Endosomal degradation is severely impaired in primary hemocytes from larvae of eye color mutants of Drosophila. Using high resolution imaging and immunofluorescence microscopy in these cells, products of eye color genes, deep-orange (dor) and carnation (car), are localized to large multivesicular Rab7-positive late endosomes containing Golgi-derived enzymes. These structures mature into small sized Dor-negative, Car-positive structures, which subsequently fuse to form tubular lysosomes. Defective endosomal degradation in mutant alleles of dor results from a failure of Golgi-derived vesicles to fuse with morphologically arrested Rab7-positive large sized endosomes, which are, however, normally acidified and mature with wild-type kinetics. This locates the site of Dor function to fusion of Golgi-derived vesicles with the large Rab7-positive endocytic compartments. In contrast, endosomal degradation is not considerably affected in car1 mutant; fusion of Golgi-derived vesicles and maturation of large sized endosomes is normal. However, removal of Dor from small sized Car-positive endosomes is slowed, and subsequent fusion with tubular lysosomes is abolished. Overexpression of Dor in car1 mutant aggravates this defect, implicating Car in the removal of Dor from endosomes. This suggests that, in addition to an independent role in fusion with tubular lysosomes, the Sec1p homologue, Car, regulates Dor function.

Introduction

Late endosomes and lysosomes are main degradative compartments within the endosomal system in metazoans, identified by their characteristic multivesicular morphology and absence of recycling receptors (Storrie and Desjardins, 1996; Luzio et al., 2000; Mullins and Bonifacino, 2001). Late endosomes are extremely dynamic organelles undergoing large scale morphological transformations (Gruenberg and Maxfield, 1995). They are capable of fusion with maturing early endosomes or carrier vesicles (Gruenberg et al., 1989), homotypic fusion to affect multivesicularization (Antonin et al., 2000), and finally heterotypic fusion to transfer contents to lysosomes (Luzio et al., 2000). In addition to endocytosed contents from early endosomes, multivesicular late endosomes also fuse with vesicles derived from the trans-Golgi network (Griffiths et al., 1988; Glickman and Kornfeld, 1993; Mullins and Bonifacino, 2001; Piper and Luzio, 2001).

This system of organelles is critical for a variety of processes in metazoans: cessation of mitogenic signaling, turnover of normal cellular proteins, disposal of abnormal proteins, antigen processing, and release of endocytosed nutrients in endolysosomes (Mullins and Bonifacino, 2001). Important clues to understanding mechanisms of development have come from analyses of animals carrying mutations in genes proposed to be involved in lysosomal delivery and degradation. Lysosomal function decides the range of action of morphogens during development by modulating the duration of intracellular signaling of ligand-bound signaling receptors (Babst et al., 2000; Dubois et al., 2001).

Genetic analysis has provided an understanding of the role of molecular players involved in secretory traffic and vacuole biogenesis in Saccharomyces cerevisiae (Horazdovsky et al., 1995; Wendland et al., 1998). Analyses of vacuolar protein sorting has led to the identification of over 50 genes involved in vacuolar biogenesis in yeast (Bryant and Stevens, 1998). These genes, referred to as vacuolar protein sorting (vps)* or vam genes (Wickner, 2002) are subdivided into six classes (A–F) based on their vacuolar phenotypes (Banta et al., 1988; Robinson et al., 1988). Development of assays for vesicle fusion in vitro has also provided insight into the biochemical functions of these genes (Wickner, 2002). Since the yeast vacuole is proposed to be functionally analogous to lysosomes in metazoans (Lloyd et al., 1998; Odorizzi et al., 1998; Dell'Angelica et al., 2000), homologues of these genes are likely to be used in constructing endolysosomes in metazoans.

To analyze the role of genes involved in endolysosomal biogenesis in a metazoan, we have been able to reproducibly derive primary cultures of hemocytes from Drosophila melanogaster. We have shown (Guha et al., 2003) that larval hemocytes from animals mutant at the shibire locus exhibit a temperature-sensitive, reversible inhibition of receptor-mediated endocytosis, faithfully reproducing temperature-sensitive paralysis phenotype observed in shi flies (Krishnan et al., 1996).

