosomal, TGN, and Golgi markers (i.e., EEA1, AP-1, and the 58-kD Golgi protein; data not shown), were not affected. These observations suggested that the morphological alterations induced by overexpression of hVam6p were specific to organelles containing lysosomal membrane and luminal proteins.
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The localization of hVam6p in transfected HeLa cells was further examined by immunoelectron microscopy of ultrathin frozen sections (Fig. 3) . hVam6p was found to be associated with an electron dense halo surrounding 0.20.6 µm vesicles that were part of large clusters (Fig. 3 A, 10-nm gold particles, arrows). All of these vesicles were also labeled for the lysosomal membrane protein, lamp-2 (Fig. 3 A, 15-nm gold particles, arrowheads). Although many hVam6p-coated vesicles did not contain cation-independent mannose 6-phosphate receptor (CI-MPR) (Fig 3 B, *), others contained both hVam6p (Fig. 3 B, 10-nm gold particles, arrows) and CI-MPR (Fig. 3 B, 15-nm gold particles, arrowhead), and some had the appearance of multivesicular bodies. Together, these observations suggest that hVam6p associates with and induces clustering of lysosomes and late endosomes.
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Ultrastructural analysis of hVam6p-induced lysosomal structures loaded with internalized HRP
We took advantage of the ability to load the hVam6p-induced lysosomal structures with fluid phase endocytic markers to analyze their ultrastructure in more detail. Untransfected or hVam6p-transfected HeLa cells were allowed to internalize HRP for 4 h. After standard fixation, diaminobenzidine development for HRP visualization, and resin embedding, cell sections were analyzed by electron microscopy. As expected, untransfected cells displayed an array of 0.20.6-µm HRP-positive vesicles scattered throughout the cytoplasm, most of which likely corresponded to late endosomes and lysosomes because of the long period of internalization (Fig. 5
A). hVam6p-transfected HeLa cells, on the other hand, contained at least three types of abnormal structures. The first type consisted of large clusters of HRP-positive 0.20.6 µM vesicles, most of which contained intraluminal vesicles or other membranous inclusions (Fig. 5, B and C), similar to those seen on ultrathin cryosections (Fig. 3). The second type of abnormal structures were large (23 µm) vacuoles (Fig. 5, B and D). Some of these vacuoles seemed empty, displaying the appearance of swollen vacuoles. Others had variable amounts of HRP-positive materials, including 0.20.6 µM vesicles, within their interior (Fig. 5 D). The third type was a combination of the former two in that clusters of 0.20.6-µM HRP-positive vesicles were docked onto the membranes of the large vacuoles (Fig. 5, B and E). Serial sectioning (not shown) revealed that these three types of structures were situated next to the nucleus, often nestled between nuclear lobes. These analyses suggested a possible series of events induced by hVam6p, in which late endosomes and lysosomes first cluster together and then undergo fusion to generate large vacuoles.
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Functional relationship of hVam6p to Rab7
The effects of hVam6p on lysosomes are reminiscent of those elicited by a constitutively active Rab7 Q67L mutant (Bucci et al., 2000). To determine whether Rab7 nucleotide cycling is necessary for the formation of hVam6p-induced lysosome clusters, we transfected cells with wild-type GFPRab7, constitutively activated GFPRab7 Q67L, or dominant-negative GFPRab7 T22N, each alone or together with hVam6p. Overexpression of wild type GFPRab7 did not affect the distribution of endogenous lamp-1 (Fig. 7, A and B)
as previously reported (Bucci et al., 2000). However, in cells transfected with GFPRab7 and hVam6p, both GFPRab7 and lamp-1 were found in large juxtanuclear clusters (Fig. 7, C and D, arrowheads). Since GFPRab7 Q67L induces an effect on lysosomes (Bucci et al., 2000) that resembles that of hVam6p, we were unable to discern any additional effect in cells expressing both of these proteins (data not shown). However, GFPRab7 T22N, which by itself causes dispersal of lysosomes from the juxtanuclear region to the periphery (Bucci et al., 2000; and Fig. 7, E and F, small arrows) did not block the coalescence of lysosomes induced by hVam6p (Fig. 7, G and H, large arrow). These findings imply that hVam6p exerts its affects either downstream of or in parallel to Rab7.
