1 Center for Basic Neuroscience, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9111, USA
2 University of Zürich, Institute of Physiology, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland
* Author for correspondence (e-mail: hkrame{at}mednet.swmed.edu)
Accepted 17 May 2005
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Endocytic trafficking, Vps-C genes, Pigment granules, Lysosome-related organelles, Autophagosomes
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
At least 16 HPS-related genes have now been identified based on mutations in humans and mice (Huizing and Gahl, 2002; Li et al., 2004
; Spritz and Oh, 1999
). Most of these genes encode proteins that assemble into one of the three biogenesis of lysosome-related organelles complexes, BLOC-I, -II, and -III (Ciciotte et al., 2003
; Gwynn et al., 2004
; Li et al., 2003a
; Martina et al., 2003
; Nazarian et al., 2003
; Starcevic and Dell'Angelica, 2004
). In addition, the HPS-2 gene encodes a subunit of the AP-3 adaptor complex (Dell'Angelica et al., 1999
).
An additional set of proteins has been implicated in HPS-related functions by a point mutation in the murine vps33A gene that causes the HPS-like phenotypes observed in buff mutants (Suzuki et al., 2003). The yeast homolog of Buff is encoded by the vacuolar protein sorting (VPS) gene Vps33/Pep14 (Banta et al., 1990
; Subramanian et al., 2004
). Vps33 proteins are members of the family of Sec1/Munc18 (SM) proteins, which are essential regulators of membrane fusion events (Jahn et al., 2003
; Toonen and Verhage, 2003
).
SM proteins have been shown to interact with SNARE proteins in three different ways. First, Munc18 binds directly to the closed form of Syntaxin I, which cannot participate in SNARE complex formation (Dulubova et al., 1999; Misura et al., 2000
). Second, yeast Sec1p binds to the syntaxin homolog Sso1p only when Sso1p is complexed with other SNARE proteins (Carr et al., 1999
). Third, Vps33p, as part of the Class C Vps protein complex, may only indirectly interact with the syntaxin homolog Vam3p (Dulubova et al., 2001
; Sato et al., 2000
).
The Class C Vps protein complex comprises four core subunits that originally were defined by mutations that severely disrupt vacuolar morphology (Raymond et al., 1992). In addition to Vps33p/Pep14p, the VPS-C complex comprises Vps18p/Pep3p, Vps11p/Pep5p and Vps16p (Rieder and Emr, 1997
). This complex has been implicated in multiple membrane fusion events including fusion of endosomes with vacuoles (Rieder and Emr, 1997
; Sato et al., 2000
), early endosome fusions (Peterson and Emr, 2001
; Richardson et al., 2004
; Srivastava et al., 2000
; Subramanian et al., 2004
) and homotypic fusion between vacuoles (Seals et al., 2000
). The Vps-C protein complex binds to Vps41p/Vam2p and Vps39/Vam6p, thus constituting the HOPS complex (for homotypic vacuole fusion and protein sorting), which acts as effector and regulator of the Rab7 homolog Ypt7 (Price et al., 2000
; Wurmser et al., 2000
).
Vps-C complexes have also been described in mammalian and Drosophila cells (Kim et al., 2001; Richardson et al., 2004
; Sevrioukov et al., 1999
; Sriram et al., 2003
). A fly homolog of Vps33p is encoded by the carnation (car) gene, and a homolog of vps18 is encoded by deep orange (dor) (Sevrioukov et al., 1999
; Shestopal et al., 1997
). Furthermore, light encodes a homolog of Vps41p (Warner et al., 1998
). Because mild loss-of-function alleles of these genes impair the biogenesis of pigment granules, these genes were originally discovered as eye-color mutants (Beadle and Ephrussi, 1936
). Stronger phenotypes, including lethality, result either from genetic interactions of mild alleles, e.g. car1 and light1 (Nickla, 1977
) or null alleles such as dor8 (Shestopal et al., 1997
). Such strong alleles revealed the additional roles of dor in membrane trafficking. For example, cells homozygous for dor8 are impaired in the delivery of internalized ligands to lysosomes (Sevrioukov et al., 1999
).
Here, we identify dVps16A as an additional subunit of the Dor/Car complex. RNAi-induced knockdown of dVps16A reveals phenotypes in lysosomal delivery and pigment granule biogenesis similar to those of a dor null allele. Furthermore, we demonstrate that a second Vps16 homolog, dVps16B, specifically interacts with a second Vps33p homolog, dVps33B. These results suggest the presence of two distinct Vps-C-like protein complexes in Drosophila.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fly work
For expression in transgenic flies, cDNAs encoding the relevant proteins were cloned into the p{UASt} vector (Brand and Perrimon, 1993). The GFP-LAMP fusion protein is designed to encode a cleavable preprolactin signal sequence followed by eGFP and a transmembrane domain and cytoplasmic tail derived from human LAMP1 (accession number CAI13797. The LAMP1 cytoplasmic tail is sufficient to direct proteins to lysosomes (data not shown) (Rohrer et al., 1996
). Flies carrying the P{UAS-Dor} construct have been described (Sevrioukov et al., 1999
). RNAi constructs containing inverted repeats of about 300 bp of dVps16A cDNA were generated in pWIZ (Lee and Carthew, 2003
). The chosen region did not contain any stretch of more than 12 bp of identity to the dVps16B cDNA. Transgenic flies were established using standard procedures (Rubin and Spradling, 1982
). Expression was driven by P{GMR-Gal4} (Hay et al., 1994
) P{ey-Gal4} (Hazelett et al., 1998
) or P{da-gal4} (Wodarz et al., 1995
), which were obtained from the Bloomington Stock center. To assess effects on pigment granules, expression in the eye was induced in a white+ background.