Here we have examined the perturbation of late endosomal trafficking and degradation in two mutants, deep-orange (dor) and carnation (car). These genes are part of the “granule group” of eye color genes in Drosophila (Lloyd et al., 1998; Odorizzi et al., 1998; Spritz, 1999), which reduce both red and brown eye pigments and encode fly homologues of yeast genes, VPS18 and VPS33, respectively (Mullins and Bonifacino, 2001). 85 mutations affecting eye color have been isolated, and a subset is proposed to be involved in pigment granule biogenesis, an organelle whose biogenesis may resemble that of lysosomes (Lloyd et al., 1998; Dell'Angelica et al., 2000). The availability of a cell culture system provides an opportunity wherein mutations in genes involved in lysosomal biogenesis that give rise to phenotypes in the animal may be analyzed at the cellular level at high resolution.

VPS18 and VPS33 are part of class C vps genes, which also includes VPS11 and VPS16 and whose products assemble as a complex called the class C complex (Sato et al., 2000; Wurmser et al., 2000). The VPS18p homologue, Dor, associates with endosomal membranes in Drosophila cells (Sevrioukov et al., 1999). Clones of a null mutant, dor8 in the compound eye of Drosophila are pigmentation deficient, and consistent with an effect on lysosomal degradation, endocytosed HRP-Boss ligand accumulates intracellularly in aberrant multivesicular structures (Sevrioukov et al., 1999). The role of Dor in lysosomal function is further suggested by the lack of degradation of overexpressed Wingless-HRP in dor8 embryos (Dubois et al., 2001). However, the reason for this defect in degradation in terms of the pathways perturbed in a metazoan system is yet unknown. The VPS33p homologue, Car, associates with Dor in vitro (Sevrioukov et al., 1999); car genetically interacts with dor (Lindsley and Zimm, 1992). These observations implicate Dor and Car in biogenesis of endolysosomes in metazoa.

To study biogenesis of endolysosomes and role(s) of Dor and Car in this process, we have followed the endocytic fate of molecules internalized by endogenously expressed anionic ligand binding receptors resembling Drosophila scavenger receptors (dSRs; unpublished data) using fluorescently labeled protein ligands for dSR together with probes for fluid phase endocytosis (e.g., fluorescently labeled dextrans) in hemocytes. We show that in Rab7-positive endosomal system, multivesicular late endosomes contain both Dor and Car and mature into small dense Dor-negative but Car-positive endosomes. These endosomes eventually fuse with tubular lysosomes. We have then addressed the role of Dor and Car in biogenesis of these different types of Rab7-positive endosomal compartments using a combination of high resolution fluorescence microscopy and EM. We provide evidence that Dor is required for fusion of Golgi-derived vesicles rich in hydrolytic enzymes with large multivesicular endocytic compartments. On the other hand, Car appears to function in removal of Dor from maturation-competent Rab7-positive late endosomes. It is also involved in fusion of small dense Dor-negative, Car-positive organelles with tubular lysosomes, providing evidence for a function independent of Dor.

Results

Morphological characterization of the endocytic pathway in larval hemocytes from wild-type animals

In a recent study (Guha et al., 2003), we have shown that fluorescently conjugated maleylated BSA (Fl-mBSA) binds specifically to endogenously expressed dSR at the cell surface of hemocytes (Fig. 1 A) and is then internalized via a dynamin (shibire)-dependent pathway into early sorting endosomes marked by the small GTPase Rab5. On the other hand, majority of the fluid phase is internalized via a shibire-independent pathway into distinct Rab5-negative endosomes. Similar to a recently described pathway in mammalian cells (Sabharanjak et al., 2002), both these populations of endosomes were not labeled by the late endosomal marker Rab7; subsequent incubation in the absence of fluorescently labeled probes for 5 min (a chase of 5 min) results in dSR ligand and fluid phase probes becoming extensively colocalized as observed in fixed cells (Fig. 1 B).