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To further assess the self-association of hVam6p in vivo, HeLa cells were cotransfected with MychVam6p and HAhVam6p (Fig. 9 B). After metabolic labeling, the cells were lysed and subjected to immunoprecipitationrecapture analysis. Sequential immunoprecipitation with antibodies to Myc and to HA demonstrated that the two epitope-tagged hVam6p proteins interacted in vivo (Fig. 9 B). The amount of total HA-tagged hVam6p coprecipitated with antibody to Myc was 1020% in several experiments. Although this percentage may seem low, several combinations of epitope-tagged proteins can be formed, namely MycMyc, HAHA, and MycHA. In addition, the immunoprecipitationrecapture procedure is not quantitative because of the presence of some SDS in the recapture step. Accordingly, the level of MycHA recaptured hVam6p probably represents a significant fraction of the total hVam6p. The coprecipitation was specific, as MychVam6p did not coprecipitate with HA-tagged JNK1 (Fig. 9 B). These results support the observations from the sucrose gradient analyses, indicating that hVam6p is a homooligomer.
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Discussion |
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Monitoring the effects of hVam6p in vivo by time-lapse imaging of fluorescently labeled lysosomes suggests a possible sequence of events. Peripheral lamp-containing vesicles (i.e., lysosomes and/or late endosomes) appear to migrate towards the juxtanuclear cytoplasm along linear tracks. These tracks likely correspond to microtubules as treatment with nocodazole impairs translocation of the vesicles. Once the vesicles arrive at the juxtanuclear area, they cluster and eventually fuse with one another as well as with preexisting juxtanuclear lysosomes. Over time, the clusters and/or vesicles give rise to giant lysosomal structures reminiscent of those found in cells from patients with Chediak-Higashi syndrome (White, 1966). Few small vesicles are seen to escape from these structures, suggesting that the vesicles are irreversibly captured into the clusters. Electron microscopy analyses show that many of the clusters are composed of a variable number of regularly shaped vesicles. Some of the clusters seem to be docked onto large vacuoles, which may precede their fusion with or engulfment into the large vacuoles.
The phenotypic effects of hVam6p overexpression are strongly suggestive of a role for hVam6p in tethering/docking events leading to fusion of lysosomes. Immunoelectron microscopy analyses show that the overexpressed hVam6p forms an electron-dense halo around vesicles containing lamp-1 and CI-MPR in the clusters. This halo resembles an exaggerated form of a filamentous coat that has been previously seen to connect the membranes of adjoining multivesicular bodies and lysosomal membranes (Futter et al., 1996). Deposition of hVam6p onto the membranes of lysosomes and late endosomes likely enhances their adhesiveness, leading to the formation of clusters. This, in turn, increases the probability of fusion resulting in the generation of large vacuoles. Thus, overexpression of hVam6p might simply augment the process by which lysosomes and late endosomes normally exchange materials through kiss-and-run (Storrie and Desjardins, 1996) or fusionfission (Mullock et al., 1998) type of interactions.
The mechanism by which hVam6p induces lysosome clustering and fusion is still unclear. One possibility is that hVam6p may be intrinsically capable of bridging the membranes of two vesicles. Once the two vesicles are closely apposed, fusion would ensue by virtue of the SNARE-based fusion machinery. Another possibility is that overexpression of hVam6p sequesters some other factor that normally functions to prevent unregulated fusion, or to effect fission. Biochemical studies of S. cerevisiae Vam6p appear to support the first hypothesis, as this protein has been shown to be a component of the 38S HOPSC-Vps complex, which mediates vacuole tethering/docking (Price et al., 2000a,b; Seals et al., 2000; Wurmser et al., 2000) and trans-SNARE pairing (Price et al., 2000b; Sato et al., 2000). HOPSC-Vps is thought to participate in these processes as an effector of the YptRab GTPase, Ypt7 (Price et al., 2000a,b), and/or a guanine nucleotide exchange factor for Ypt7 (Wurmser et al., 2000).