Antibodies
To raise anti-dVps16A antibodies, a GST-fusion protein containing aa 663-833 of dVps16A, GST-dVps16C2, was expressed in bacteria, purified and injected into rabbits. Specific antibodies were affinity-purified from sera using the GST-dVps16C2 fusion proteins coupled to agarose beads (Affigel 10, Bio-Rad) as described (Harlow and Lane, 1988). An antibody recognizing the C-terminus of Dor (anti-DorY81) was raised in guinea pigs using a peptide encompassing aa 987-1002. For detection of endogenous proteins by western blots we used anti-dVps16A (1:3000), anti-Car (1:2000) (Sevrioukov et al., 1999
), anti-DorY81 (1:1000), anti-HA 12CA5 (BABCO, 1:500), anti-Myc 9E10 (Sigma, 1:2000) anti-tubulin DM1A (Sigma, 1:3000), dSyn7-45 (1:5000) and anti-Hook (1:2000) (Krämer and Phistry, 1996
).
Tissue culture and biochemistry
cDNAs encoding full-length or truncated tagged forms of Dor and Car (Sevrioukov et al., 1999), Rop (Wu et al., 1998
) dVps16A, dVps16B, or dVps33B were transfected into S2 cells and expression was induced using 0.7 µM CuSO4 (Krämer and Phistry, 1996
). After 16 hours, S2-cell extracts were used for co-immunoprecipitations as described (Sevrioukov et al., 1999
).
For analysis of endogenous proteins, Drosophila cytosol (12 µg protein µl1) was prepared from S2 cells (Walenta et al., 2001
). For sucrose density gradient centrifugation (10-20%), 50 µl cytosol was fractionated and sedimentation coefficients (S20w) determined as described (Sevrioukov et al., 1999
). Individual fractions were separated by SDS-PAGE and probed with the indicated antibodies. Extracts for developmental western blots were prepared from different developmental stages as described (Sevrioukov et al., 1999
).
For western blots of dVps16A-RNAi larvae, the homozygous da-Gal4 driver was crossed to a line homozygous for the dVps16A-RNAi transgene. Larvae hemizygous for dor8 were recognized by the absence of the GFP-tagged FM7 balancer (Casso et al., 2000).
Histology
Eye imaginal discs were stained as described using anti-Boss (Krämer et al., 1991) or anti-Sca1 antibodies (Lee et al., 1996
). Alexa-488- or Alexa-568-conjugated secondary antibodies were detected with a Leica TCS SP2 confocal microscope with a 63x NA 1.32 lens. All digital images were imported into Photoshop and adjusted for gain and contrast.
For the quantification of LAMP1-GFP levels, third instar eye discs were stained with antibodies against Boss using Alexa-568-conjugated secondary antibodies and imaged on a Leica TCS SP2 confocal microscope as described above. A Z-stack of images 120 µm x 120 µm in size were obtained to a depth of about 10 µm, with images taken every 0.5 µm. Boss staining on the apical surface of R8 cells was used as a reference to ensure that images were collected at the same depth and distance from the morphogenetic furrow. Image stacks were imported into Volocity software for thresholding and quantification of GFP levels. The intensity of each LAMP1-GFP pixel was determined and the sum of pixel intensities in the Z-stack was determined to be the total GFP fluorescence for each sample. For electron microscopy, adult heads were fixed, embedded in plastic and sectioned as described (Van Vactor et al., 1991).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
dVps16A is part of the Vps-C complex and binds to Car and Dor
When S2 cytosol was separated in a 10-20% sucrose density gradient, the majority of dVps16A was found in the same fractions in which the level of Dor and Car proteins peaked (Fig. 1A). To test for a physical interaction between dVps16A and the Dor/Car complex we used co-immunoprecipitation of endogenous proteins. Antibodies against Dor, Car and dVps16A, or control antibodies, were used to immunoprecipitate proteins from Drosophila S2 cell cytosol. Immunoprecipitates were separated by SDS-PAGE and probed for the presence of the three proteins for which antibodies are available: Dor, Car and dVps16A. Antibodies against each of these proteins, but not control antibodies, precipitated each of the three proteins (Fig. 1B) consistent with the model that the Drosophila homologs of the C-class Vps proteins form a stable complex.
|
Co-immunoprecipitation experiments between Car and different dVps16A truncations were used to define the domain within dVps16A that is responsible for its binding to Car (Fig. 3). After immunoprecipitation of Car from co-transfected cells, the C-terminal domain of dVps16A was pulled down, but not the N-terminus or the well-conserved central part (Fig. 3A). We noted that in these experiments that the C-terminal domain was consistently higher expressed than other truncations; however, even 10-fold longer exposure did not detect Vps16A-N or -M in the immunoprecipitates (data not shown). Because in these experiments Car and the respective dVps16A truncations were both HA-tagged, the levels of co-immunoprecipitated proteins could be directly compared. Full-length dVps16A as well as the dVps16A-C domain bound Car in a ratio close to 1:1, indicating this is a direct interaction between the over-expressed proteins (Fig. 3A,B). The specificity of the interaction with SM proteins is encoded within the C-terminal domain of dVps16A, which only pulled down Car but not dVps33B or Rop (Fig. 3C). In these co-expression experiments, we consistently noticed that the level of dVps16A-C in the cytosol was increased in response to the co-expression of Car but not other SM proteins (Fig. 3C). This adds further weight to the notion that the Vps16-C domain determines the specific interaction of Vps16 proteins with SM protein family members. Further truncation analysis indicated that aa 489-555 of dVps16A are necessary for its binding to Car (Fig. 3B,D,E).