In live hemocytes, upon incubation with fluorescently labeled probes for 5 min (5-min pulse) the majority of Fl-mBSA and lissamine rhodamine–labeled dextran (LR-Dex) are colocalized in large endosomal compartments often near the center of the cell (Fig. 1 C, open arrowheads). Lumenal connectivity of Fl-mBSA and a fluid phase probe in these large (1–2 μm) endosomes at 5 min was confirmed by HRP-mediated quenching (Mayor et al., 1998) of Fl-mBSA fluorescence by diaminobenzidine (DAB) radicals generated from HRP cointernalized via the fluid phase (unpublished data). In some endosomes, dSR ligands label only the limiting membrane, whereas the fluid tracer labels the lumen of the organelle (Fig. 1 C, open arrowheads and top inset). Ultrastructure analyses by EM of endosomes labeled by a 10-min pulse of fluid phase HRP (Fig. 1 G i) revealed a multivesicular morphology; HRP labels the lumen of the compartment and is excluded from luminal vesicles (Fig. 1 G ii, arrows). These criteria establish that endosomes accessed by a 5-min pulse of the two probes are similar to late endosomes (multivesicular bodies [MVBs]) observed in mammalian cells (Piper and Luzio, 2001). To confirm the identity of the 5-min-labeled organelles as late endosomes, we used antibodies generated against mammalian Rab7 (Chavrier et al., 1990), which recognizes Drosophila Rab7 expressed in these cells (Fig. S1); mAb against lysobisphosphatidic acid (anti-LBPA, 6C4; Kobayashi et al., 1998) also labels the same endosomes (Fig. S1). In addition, endosomes accessed by a 5-min pulse of Fl-mBSA are also labeled by antiserum to Hrs and Hook proteins (Fig. S2), markers of late endosomal compartments identified previously in garland cells in Drosophila (Kramer and Phistry, 1996; Lloyd et al., 2002).

After a 5-min pulse, increasing chase times (Fig. 1, D–F) result in major morphological changes in endosomal compartments accessed by Fl-mBSA and LR-Dex, clearly distinguishing them from MVBs observed at 5 min. After a 15-min chase period, endosomes appear smaller (0.5–1.0 μm) and show complete colocalization of Fl-mBSA and fluid tracer within the endosome (Fig. 1 D, arrows and inset). EM analysis confirms the formation of small HRP-filled electron-dense structures (Fig. 1 G iii). After a 1-h chase, endosomal morphology is tubular-vesicular (Fig. 1 E), and this persists for 2 (Fig. 1 F) to 4 h (data not depicted). This tubular morphology is sensitive to osmotic stress (unpublished data), analogous to tubular lysosomes observed in mammalian bone marrow–derived macrophages labeled with the fluid tracer lucifer yellow (Knapp and Swanson, 1990) and conventional fixing protocols. Tubules are not observed by fluorescence (Fig. 3 C) and EM analyses (Fig. 1 G iv) of fixed samples. Comparison of the morphology of endosomes as a function of chase times in live cells versus those obtained in fixed cells is shown in Fig. 2.

To address the identity of the morphologically distinct compartments accessed by the endocytic probes at different chase times, we first asked whether they are marked by Rab7. The fraction of FITC-dextran (F-Dex)–containing endosomes (at different chase times after a 15-min pulse of the endocytic probe) labeled by Rab7 was quantified as described (see Materials and methods). Quantitative analysis shows that majority of F-Dex–containing endosomes at chase times described above are positive for Rab7 (Fig. 3).

Together with observations made in a separate study (Guha et al., 2003), these observations show that endocytosed probes in larval hemocytes are first delivered to a collection of Rab7-negative early endosomes followed by subsequent delivery to Rab7-positive multivesicular late endosomes. After a chase of 1 h (or 2 h), probes are visualized in Rab7-positive lysosomal network that is a tubular-vesicular system in live cells (Fig. 2). Although, these endosomal stages are persistently labeled by Rab7, they exhibit a characteristic dynamic of multiple molecular players as detailed below (see Fig. 11).

Multivesicular large sized endosomes in wild-type cells mature into fusion-inaccessible small dense organelles

We next probed the process by which endosomal cargo is trafficked between large sized endosomes and small dense endosomes formed in wild-type cells. There are mainly two ways this trafficking can happen: (1) via transformation of the large sized endosomes into the small dense endosomes (maturation process) or (2) vesicle budding from large sized endosomes, fusing with small dense endosomes (vesicle shuttle) (Fig. 4 B). These processes have distinct predictions for the mixing of endosomal contents between two temporally separated endocytic probe pulses (Fig. 4 A). Measuring the ratio of the amount of the two probes in colocalized endosomal compartments at different chase times provides a method of distinguishing between the vesicle shuttle and maturation models of endosomal trafficking between two types of organelles (Stoorvogel et al., 1991; Dunn and Maxfield, 1992).

