Identification and domain organization of human Vam6p
A novel human cDNA encoding a protein homologous to the S
vacuolar protein sorting gene product Vam6p/Vps39p (Nakamura et al., 1997
) was identified through searches of DNA databases and 5′ RACE-PCR (
A). This cDNA encodes a protein designated hVam6p of 886 amino acid residues and a predicted molecular mass of ~100 kD. hVam6p bears 26% identity and 42% overall amino acid sequence homology to the COOH-terminal region of S
Vam6p (). Homologues of hVam6p were also identified in Drosophila melanogaster
(35% identity, 53% homology), Caenorhabditis elegans
(30% identity, 50% homology) (), and Schizosaccharomyces pombe
(24% identity, 41% homology) ( B). Our searches also identified a second human homologue of S
Vam6p that had already been characterized as the TGF-β receptor-I–associated protein-1 (TRAP-1) (21% identity, 40% homology) (Charng et al., 1998
) ( B).
Figure 1. Sequence homology, domain organization, and expression of hVam6p. (A) Full-length amino acid sequences of human (Homo sapiens; Hs) Vam6p (hVam6p), a D. melanogaster (Dm) homologue, and a C. elegans (Ce) homologue were aligned together with residues (more ...)
Theoretical analyses predict the presence of a CNH domain (Madaule et al., 1995
) near the NH2
terminus of hVam6p, TRAP-1, and their S
, and C
homologues (). Although these proteins exhibit significant homology to hVam6p throughout the entire protein ( B, gray shaded blocks), the S
Vam6p lacks a defined CNH domain, and the region of significant homology commences only after the first 450 residues. CNH domains also exist near the COOH terminus of several kinases involved in the jun kinase (JNK) activation pathway (e.g., NIK and TNIK) and of the S
Rho guanine nucleotide exchange factor Rom1p ( B). Little is known about the function of this domain, although it has been proposed to regulate kinase activity (Fu et al., 1999
; Dan et al., 2000
) and to mediate binding to the GTP-bound forms of Rac and Rho (Madaule et al., 1995
hVam6p and its homologues also contain a CLH motif () 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
vacuolar sorting proteins Vam2p/Vps41p ( B), Vps18p, Vps11, and Vps8p. These motifs have been proposed to mediate protein–protein 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 ( C), indicating that the protein may be widely expressed.
Coalescence of lysosomes and late endosomes caused by overexpression of hVam6p
Vam6p has been implicated in tethering and/or docking events that precede homotypic vacuole fusion (Eitzen et al., 2000
; Price et al., 2000a
; 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 epitope–tagged hVam6p (Myc–hVam6p) 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 (
, A–C), lamp-2 (, D–F), and CD63 (, G–I). 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 (, A–I). In contrast, cells overexpressing hVam6p displayed a striking coalescence of lysosomes into a few large juxtanuclear structures (, A–I, 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 (, J–L, arrows). The distributions of the early endosomal marker, transferrin receptor, in cells overexpressing hVam6p (, M–O, 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.
Figure 2. Overexpression of hVam6p induces coalescence of lysosomal vesicles. (A–O) HeLa cells were transiently transfected with a plasmid encoding Myc–hVam6p. Cells were fixed, permeabilized, and incubated with rabbit polyclonal antibodies to the (more ...)
The exogenously expressed hVam6p appeared to be mostly cytosolic (). To determine whether the cytosolic pool could have masked a population of membrane-associated hVam6p, we performed immunofluorescence microscopy of hVam6p-expressing HeLa cells after extraction of the cytosol with 0.05% wt/vol saponin before fixation. This procedure uncovered the presence of hVam6p on the same large lysosomal structures that contained lamp-1 (, P–R).
The localization of hVam6p in transfected HeLa cells was further examined by immunoelectron microscopy of ultrathin frozen sections ()
. hVam6p was found to be associated with an electron dense halo surrounding 0.2–0.6 μm vesicles that were part of large clusters ( A, 10-nm gold particles, arrows). All of these vesicles were also labeled for the lysosomal membrane protein, lamp-2 ( A, 15-nm gold particles, arrowheads). Although many hVam6p-coated vesicles did not contain cation-independent mannose 6-phosphate receptor (CI-MPR) ( B, *), others contained both hVam6p ( B, 10-nm gold particles, arrows) and CI-MPR ( 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.
Figure 3. Association of hVam6p with clusters of lysosomes and late endosomes. HeLa cells were transiently transfected with HA–hVam6p and processed for immunoelectron microscopy. Ultra-thin frozen sections were labeled with antibodies to HA (to detect hVam6p) (more ...)
Functional assessment of hVam6p-induced lysosomal structures
The hVam6p-induced lysosomal structures could be labeled with the acidotropic fluorescent probe, LysoTracker red, indicating that they were acidic (
, A–C, arrow). To determine if they were also accessible to fluid-phase markers, HeLa cells expressing Myc–hVam6p were allowed to internalize the fluid phase marker rhodamine–dextran for various time periods. Cells were then fixed permeabilized cells and subjected to indirect immunofluorescence to detect hVam6p-expressing cells. At time points ranging from 15 min (not shown) to 1 h (, D–F), rhodamine–dextran was found to be internalized equally well into typical lysosomes in untransfected cells and lysosomal clusters in hVam6p-transfected cells ( D, arrows), suggesting no major change in accessibility to fluid phase markers upon expression of hVam6p.