We also probed the interaction between Dor and the Vps16 homologs using co-immunoprecipitation of epitope-tagged Vps-C subunits. Western blots of immunoprecipitates revealed that Myc-tagged Dor pulled down full-length dVps16A or its C-terminal domain, dVps16A-C (Fig. 4A). C-terminal truncations narrowed the region of dVps16A that is required for binding to Dor at aa 489-633 (Fig. 4B,C). This region is partially overlapping with the Car-binding domain, but the presence of all three endogenous proteins in a single complex (Fig. 1) suggests that these interactions are not competitive.
|
Interestingly, the Dor-binding domain of dVps16A is the most similar to dVps16B. We therefore tested its interaction with Dor and found that after co-expression in S2 cells Dor pulled down dVps16B (Fig. 4D). Together, these experiments suggested that the two distinct Vps-C complexes in Drosophila use Dor as a subunit.
dVps16A knockdown causes loss of Dor
Because no mutation has yet been identified in the dVps16A gene, we used RNAi to analyze its loss-of-function phenotypes. Two snap-back dVps16A-RNAi constructs were placed under UAS control. One of these constructs was efficient in knocking-down dVps16A after expression in transgenic flies and is further characterized below. In wild-type Drosophila tissue, affinity-purified anti-dVpsd16A antibodies recognized a major band of about 100 kDa (Fig. 5A), close to the predicted mass of dVps16A. This band was not detected in extracts from larvae in which the dVps16A-RNAi transgene was ubiquitously expressed using the da-Gal4 driver (Fig. 5B). These larvae developed into pupae but no flies emerged, indicating that dVps16A is an essential gene. No changes in the levels of Car, dSyntaxin-7 or tubulin proteins were detected in larvae and pupae after dVps16A knockdown, but the level of Dor protein was dramatically reduced (Fig. 5B).
|
dVps16A is required for the formation of the compound eye
To analyze the cellular consequences of loss of dVps16A function, we focused on the compound eye. Eye-specific expression of the dVps16A-RNAi construct was induced with the combined ey-Gal4 (Hazelett et al., 1998) and GMR-Gal4 drivers (Hay et al., 1994
), which initiate expression early in eye development and maintain high levels in the adult eye. Externally, the most obvious change in response to dVps16A knockdown was a dramatic reduction of eye pigmentation compared with wild-type flies (Fig. 6A-C). The resulting eye color exhibited a gradient with the most anterior ommatidia having lost most of their pigmentation (Fig. 6B). This gradient reflects the expression pattern of the ey-Gal4 driver, as we consistently see such gradients with this driver using different RNAi transgenes (T. Endicott and H.K., unpublished). The eye-color phenotype of dVps16A knockdown flies was not rescued by over-expressing Dor or Car protein using the uas/Gal4 systems with the combined ey-Gal4 and GMR-Gal4 drivers (data not shown).
|
|
Different cell types exhibited additional specific defects. Lenses overlaying each ommatidium (`L' in Fig. 6D and Fig. 7A,B) appeared correctly formed, but the pseudocones just below them (`P' in Fig. 6D and Fig. 7A,B) were essentially absent after dVps16A knockdown (Fig. 6E and Fig. 7B). Pseudocones contain a transparent fibrous material that is secreted by cone cells (Cagan and Ready, 1989). After dVps16A knockdown, the remnants of the pseudocones were surrounded by a dense structure (arrow in Fig. 6E) that EM revealed to be composed of vesicles and vacuolar structures containing internal vesicles (Fig. 7B). Furthermore, the smaller type I pigment granules normally found in primary pigment cells (PPC in Fig. 7A) as well as the larger type II pigment granules characteristic for secondary pigment cells (SPC) were absent (Fig. 7B,G). The accumulation of autophagosomes in pigment cells paralleled their expansion from the typical width of 1-2 µm to often more than 5 µm (Fig. 7G).
In plastic sections, photoreceptor cells are easily identified based on their darkly stained rhabdomeres (arrowheads in Fig. 6G-I). Upon dVps16A knockdown, however, most photoreceptor cells contained only remnants of rhabdomeres (e.g. arrowhead in Fig. 6H and arrows in Fig. 7D). The loss of rhabdomeres was accompanied by the accumulation of autophagosomes and vacuoles. Together the different ultrastructural phenotypes support a requirement for dVps16A in the trafficking to lysosomes and pigment granules.
dVps16A is required for lysosomal delivery
Because the retinal degeneration detected in the adult compound eyes might be an indirect consequence of defects in endocytosis or secretion we looked earlier in development for defects in either pathway. For this purpose, we analyzed the trafficking of two ligands, Boss and Scabrous, in the developing eye disc. Boss is targeted to the apical surface of R8 cells and, upon binding to the sevenless receptor, internalized into R7 cells (Fig. 7A) (Krämer et al., 1991). The pattern of Boss expression on the R8 cell surface is diagnostic for several developmental decisions in eye development (e.g. Van Vactor et al., 1991
). Vps16A knockdown did not change this pattern indicating that R8 cell specification was not affected.