Several lines of evidence suggest that hVam6p may act downstream or independently of Rab7: (a) overexpression of hVam6p induces a more dramatic effect on lysosomes than overexpression of the constitutively activated Rab7 protein (unpublished data), (b) hVam6p induces lysosome clustering and fusion even in the presence of overexpressed dominant-negative Rab7 T22N, and (c) hVam6p does not interact with the constitutively activated form of Rab7. Although by analogy with yeast Vam6p, hVam6p would be expected to be a component of a putative human HOPSC-Vps complex, data from sucrose gradients, coimmunoprecipitations, and two-hybrid analyses suggest that cytosolic hVam6p is a homooligomer. In addition, we could not detect association of hVam6p with endogenous hVam2p/Vps41p in the cytosol, and overexpression of the human Vam2p/Vps41p fails to induce effects similar to hVam6p (unpublished data). Since yeast Vam2p/Vps41p also exists as a homooligomer in the cytosol (Darsow et al., 2001), it is possible that Vam6p and Vam2p may become part of the HOPSC-Vps complex only upon association with the vacuolar/lysosomal membrane. Based on our observations, we speculate that hVam6p may subserve the tethering/docking function of the HOPSC-Vps complex.
Our studies have identified two functionally important domains within hVam6p: the CNH and CLH domains. The CNH domain is required for association with lysosomes and overexpression-induced lysosome clustering and fusion (Fig. 9). This suggests that the CNH domain could interact with a docking protein or with lipids on the lysosomal membrane. Other CNH domains have been shown to mediate interactions with the GTP-bound forms of the Rac and Rho GTPases (Madaule et al., 1995); although, our two-hybrid and coimmunoprecipitation analyses did not demonstrate such binding for hVam6p (unpublished data).
The CLH domain is also required for association of hVam6p with lysosomes and overexpression-induced lysosome clustering and fusion (Fig. 9). These requirements are probably related to the ability of the CLH domain to mediate homooligomerization of hVam6p (Fig. 9). CLH domains in the clathrin heavy chain (Ybe et al., 1999) and S. cerevisiae Vam2p (Darsow et al., 2001) have also been implicated in homooligomerization, suggesting that this may be the primary function of this domain. Thus, the effects of hVam6p on lysosomes appear to require homooligomerization of the protein.
In conclusion, our studies implicate hVam6p as a mammalian specific tethering/docking factor that has intrinsic ability to promote fusion of lysosomes and late endosomes in vivo. Further studies of this protein and other mammalian homologues of yeast components of the HOPSC-Vps complex are likely to provide a greater understanding of the mechanisms involved in lysosome fusion and biogenesis.
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Materials and methods |
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Recombinant DNA constructs
Epitope tagging at the NH2 terminus of hVam6p was performed by PCR amplification of the full-length hVam6p using 5' primers containing the nucleotide sequences for the HA or Myc epitopes. These products were cloned into the EcoRV-KpnI sites of the pXS vector to yield HAhVam6p and MychVam6p constructs, respectively. A Myc-tagged construct lacking residues 1382 (MycCNH) was obtained by PCR amplification using a 5' primer containing the Myc epitope nucleotide sequence and beginning with residue 383. Myc- and HA-tagged constructs encoding residues 1560 (Myc
CT) and 1372 (HA
CT+CLH) were obtained by PCR amplification using the same 5' primer as done for MychVam6p, but with a 3' primer encoding a stop site after residues 560 and 372, respectively. For yeast two-hybrid assays, the GAL4adhVam6p and the GAL4bdhVam6p constructs were prepared by ligating NdeI-BamHI PCR fragments into the multiple cloning sites of the pGADT7 (LEU2) and pGBKT7 (TRP1) vectors (CLONTECH Laboratories, Inc.). The GAL4adRab7 Q67L and GAL4bdRILP were provided by Dr. V. Deretic (University of Michigan Medical School, Ann Arbor, Michigan). GFPlgp120 (rat lamp-1) was a gift of R. Lodge and G. Patterson (National Institutes of Health, Bethesda, MD).
Antibodies
The following monoclonal antibodies were used: HA.11 antibody to the HA epitope and 9E10 antibody to the Myc epitope (Covance), H4A3, H4B4, and anti-CD63 antibodies to lamp-1, lamp-2 and CD63, respectively (Developmental Studies Hybridoma Bank), antibody to the CI-MPR (Affinity Bioreagents), 7G7.B6 antibodies directed against the Tac epitope (American Type Culture Collection), B3/25 antibody to the human transferrin receptor (Roche Molecular Biochemicals), antitubulin antibodies, and 100/2 antibody to AP-2 (Sigma-Aldrich). Rabbit polyclonal antibodies to cathepsin D (Upstate Biotechnology) or HA and Myc epitopes (Covance) were also used.