Figure 4. Functional characterization of hVam6p-induced lysosomal clusters. (A–F) Live HeLa cells transiently transfected with Myc–hVam6p were incubated with LysoTracker red (A–C) or rhodamine–dextran (D–F) for 1 h, and then (more ...)
To determine whether hVam6p-induced structures were also capable of degrading protein cargo, we cotransfected cells with a Tac fusion protein containing a dileucine-based endocytic and lysosomal targeting signal, Tac-DKQTLL, (Letourneur and Klausner, 1992
), together with either hVam6p or nonmyristylated Arf6 as a control. Pulse–chase analysis of Tac-DKQTLL subsequent to both metabolic and cell surface labeling demonstrated that cells transfected with hVam6p were capable of degrading proteins with kinetics similar to normal cells ( G). All of these observations suggested that hVam6p-induced lysosomal structures, despite their altered morphology, were still capable of receiving and degrading materials delivered from the endocytic system.
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 0.2–0.6-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 (
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 0.2–0.6 μM vesicles, most of which contained intraluminal vesicles or other membranous inclusions (), similar to those seen on ultrathin cryosections (). The second type of abnormal structures were large (2–3 μm) vacuoles (). Some of these vacuoles seemed empty, displaying the appearance of swollen vacuoles. Others had variable amounts of HRP-positive materials, including 0.2–0.6 μM vesicles, within their interior ( D). The third type was a combination of the former two in that clusters of 0.2–0.6-μM HRP-positive vesicles were docked onto the membranes of the large vacuoles (). 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.
Figure 5. Ultrastructural analysis of hVam6p-induced lysosomal structures labeled with internalized HRP. Untransfected (A) or hVam6p-transfected (B–E) HeLa cells were subjected to a continuous 4 h fluid phase uptake of HRP 24 h after transfection. (A) In (more ...)
Dynamics of lysosome clustering and fusion induced by hVam6p
To investigate the processes that led to the formation of lysosomal clusters and large vacuoles over time, we monitored the movement of lysosomes in live cells by time-lapse fluorescence microscopy. To this aim, COS-7 and HeLa cells were transfected with plasmids encoding green fluorescent protein (GFP)–lamp-1 alone or GFP–lamp-1 and hVam6p. In cells transfected with GFP–lamp-1 alone, lysosomes and/or late endosomes exhibited bidirectional movement between the juxtanuclear area and sites in the cell periphery (data not shown). In the doubly transfected cells, the effects of hVam6p on lysosomes were concurrent with the expression of GFP–lamp-1, which made it necessary to image cells already displaying incipient lysosome clustering. At early time points, we could observe peripheral lysosomes moving centripetally toward the center of the cell and attaching to or merging with other lysosomes (
A, arrow, Video 1). More centrally located lysosomes were also observed to cluster and/or fuse with one another ( A, arrowhead, and B; Videos 1 and 2). Over time, these clustering and fusion events lead to the accumulation of large lysosomal structures in the juxtanuclear area ( C; Video 3). These observations suggested that hVam6p causes lysosomes and late endosomes to stick together in tight clusters and fuse, thus preventing them from migrating back to the cell periphery. (Videos 1, 2, and 3 are available at http://www.jcb.org/cgi/content/full/200102142/DC1
Figure 6. Dynamics of hVam6p-induced lysosome clustering and fusion. COS-7 (A and C) or HeLa (B) cells were transfected with plasmids encoding GFP–lamp-1 (GFP–lgp120) and HA–hVam6p. 10 h after transfection, live cells were incubated at 37°C (more ...)
Treatment of HeLa cells with 0.5 μM nocodazole immediately after transfection with an hVam6p-encoding plasmid did not impede the coalescence of lamp-1–containing structures in the cell periphery but did prevent their translocation to the juxtanuclear area of the cell ( D). This indicated that microtubules are not involved in the clustering process itself but in the localization of the clusters to the juxtanuclear area.
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 GFP–Rab7, constitutively activated GFP–Rab7 Q67L, or dominant-negative GFP–Rab7 T22N, each alone or together with hVam6p. Overexpression of wild type GFP–Rab7 did not affect the distribution of endogenous lamp-1 ()
as previously reported (Bucci et al., 2000
). However, in cells transfected with GFP–Rab7 and hVam6p, both GFP–Rab7 and lamp-1 were found in large juxtanuclear clusters (, arrowheads). Since GFP–Rab7 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, GFP–Rab7 T22N, which by itself causes dispersal of lysosomes from the juxtanuclear region to the periphery (Bucci et al., 2000
; and , small arrows) did not block the coalescence of lysosomes induced by hVam6p (, large arrow). These findings imply that hVam6p exerts its affects either downstream of or in parallel to Rab7.