The level of Boss protein detectable in R7 cells is sensitive to mutations that perturb endocytic trafficking (Chang et al., 2002; Sevrioukov et al., 1999
; Sunio et al., 1999
). After dVps16A knockdown using the da-Gal4 driver, the level of Boss expressed on the R8 cell surface was not significantly altered, indicating no change in secretion. By contrast, the level of Boss protein in R7 cells was increased (Fig. 8A,B), consistent with an inhibition of lysosomal delivery.
|
We also tested the effect of dVps16A knockdown on lysosomal trafficking using a GFP-LAMP fusion protein. In Drosophila cells, the LAMP1-derived cytoplasmic tail is sufficient to target this fusion protein from the Golgi to late endosomes and lysosomes, where hydrolases degrade GFP (data not shown) (Rohrer et al., 1996). Thus, GFP-LAMP did not accumulate to high levels in wild-type eye discs (Fig. 8E). By contrast, massive accumulation of GFP-LAMP in dVps16A knockdown flies was observed. Quantification revealed a more than 12-fold increase in average GFP-LAMP fluorescence after dVps16A knockdown (12.4x107±5.5; n=5) compared with wild-type controls (1.0x107±0.4; n=3). Because the number of GFP-positive vesicles was at least threefold increased this is likely to reflect the accumulation of endosomal intermediates due to the inhibited fusion with lysosomes and earlier intermediates in the pathway. This demonstrated that delivery to lysosomes is inhibited for Golgi-derived biosynthetic cargo, along with the internalized ligands described above.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Most phenotypes after dVps16A knockdown were similar to those observed for dor mutations, which is consistent with the loss of Dor protein after dVps16A knockdown (Fig. 5). Both cause deficits in lysosomal delivery as well as eye-color defects due the loss of pigment granules. Additionally, we observed a loss of pseudocones, whose gelatinous content is secreted by cone cells starting 110 hours after pupariation (Cagan and Ready, 1989
). Although it is possible that the loss of pseudocones indicates a primary role of dVps16A in secretion, it is likely that this secretory defect is a secondary consequence of the massive changes we notice in cells after dVps16A knockdown (Fig. 7). For example, the lenses overlaying each ommatidium are secreted by cone cells and primary pigment cells about 1 day before the pseudocones (
70 hours after pupariation) (Cagan and Ready, 1989
). At this stage, secretion is normal as indicated by the fully formed lenses in dVps16A knockdown flies. Furthermore, 3 days earlier in development, delivery of endocytosed ligands and Golgi-derived biosynthetic cargo to lysosomes is inhibited in eye discs, yet secretion of Boss to the R8 cell surface is unchanged (Fig. 8), indicating that the primary defect is in endosomal trafficking. Finally, indirect effects on secretion have also been observed in loss-of-function mutations in VACUOLELESS1, which encodes an Arabidopsis homolog of Vps16 (Rojo et al., 2001
).
Another phenotype of VACUOLELESS1 mutant cells that we also observed in dVps16A knockdown flies is the accumulation of autophagosomes. Autophagy is a process in which cytosol and organelles are engulfed by double-membrane bounded vacuoles. These vacuoles fuse with lysosomes, which exposes their content to lysosomal hydrolases for degradation (Klionsky, 2005). In Drosophila, as in other organisms, autophagy is induced by starvation (Scott et al., 2004
) and is observed during developmentally induced cell death (Baehrecke, 2003
; Rusten et al., 2004
). The accumulation of autophagosomes after dVps16A knockdown indicates that the dVps16A containing complex is required for the fusion of autophagosomes to lysosomes, similar to the function of the Vps-C complex in yeast (Klionsky, 2005
; Rieder and Emr, 1997
).
Because dVps16A binds tightly and specifically to Car (Figs 2 and 3), it is interesting to compare the dVps16A phenotypes with those of car and its mammalian ortholog buff, which causes some HPS-like phenotypes in mice (Suzuki et al., 2003). Similar to the eye-color defect observed in Vps16A knockdown and car mutant flies, the buff mutation causes strong pigmentation defects in the skin and the eye. Knockdown of dVps16A also causes defects in lysosomal delivery. Similar defects in HPS-related mutations are indicated by the deficiency to secrete lysosomal enzymes into urine (Li et al., 2004
). However, little or no secretion deficiency was observed in buff mice (Suzuki et al., 2003
). In Drosophila, car1 exhibits only a slightly reduced rate of degradation for internalized cargo (Sriram et al., 2003
). Some of the mild phenotypes observed for car and buff are likely due to the fact that both mutations are single amino acid changes. Complete loss-of-function mutations will be necessary to define their functions more precisely.