Live imaging by time-lapse fluorescence microscopy
HeLa or COS-7 cells grown on a Lab-Tek chambered coverglass system (Nunc) were transfected with plasmids encoding either GFPlgp120 alone or both GFPlgp120 and HAhVam6p, as described above. 10 h after transfection, 25 mM Hepes, pH 7.4, was added to the media, and live images of the GFP-expressing cells were obtained on a ZEISS LSM 410 confocal microscope. Temperature was maintained at 37°C with a Nev-Tek airstream stage incubator. GFP molecules were excited with the 488-nm line of a kryptonargon laser and imaged with a 527 filter. Image acquisition, processing, and automatic and manual data collection were performed using NIH Image v1.62 (Wayne Rasband Analytics). Live images are shown inverted to facilitate analysis.
Electron microscopy
For HRP-uptake studies, HeLa cells were transiently transfected on coverslips with HAhVam6p and incubated 24 h later with 6 mg/ml HRP (Fraction VI; Sigma-Aldrich) diluted in culture medium for 4 h. Cells were fixed with 2% glutaraldehyde in 100 mM Hepes buffer (pH 7.4). HRP enzymatic activity was developed with 1% diaminobenzidine as previously described (van der Sluijs et al., 1992). Cells on coverslips were treated with reduced osmium tetroxide, dehydrated, and embedded in epoxy resin. Sections were viewed with a Philips CM-10 transmission electron microscope. For immunoelectron microscopy studies, HeLa cells were transiently transfected, fixed with 4% formaldehyde, 0.2% glutaraldehyde in 100 mM Hepes buffer (pH 7.4), and prepared for ultra-thin frozen sectioning as described (Slot et al., 1991).
Other procedures
Human HeLa cells were grown on glass coverslips, transfected using FUGENE-6 (Roche Molecular Biochemicals), and either fixed and processed for immunofluorescence (Dell'Angelica et al., 1997), or used directly for analysis of rhodaminedextran or LysoTrackerTM (Molecular Probes) uptake, followed by fixation and processing, as described above. Microtubule disruption was induced by treatment with 0.5 µM nocodazole (Sigma-Aldrich) for 16 h, immediately after transfection. All images were obtained using a ZEISS LSM 410 confocal microscope. Northern blot analysis (Dell'Angelica et al., 1997), metabolic labeling, cell surface biotinylation (Caplan et al., 2000), sedimentation analysis (Dell'Angelica et al., 1997), and yeast two-hybrid assays (Aguilar et al., 1997) using the strain AH109 (CLONTECH Laboratories, Inc.) were performed as previously described.
Online supplemental material
Online supplemental materials are available at http://www.jcb.org/cgi/content/full/200102142/DC1. Video 1 corresponds to Fig. 6 A and contains a Quicktime movie sequence depicting the centripetal movement and fusion of GFPlamp-1-containing vesicles in a COS-7 cell also expressing hVam6p. Images were captured every 30 s over the course of 31 min. Video 2 corresponds to Fig. 6 B and contains a Quicktime movie sequence demonstrating fusion of a peripheral GFPlamp-1-containing organelle with a large juxtanuclear lysosomal structure in a HeLa cell also expressing hVam6p. Images were captured every 30 s over the course of 200 min. Video 3 corresponds to Fig. 6 C and contains a Quicktime movie sequence illustrating the centripetal movement and alignment of GFPlamp-1 structures from the periphery along the juxtanuclear axis of a COS-7 cell expressing hVam6p. Images were captured every minute over the course of 388 min.
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Footnotes |
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* Abbreviations used in this paper: CI-MPR, cation-independent mannose-6-phosphate receptor; CLH, clathrin homology; CNH, citron homology; GAL4ad, GAL4 transcription activation domain; GAL4bd, GAL4 DNAbinding domain; GFP, green fluorescent protein; HOPS, homotypic fusion and vacuole protein sorting; TRAP-1, TGF-ß receptor-Iassociated protein-1.
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Acknowledgments |
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S. Caplan is the recipient of a long-term Human Frontiers Science Program Fellowship.
Submitted: 27 February 2001
Revised: 4 May 2001
Accepted: 25 May 2001
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References |
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