Figure 7. hVam6p affects lysosomal morphology independently of Rab7 nucleotide cycling. HeLa cells were transfected with either GFP–Rab7 (A and B, control) or dominant-negative GFP–Rab7 T22N (E and F, control), or were cotransfected with Myc–hVam6p (more ...)
Homooligomerization of hVam6p
A novel Rab7 effector termed RILP (Cantalupo et al., 2001
) has recently been identified through its binding to the constitutively active Rab7 Q67L and has been demonstrated to mediate the effects of Rab7 on lysosome biogenesis. To determine whether hVam6p interacts with either Rab7 Q67L or RILP, we used a yeast two-hybrid approach. As expected, Rab7 Q67L and RILP interacted with one another (Cantalupo et al., 2001
A). However, neither Rab7 Q67L nor RILP displayed an interaction with hVam6p, consistent with the idea that hVam6p acts independently of Rab7. A strong interaction was observed upon coexpression of GAL4 transcription activation domain (GAL4ad)–hVam6p and GAL4 DNA–binding domain (GAL4bd)–hVam6p ( A), however, suggesting that hVam6p may be able to self-assemble.
Figure 8. Homooligomerization of hVam6p. (A) The S. cerevisiae yeast strain AH109 was cotransformed with the following GAL4ad fusion constructs: GAL4ad–hVam6p, GAL4ad–Rab7 Q67L, and GAL4ad–pVA3 (murine p53 control), together with the GAL4bd (more ...)
To determine the size of potential hVam6p oligomers in vivo, HA–hVam6p-transfected HeLa cells were labeled for 8 h with [35S]methionine, extracted with detergent, fractionated by ultracentrifugation on a 5–20% sucrose gradient, and analyzed by immunoprecipitation with anti-HA antibodies ( B). A major species of ~105 kD was detected on SDS-PAGE of anti-HA immunoprecipitates, in good agreement with the predicted molecular mass of hVam6p (~100 kD). HA–hVam6p peaked in gradient fractions corresponding to a molecular mass of ~317 kD, higher than the molecular mass of the adaptor protein complex AP-2 (~270 kD). This indicated that hVam6p is a component of an oligomeric complex. Since no other major bands were detected in the immunoprecipitates ( B), these results suggest that hVam6p is a homooligomer, most likely a homotrimer.
To further assess the self-association of hVam6p in vivo, HeLa cells were cotransfected with Myc–hVam6p and HA–hVam6p (
B). After metabolic labeling, the cells were lysed and subjected to immunoprecipitation–recapture analysis. Sequential immunoprecipitation with antibodies to Myc and to HA demonstrated that the two epitope-tagged hVam6p proteins interacted in vivo ( B). The amount of total HA-tagged hVam6p coprecipitated with antibody to Myc was 10–20% in several experiments. Although this percentage may seem low, several combinations of epitope-tagged proteins can be formed, namely Myc–Myc, HA–HA, and Myc–HA. In addition, the immunoprecipitation–recapture procedure is not quantitative because of the presence of some SDS in the recapture step. Accordingly, the level of Myc–HA recaptured hVam6p probably represents a significant fraction of the total hVam6p. The coprecipitation was specific, as Myc–hVam6p did not coprecipitate with HA-tagged JNK1 ( B). These results support the observations from the sucrose gradient analyses, indicating that hVam6p is a homooligomer.
Figure 9. Delineation of functional domains in hVam6p. (A) Schematic representation of full-length hVam6p and various deletion constructs used in these experiments. (B) HeLa cells were cotranfected with plasmids encoding Myc– and HA–hVam6p, or hVam6p (more ...)
Delineation of functional domains in hVam6p
To determine the region of hVam6p necessary for homooligomerization, a series of truncations and deletions were prepared ( A). HeLa cells were cotransfected with the Myc- and HA-tagged hVam6p constructs, labeled with [35S]methionine, and analyzed by immunoprecipitation–recapture ( B). Full-length hVam6p was found to interact with truncated hVam6p constructs lacking either the CNH domain (ΔCNH) or a COOH-terminal segment (ΔCT). However, truncation of the CLH domain in addition to the COOH-terminal segment (ΔCLH+CT) resulted in a loss of interaction with the full-length protein. These data point to a role for the CLH domain in self-assembly of hVam6p. To assess the role of the hVam6p domains in promoting lysosome clustering and fusion, we transfected Hela cells with Myc-tagged versions of full-length, ΔCT, ΔCNH, and ΔCLH+CT constructs (, C–N). Indirect immunofluorescence microscopy for lamp-1 demonstrated that, in addition to the full-length hVam6p (, C–E), the ΔCT protein (, F–H) also induced coalescence of lysosomes (green channel, arrows in D and G). However, loss of either the CNH domain (, I–K), or the COOH-terminal segment plus CLH domain of the protein (, L–N) completely abrogated the induction of lysosome clustering and fusion (, I–N). Double label immunoelectron microscopy analysis showed that both ΔCNH and ΔCLH+CT proteins lost their ability to localize to lysosomes (unpublished data), suggesting that both association with lysosomes and homo-oligomerization are requisites for inducing lysosome clustering and fusion.