In addition to dVps16A, we have described a second Vps16 homolog, encoded by CG18112. We have called this protein dVps16B based on its similarity to Vps16 proteins and its binding to the second Vps33 homolog in flies, dVps33B. Homologs of dVps16B are encoded by other insect and vertebrate genomes (Fig. 2). The presence of two Vps16 homologs appears to parallel the presence of two Vps33 homologs in metazoans consistent with the formation of two distinct Vps-C complexes. No mutation has yet been identified in dVps16B or dVps33B, but an indication for their function comes from the phenotypes observed in humans with mutations in VPS33B. These patients suffer from arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome (Gissen et al., 2004). The hallmark of this syndrome is occurrence of muscle atrophy that limits the motion of joints, in conjunction with renal dysfunction and liver defects that prevent bile secretion (Eastham et al., 2001
). Cellular phenotypes in the liver and kidney of ARC patients suggest an underlying deficiency in the targeting of proteins to the apical cell surface: proteins normally restricted to the apical domain are distributed over the entire cell surface of hepatocytes and kidney cells (Gissen et al., 2004
). Because endosomes play a key role in the maintenance of apical-basal polarity (Nelson and Rodriguez-Boulan, 2004
) these phenotypes are consistent with a role of Vps33B proteins in endosomes.
Although the distinct phenotypes suggest non-overlapping function between the two Vps33 homologs in mammals, an interaction with other VpsC proteins was suggested by overexpression of human Vps33B, which resulted in the clustering of lysosomes (Gissen et al., 2004). This phenotype mimics closely the consequences of overexpression of mammalian homologs of Vps18p (Poupon et al., 2003
). Similar lysosome clustering has also been observed upon expression of human Vam6p (Caplan et al., 2001
). In yeast, Vam6/Vps39 is part of the HOPS complex (Seals et al., 2000
; Wurmser et al., 2000
). A connection between the Vps33B/Vps16B complex and other subunits of the Vps-C complex is also supported by our detection of a physical interaction between dVps16B and Dor (Fig. 4D). To define to which extend the two Vps-C complexes have distinct and overlapping functions in different membrane fusion events the consequences of complete loss-of-function mutations need to be analyzed in a single organism.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adams, M. D., Celniker, S. E., Holt, R. A., Evans, C. A., Gocayne, J. D., Amanatides, P. G., Scherer, S. E., Li, P. W., Hoskins, R. A., Galle, R. F. et al. (2000). The genome sequence of Drosophila melanogaster. Science 287, 2185-2195.
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. and Struhl, K. (1994). Current Protocols in Molecular Biology. New York, NY, USA: John Wiley & Sons.
Baehrecke, E. H. (2003). Autophagic programmed cell death in Drosophila. Cell Death Differ. 10, 940-945.[CrossRef][Medline]
Banta, L. M., Vida, T. A., Herman, P. K. and Emr, S. D. (1990). Characterization of yeast Vps33p, a protein required for vacuolar protein sorting and vacuole biogenesis. Mol. Cell. Biol. 10, 4638-4649.[Medline]
Beadle, G. and Ephrussi, B. (1936). The differentiation of eye pigments in Drosophila as studied by transplantation. Genetics 21, 225-247.
Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401-415.
Bunch, T. A., Grinblat, Y. and Goldstein, L. S. (1988). Characterization and use of the Drosophila metallothionein promoter in cultured Drosophila melanogaster cells. Nucleic Acids Res. 16, 1043-1061.[Abstract]
Cagan, R. L. and Ready, D. F. (1989). The emergence of order in the Drosophila pupal retina. Dev. Biol. 136, 346-362.[CrossRef][Medline]
Caplan, S., Hartnell, L. M., Aguilar, R. C., Naslavsky, N. and Bonifacino, J. S. (2001). Human Vam6p promotes lysosome clustering and fusion in vivo. J. Cell Biol. 154, 109-122.
Carr, C. M., Grote, E., Munson, M., Hughson, F. M. and Novick, P. J. (1999). Sec1p binds to SNARE complexes and concentrates at sites of secretion. J. Cell Biol. 146, 333-344.
Casso, D., Ramirez-Weber, F. and Kornberg, T. B. (2000). GFP-tagged balancer chromosomes for Drosophila melanogaster. Mech. Dev. 91, 451-454.[CrossRef][Medline]
Chang, H. C., Newmyer, S. L., Hull, M. J., Ebersold, M., Schmid, S. L. and Mellman, I. (2002). Hsc70 is required for endocytosis and clathrin function in Drosophila. J. Cell Biol. 159, 477-487.
Ciciotte, S. L., Gwynn, B., Moriyama, K., Huizing, M., Gahl, W. A., Bonifacino, J. S. and Peters, L. L. (2003). Cappuccino, a mouse model of Hermansky-Pudlak syndrome, encodes a novel protein that is part of the pallidin-muted complex (BLOC-1). Blood 101, 4402-4407.
Dell'Angelica, E. C., Shotelersuk, V., Aguilar, R. C., Gahl, W. A. and Bonifacino, J. S. (1999). Altered trafficking of lysosomal proteins in Hermansky-Pudlak syndrome due to mutations in the beta 3A subunit of the AP-3 adaptor. Mol. Cell 3, 11-21.[CrossRef][Medline]
Dulubova, I., Sugita, S., Hill, S., Hosaka, M., Fernandez, I., Sudhof, T. C. and Rizo, J. (1999). A conformational switch in syntaxin during exocytosis: role of munc18. EMBO J. 18, 4372-4382.