Using F-Dex as the first pulse (Fig. 4, A, C, and D, green) and Cy3-mBSA as the second pulse (Fig. 4, A, C, and D, red), we monitored the extent of fusion accessibility of the first pulse–containing endosomes to the second pulse with increasing chase times between the two pulses (Fig. 4, A, C, and D). As a control, we ensured that Cy3-mBSA and F-Dex label the same late endocytic compartments with comparable kinetics when pulsed in together (Fig. 1). The endosomes containing first pulse are completely accessible to the second pulse at short chase times (Fig. 4 C, open arrowhead and arrow). A longer chase of first pulse–containing endosomes results in morphological transformation to small dense endosomes (Fig. 1) becoming fusion inaccessible (Fig. 4 D, arrow); the first pulse loses fusion accessibility with a half time of ∼12 min (Fig. 4 E). After a chase time of 5 min, fusion accessibility of the Rab7-positive large endosomes (Figs. 1 and 3) is corroborated by complete quenching (∼80 ± 10%) of first pulse of Cy3-mBSA fluorescence by DAB-mediated radicals generated from a second pulse of HRP. Consistent with the loss of colocalization, after a chase of 45 min Cy3-mBSA fluorescence (first pulse) is protected (only ∼40 ± 20% is quenched) from HRP second pulse.

The mere loss of fusion accessibility does not discriminate between the two models. If a vesicle shuttle mechanism is involved in traffic between large endosomes and small compartments (or if small compartments represent vesicles in transit between two stages of the endocytic pathway), with increasing chase times (Fig. 4 B) the endosomes accessible to the second pulse will gradually lose the first pulse to the next set of compartments. However, in a maturation process the endosomes become the next set of compartments, thus those endosomes accessible to the second pulse would always contain similar levels of the first pulse during the entire chase period. These processes may be distinguished by measuring the ratio of the amount of first pulse to second pulse in colocalized compartments with increasing chase times (Dunn and Maxfield, 1992).

After a 45-min chase period, although most of the endosomes labeled by the first pulse are fusion inaccessible (Fig. 4, C and E), there are a few endosomal structures where the first pulse colocalizes with the second pulse. The ratio of the amount of the first pulse in endosomes marked by the second pulse remains constant throughout the chase period (Fig. 4 F), consistent with a maturation process (Fig. 4 B). This demonstrates that Rab7-positive late endosomes of large multivesicular morphology transform into small dense organelles via a maturation process. After maturation into fusion-inaccessible endosomes, the small sized endosomes finally mix their contents in tubular lysosomes (for example, if cells treated as in Fig. 4 D are further chased for 1 h; data not depicted). Note that these endosomal-morphological transformations are not necessary for the maturation process (Fig. S3).

Endosomal degradation in hemocytes from wild-type and eye color mutant alleles of dor and car

To determine the extent of degradation of endocytosed protein ligands in the endolysosomal system in wild-type (Canton-S strain) cells, Cy3-mBSA was pulsed into cells for 5 min (Fig. 5 A) and chased for different times. Quantification of total cell (Fig. 5 B) and endosome-associated (unpublished data) fluorescence at the end of each chase time showed a dramatic reduction (∼80–85%) between 1 and 2 h. At this time, the endocytosed probes are in tubular, Rab7-positive endosomes (Figs. 13), indicating this compartment as a major site for endosomal degradation. The reduction in fluorescence is inhibited by a protease-inhibitor cocktail (Fig. 5 C), confirming that loss in fluorescence is a measure of protein degradation in these endosomal compartments.

To determine the effect of mutations in eye color genes, we measured the extent of degradation in cells from wild-type and mutant alleles of dor and car. Degradation in mutant alleles was significantly reduced compared with wild-type cells (Fig. 5 C); this reduction correlated with severity of the eye color defect (dor1 > dor4). The defect in degradation in the dor4 mutant cells is rescued by a duplication of the dor gene Dp(1:Y)1E (Ydor+; Narayanan et al., 2000); the fraction of Cy3-mBSA fluorescence remaining after a 2-h chase of a 5-min pulse with respect to the beginning of the chase in dor4/Ydor+ is 0.25 ± 0.08 compared with 0.52 ± 0.11 in the dor4 mutant cells. This is consistent with Ydor+ rescuing the eye color defect in dor4 mutant animals (unpublished data). These data provide evidence that dor is involved in endosomal degradation, consistent with a lack of down-regulation of internalized wingless protein in embryos of the null allele, dor8 (Dubois et al., 2001), and a HRP–Boss fusion protein in dor8 clone of cells in the Drosophila eye (Sevrioukov et al., 1999).