Dulubova, I., Yamaguchi, T., Wang, Y., Sudhof, T. C. and Rizo, J. (2001). Vam3p structure reveals conserved and divergent properties of syntaxins. Nat. Struct. Biol. 8, 258-264.[CrossRef][Medline]
Eastham, K. M., McKiernan, P. J., Milford, D. V., Ramani, P., Wyllie, J., van't Hoff, W., Lynch, S. A. and Morris, A. A. (2001). ARC syndrome: an expanding range of phenotypes. Arch. Dis. Child. 85, 415-420.
Gahl, W. A., Brantly, M., Kaiser-Kupfer, M. I., Iwata, F., Hazelwood, S., Shotelersuk, V., Duffy, L. F., Kuehl, E. M., Troendle, J. and Bernardini, I. (1998). Genetic defects and clinical characteristics of patients with a form of oculocutaneous albinism (Hermansky-Pudlak syndrome). New Engl. J. Med. 338, 1258-1264.
Giot, L., Bader, J. S., Brouwer, C., Chaudhuri, A., Kuang, B., Li, Y., Hao, Y. L., Ooi, C. E., Godwin, B., Vitols, E. et al. (2003). A protein interaction map of Drosophila melanogaster. Science 302, 1727-1736.
Gissen, P., Johnson, C. A., Morgan, N. V., Stapelbroek, J. M., Forshew, T., Cooper, W. N., McKiernan, P. J., Klomp, L. W., Morris, A. A., Wraith, J. E. et al. (2004). Mutations in VPS33B, encoding a regulator of SNARE-dependent membrane fusion, cause arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome. Nat. Genet. 36, 400-404.[CrossRef][Medline]
Gwynn, B., Martina, J. A., Bonifacino, J. S., Sviderskaya, E. V., Lamoreux, M. L., Bennett, D. C., Moriyama, K., Huizing, M., Helip-Wooley, A., Gahl, W. A. et al. (2004). Reduced pigmentation (rp), a mouse model of Hermansky-Pudlak syndrome, encodes a novel component of the BLOC-1 complex. Blood 104, 3181-3189.
Harlow, E. and Lane, D. (1988). Antibodies: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
Hay, B. A., Wolff, T. and Rubin, G. M. (1994). Expression of baculovirus P35 prevents cell death in Drosophila. Development 120, 2121-2129.
Hazelett, D. J., Bourouis, M., Walldorf, U. and Treisman, J. E. (1998). decapentaplegic and wingless are regulated by eyes absent and eyegone and interact to direct the pattern of retinal differentiation in the eye disc. Development 125, 3741-3751.
Horazdovsky, B. F. and Emr, S. D. (1993). The VPS16 gene product associates with a sedimentable protein complex and is essential for vacuolar protein sorting in yeast. J. Biol. Chem. 268, 4953-4962.
Huizing, M. and Gahl, W. A. (2002). Disorders of vesicles of lysosomal lineage: the Hermansky-Pudlak syndromes. Curr. Mol. Med. 2, 451-467.[CrossRef][Medline]
Huizing, M., Didier, A., Walenta, J., Anikster, Y., Gahl, W. A. and Krämer, H. (2001). Molecular cloning and characterization of human VPS18, VPS 11, VPS16, and VPS33. Gene 264, 241-247.[CrossRef][Medline]
Jahn, R., Lang, T. and Sudhof, T. C. (2003). Membrane fusion. Cell 112, 519-533.[CrossRef][Medline]
Kim, B. Y., Krämer, H., Yamamoto, A., Kominami, E., Kohsaka, S. and Akazawa, C. (2001). Molecular characterization of mammalian homologues of class C Vps proteins that interact with syntaxin-7. J. Biol. Chem. 276, 29393-29402.
Klionsky, D. J. (2005). The molecular machinery of autophagy: unanswered questions. J. Cell Sci. 118, 7-18.
Krämer, H. and Phistry, M. (1996). Mutations in the Drosophila hook gene inhibit endocytosis of the boss transmembrane ligand into multivesicular bodies. J. Cell Biol. 133, 1205-1215.[Abstract]
Krämer, H., Cagan, R. L. and Zipursky, S. L. (1991). Interaction of bride of sevenless membrane-bound ligand and the sevenless tyrosine-kinase receptor. Nature 352, 207-212.[CrossRef][Medline]
Lee, E. C., Hu, X., Yu, S. Y. and Baker, N. E. (1996). The scabrous gene encodes a secreted glycoprotein dimer and regulates proneural development in Drosophila eyes. Mol. Cell. Biol. 16, 1179-1188.[Abstract]
Lee, Y. S. and Carthew, R. W. (2003). Making a better RNAi vector for Drosophila: use of intron spacers. Methods 30, 322-329.[CrossRef][Medline]
Li, W., Zhang, Q., Oiso, N., Novak, E. K., Gautam, R., O'Brien, E. P., Tinsley, C. L., Blake, D. J., Spritz, R. A., Copeland, N. G. et al. (2003a). Hermansky-Pudlak syndrome type 7 (HPS-7) results from mutant dysbindin, a member of the biogenesis of lysosome-related organelles complex 1 (BLOC-1). Nat. Genet. 35, 84-89.[CrossRef][Medline]
Li, W., Rusiniak, M. E., Chintala, S., Gautam, R., Novak, E. K. and Swank, R. T. (2004). Murine Hermansky-Pudlak syndrome genes: regulators of lysosome-related organelles. BioEssays 26, 616-628.[CrossRef][Medline]
Li, Y., Fetchko, M., Lai, Z. C. and Baker, N. E. (2003b). Scabrous and Gp150 are endosomal proteins that regulate Notch activity. Development 130, 2819-2827.