Cells from the only available allele of car, car1 showed a small but significant (P < 0.0001) impairment in degradation of Cy3-mBSA after a 2-h chase (Fig. 5 C), suggesting a role for Car in degradation. Although Car associates with Dor in the same protein complex (Sevrioukov et al., 1999), its role in endosomal function is yet to be determined. To test whether car is involved with dor in endosomal degradation, we used a double mutant of dor and car (dor1car1), which does not survive beyond the prepupal stage as reported previously (Lindsley and Zimm, 1992). The cell culture system that we developed afforded us an opportunity to analyze endosomal trafficking in this mutant combination. Cells from dor1car1 showed the most severe defect in endosomal degradation (Fig. 5 A, bottom, and C). There is a small but significant (P < 0.05) difference between the degradation defect in dor1car1 and dor1, consistent with a role for both Car and Dor in endosomal degradation.

The impairment of degradation in cells from mutant animals may be due to an acidification defect in mutant cells, causing a perturbation of lysosomal degradation. This possibility is ruled out, since the extent of endosomal acidification in compartments labeled by a 5-min pulse or a subsequent 2-h chase in wild-type and mutant cells are comparable (Fig. 5 D). Another possibility may be due to differences in the extent of internalization of Cy3-mBSA. However, endosomal fluorescence in hemocytes from wild-type and dor1car1 is comparable after the 5-min pulse (Fig. 5 A, compare left panels). Thus, the degradation defects observed in the mutant cells are likely to be due to alterations in biogenesis of late endosomes or lysosomes.

Mutant alleles of dor and car show blockage in morphological transformation at distinct stages of endolysosomal biogenesis but mature with normal kinetics

We next examined the morphology of endosomal compartments accessed by dSR ligands and fluid phase in live hemocytes from dor4, dor1, car1, and dor1car1. Following a similar pulse–chase protocol outlined earlier (Fig. 1), we find that the net internalization of probes was not affected in any of the mutants studied (Fig. 5). The probes are delivered to Rab7-positive (unpublished data) large endosomes in all alleles (Fig. 6). Distinct from cells from wild-type (Fig. 1 D) and car1 animals (Fig. 6 E), the large endosomes in mutant dor alleles appear blocked in progression to later stages (Fig. 6, A and B, dor1; C, dor4; H, dor1car1). This defect is completely rescued by overexpression of Dor; in cells from dor4/Ydor+ endosomal progression is similar to wild type (Fig. 6 I). Ultrastructure analysis using HRP as a fluid phase probe showed no difference in the morphology and formation of the MVB and the small dense organelle between car1 and Canton-S cells (unpublished data). In the cells from the synthetic lethal mutant dor1car1, after a 2-h chase, many endosomes showed an aberrant distribution of endocytosed probes wherein the dSR ligand is often distributed on the endosomal membrane and the fluid tracer is present in the lumen of exaggerated large sized endosomes (3–4 μm; Fig. 6 H, inset). Cells from car1 animals exhibited a block in transition of small dense vesicles to tubular-vesicular compartments; even after a chase of 2 h (Fig. 6 G compared with Fig. 1 F) or 4 h (data not depicted) only small vesicular structures are observed.

The distinct effects of mutants of dor and car on endosomal morphology and progression of endocytosed probes along the pathway described above prompted us to examine the kinetics of loss of fusion accessibility and mechanism of endosomal trafficking in the mutant cells using the protocol established above (Fig. 4). Endosomes in cells from mutant alleles of dor and dor1car1, although incapable of a morphological transition to small dense vesicles, are capable of fusion at early times and normal maturation to fusion inaccessible structures (Fig. S3). Endosomes in cells from the car1 allele also exhibit wild-type fusion characteristics and mature into fusion-inaccessible small dense structures (Fig. S3). Thus, morphological transformation of endosomes is not essential for normal maturation.

Staining of intracellular organelles with an acidophillic dye in live cells from wild-type and mutant animals is consistent with this interpretation. Lysotracker™ labels all endocytically accessible compartments in wild-type cells, whereas in addition to endocytically accessible organelles, large sized endocytically inaccessible structures are also labeled in dor1car1. Most of the endocytically inaccessible Lysotracker™-labeled structures in this mutant correspond to phase-lucent vesicles that accumulate inside cells (unpublished data). This suggests that endosomes in mutant cells mature into fusion-inaccessible acidic endosomes, incapable of lateral fusion with each other.