Marchler-Bauer, A., Anderson, J. B., Cherukuri, P. F., DeWeese-Scott, C., Geer, L. Y., Gwadz, M., He, S., Hurwitz, D. I., Jackson, J. D., Ke, Z. et al. (2005). CDD: a conserved domain database for protein classification. Nucleic Acids Res. 30, D192-D196.
Martina, J. A., Moriyama, K. and Bonifacino, J. S. (2003). BLOC-3, a protein complex containing the Hermansky-Pudlak syndrome gene products HPS1 and HPS4. J. Biol. Chem. 278, 29376-29384.
Misura, K. M., Scheller, R. H. and Weis, W. I. (2000). Three-dimensional structure of the neuronal-Sec1-syntaxin 1a complex. Nature 404, 355-362.[CrossRef][Medline]
Nazarian, R., Falcon-Perez, J. M. and Dell'Angelica, E. C. (2003). Biogenesis of lysosome-related organelles complex 3 (BLOC-3): a complex containing the Hermansky-Pudlak syndrome (HPS) proteins HPS1 and HPS4. Proc. Natl. Acad. Sci. USA 100, 8770-8775.
Nelson, W. J. and Rodriguez-Boulan, E. (2004). Unravelling protein sorting. Nat. Cell Biol. 6, 282-284.[CrossRef][Medline]
Nguyen, T. and Wei, M. L. (2004). Characterization of melanosomes in murine Hermansky-Pudlak syndrome: mechanisms of hypopigmentation. J. Invest. Dermatol. 122, 452-460.[CrossRef][Medline]
Nguyen, T., Novak, E. K., Kermani, M., Fluhr, J., Peters, L. L., Swank, R. T. and Wei, M. L. (2002). Melanosome morphologies in murine models of hermansky-pudlak syndrome reflect blocks in organelle development. J. Invest. Dermatol. 119, 1156-1164.[CrossRef][Medline]
Nickla, H. (1977). Maternal effects determine effective lethal phase of carnation-light synthetic lethal in Drosophila melanogaster. Nature 268, 638-639.[CrossRef][Medline]
Peterson, M. R. and Emr, S. D. (2001). The class C Vps complex functions at multiple stages of the vacuolar transport pathway. Traffic 2, 476-486.[CrossRef][Medline]
Poupon, V., Stewart, A., Gray, S. R., Piper, R. C. and Luzio, J. P. (2003). The role of HmVps18p in clustering, fusion, and intracellular localization of late endocytic organelles. Mol. Biol. Cell 14, 4015-4027.
Price, A., Seals, D., Wickner, W. and Ungermann, C. P. (2000). The docking stage of yeast vacuole fusion requires the transfer of proteins from a cis-SNARE complex to a Rab/Ypt protein. J. Cell Biol. 148, 1231-1238.
Raymond, C. K., Howald-Stevenson, I., Vater, C. A. and Stevens, T. H. (1992). Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E VPS mutants. Mol. Biol. Cell 3, 1389-1402.[Abstract]
Richardson, S. C., Winistorfer, S. C., Poupon, V., Luzio, J. P. and Piper, R. C. (2004). Mammalian late vacuole protein sorting orthologues participate in early endosomal fusion and interact with the cytoskeleton. Mol. Biol. Cell 15, 1197-1210.
Rieder, S. E. and Emr, S. D. (1997). A novel RING finger protein complex essential for a late step in protein transport to the yeast vacuole. Mol. Biol. Cell 8, 2307-2327.
Robinson, J. S., Klionsky, D. J., Banta, L. M. and Emr, S. D. (1988). Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases. Mol. Cell. Biol. 8, 4936-4948.[Medline]
Rohrer, J., Schweizer, A., Russell, D. and Kornfeld, S. (1996). The targeting of Lamp1 to lysosomes is dependent on the spacing of its cytoplasmic tail tyrosine sorting motif relative to the membrane. J. Cell Biol. 132, 565-576.[Abstract]
Rojo, E., Gillmor, C. S., Kovaleva, V., Somerville, C. R. and Raikhel, N. V. (2001). VACUOLELESS1 is an essential gene required for vacuole formation and morphogenesis in Arabidopsis. Dev. Cell 1, 303-310.[CrossRef][Medline]
Rubin, G. M. and Spradling, A. C. (1982). Genetic transformation of Drosophila with transposable element vectors. Science 218, 348-353.[Medline]
Rubin, G. M., Hong, L., Brokstein, P., Evans-Holm, M., Frise, E., Stapleton, M. and Harvey, D. A. (2000). A Drosophila complementary DNA resource. Science 287, 2222-2224.
Rusten, T. E., Lindmo, K., Juhasz, G., Sass, M., Seglen, P. O., Brech, A. and Stenmark, H. (2004). Programmed autophagy in the Drosophila fat body is induced by ecdysone through regulation of the PI3K pathway. Dev. Cell 7, 179-192.[CrossRef][Medline]
Sato, T. K., Rehling, P., Peterson, M. R. and Emr, S. D. (2000). Class C Vps protein complex regulates vacuolar SNARE pairing and is required for vesicle docking/fusion. Mol. Cell 6, 661-671.[CrossRef][Medline]
Scott, R. C., Schuldiner, O. and Neufeld, T. P. (2004). Role and regulation of starvation-induced autophagy in the Drosophila fat body. Dev. Cell 7, 167-178.[CrossRef][Medline]
Seals, D. F., Eitzen, G., Margolis, N., Wickner, W. T. and Price, A. (2000). A Ypt/Rab effector complex containing the Sec1 homolog Vps33p is required for homotypic vacuole fusion. Proc. Natl. Acad. Sci. USA 97, 9402-9407.
Sevrioukov, E. A., He, J. P., Moghrabi, N., Sunio, A. and Krämer, H. (1999). A role for the deep orange and carnation eye color genes in lysosomal delivery in Drosophila. Mol. Cell 4, 479-486.[CrossRef][Medline]
Shestopal, S. A., Makunin, I. V., Belyaeva, E. S., Ashburner, M. and Zhimulev, I. F. (1997). Molecular characterization of the deep orange (dor) gene of Drosophila melanogaster. Mol. Gen. Genet. 253, 642-648.[CrossRef][Medline]
Spritz, R. A. and Oh, J. (1999). HPS gene mutations in Hermansky-Pudlak syndrome. Am. J. Hum. Genet. 64, 658.[CrossRef][Medline]
Sriram, V., Krishnan, K. S. and Mayor, S. (2003). deep-orange and carnation define distinct stages in late endosomal biogenesis in Drosophila melanogaster. J. Cell Biol. 161, 593-607.
Srivastava, A., Woolford, C. A. and Jones, E. W. (2000). Pep3p/Pep5p complex: a putative docking factor at multiple steps of vesicular transport to the vacuole of Saccharomyces cerevisiae. Genetics 156, 105-122.
Starcevic, M. and Dell'Angelica, E. C. (2004). Identification of snapin and three novel proteins (BLOS1, BLOS2 and BLOS3/reduced pigmentation) as subunits of biogenesis of lysosome-related organelles complex-1 (BLOC-1). J. Biol. Chem. 279, 28393-28401.
Stinchcombe, J., Bossi, G. and Griffiths, G. M. (2004). Linking albinism and immunity: the secrets of secretory lysosomes. Science 305, 55-59.
Subramanian, S., Woolford, C. A. and Jones, E. W. (2004). The Sec1/Munc18 protein, Vps33p, functions at the endosome and the vacuole of Saccharomyces cerevisiae. Mol. Biol. Cell 15, 2593-2605.
Sunio, A., Metcalf, A. B. and Krämer, H. (1999). Genetic dissection of endocytic trafficking in Drosophila using a horseradish peroxidase-bride of sevenless chimera: hook is required for normal maturation of multivesicular endosomes. Mol. Biol. Cell 10, 847-859.
Suzuki, T., Oiso, N., Gautam, R., Novak, E. K., Panthier, J. J., Suprabha, P. G., Vida, T., Swank, R. T. and Spritz, R. A. (2003). The mouse organellar biogenesis mutant buff results from a mutation in Vps33a, a homologue of yeast vps33 and Drosophila carnation. Proc. Natl. Acad. Sci. USA 100, 1146-1150.
Toonen, R. F. and Verhage, M. (2003). Vesicle trafficking: pleasure and pain from SM genes. Trends Cell Biol. 13, 177-186.[CrossRef][Medline]
Van Vactor, D. L., Jr, Cagan, R. L., Krämer, H. and Zipursky, S. L. (1991). Induction in the developing compound eye of Drosophila: multiple mechanisms restrict R7 induction to a single retinal precursor cell. Cell 67, 1145-1155.[CrossRef][Medline]
Walenta, J. H., Didier, A. J., Liu, X. and Krämer, H. (2001). The Golgi-associated hook3 protein is a member of a novel family of microtubule-binding proteins. J. Cell Biol. 152, 923-934.
Warner, T. S., Sinclair, D. A., Fitzpatrick, K. A., Singh, M., Devlin, R. H. and Honda, B. M. (1998). The light gene of Drosophila melanogaster encodes a homologue of VPS41, a yeast gene involved in cellular-protein trafficking. Genome 41, 236-243.[CrossRef][Medline]
White, J. G. (1982). Membrane defects in inherited disorders of platelet function. Am. J. Pediatr. Hematol. Oncol. 4, 83-94.[Medline]
Wodarz, A., Hinz, U., Engelbert, M. and Knust, E. (1995). Expression of crumbs confers apical character on plasma membrane domains of ectodermal epithelia of Drosophila. Cell 82, 67-76.[CrossRef][Medline]
Wu, M. N., Littleton, J. T., Bhat, M. A., Prokop, A. and Bellen, H. J. (1998). ROP, the Drosophila Sec1 homolog, interacts with syntaxin and regulates neurotransmitter release in a dosage-dependent manner. EMBO J. 17, 127-139.
Wurmser, A. E., Sato, T. K. and Emr, S. D. (2000). New component of the vacuolar class C-Vps complex couples nucleotide exchange on the Ypt7 GTPase to SNARE-dependent docking and fusion. J. Cell Biol. 151, 551-562.
|