Deep-orange is involved in the delivery of Golgi-derived enzymes to late endosomal compartments

The effects of mutant alleles of dor characterized thus far do not provide an explanation for reduced endosomal degradation observed in cells from mutant animals. To determine whether defects in endosomal degradation observed in cells from dor alleles are due to alterations in specific trafficking steps, we monitored the delivery of Golgi-derived hydrolases to the endolysosomal system. Cysteine proteinases like cathepsin L are important constituents of the lytic system in lysosomes (Turk et al., 2001; Zwad et al., 2002) and are delivered directly from the Golgi to MVBs and lysosomes in the pro form where they undergo cleavage to yield the mature proteinase (Turk et al., 2001). To detect the delivery of Golgi cargo we have used antiserum generated against Sacrophaga peregrina pro–cathepsin L that cross-reacts with Drosophila cathepsin L–like enzyme encoded by cp1 (Tryselius and Hultmark, 1997) and monitored the time course of intersection of endocytosed F-Dex with this immunoreactivity. At the earliest time monitored (5 min), a fraction (∼20%) of large sized F-Dex–containing endosomes is labeled by antiserum against pro–cathepsin L in cells from wild-type (Fig. 7 A, open arrowhead) and car1 animals (unpublished data). This suggests that at least some of Golgi-derived pro–cathepsin L is delivered to the 5 min, Rab7-positive large sized endosomes. At this time, endosomes in cells from dor alleles and dor1car1 cells do not show any detectable pro–cathepsin L staining (unpublished data). When a 15-min pulse of F-Dex is chased for longer times in cells from wild-type and car1 animals an increasing fraction of F-Dex–containing endosomes shows staining for pro–cathepsin L as seen qualitatively in Fig. 7, B and C, and quantitatively in Fig. 7 F. However, in none of the dor alleles, is significant pro–cathepsin L staining detected even at late times (Fig. 7, D–F; dor4, unpublished data). This defect is completely rescued in cells overexpressing Dor (dor4/Ydor+; Fig. 7 F and Fig. S4).

Unlike in wild-type and car1 cells, wherein pro–cathepsin L is localized to endocytically accessible small and large organelles (Fig. 7, A–C), in all dor alleles this protein is present in altogether novel, large ring-like organelles, inaccessible to endosomal probes (Fig. 7, D and E, bold arrows). To check whether these structures represent an altered Golgi from which exit of pro–cathepsin L is blocked, we examined the distribution of a bonafide Golgi marker, lava lamp (Sisson et al., 2000). In all cells, Golgi distribution and morphology, as outlined by lava lamp, is unperturbed (Fig. S5) and distinct from the ring-like appearance of pro–cathepsin L structures in the dor mutants (Fig. 7, D and E). Furthermore, there is no obvious defect in secretion since dSR-binding capacity at the surface of both mutant and wild-type cells is unaltered (unpublished data). This suggests that both Golgi morphology and secretory function are unperturbed in eye color mutants. The ring-like pro–cathepsin L–containing organelles are likely to represent Golgi-derived vesicles, accumulating in dor mutants due to an inhibition of fusion of Golgi vesicles with the late endosomal system. Together, these data suggest that the primary reason for the endosomal degradation defect in mutant alleles of dor and in dor1car1 is the inability of Golgi-derived (pro–cathepsin L–containing) vesicles to fuse with endocytic compartments, a defect not seen in car1 cells.

Eye color mutants affect the removal of Dor and Car from Rab7-positive endosomes

To obtain a mechanistic understanding of Dor and Car in biogenesis of endolysosomes, we examined the localization of Dor and Car on different Rab7-positive endosomal compartments in wild-type cells. F-Dex was pulsed and chased into morphologically distinct stages of the endosomal system as before (Fig. 2), and Dor and Car immunoreactivity associated with corresponding compartments was analyzed. Dor is associated with compartments labeled by a 15-min pulse of F-Dex (Fig. 8 A, inset). However, endosomal compartments accessed by a 15-min pulse followed by a 1-h chase are devoid of Dor immunoreactivity (Fig. 8 B, arrowheads). This is similar to observations made in garland cells wherein dextran beads are first seen in Dor-positive compartments and subsequently in Dor-negative compartments (Sevrioukov et al., 1999). Quantitative analysis shows that immediately after a 15-min pulse of F-Dex, 90% of all F-Dex–containing endosomes are α-Dor positive, whereas almost all endosomes accessed after a 1-h chase are devoid of detectable Dor immunoreactivity (Fig. 8 F). In a more detailed temporal analysis, after a pulse of 5 min wherein a majority of endosomes are large sized and Dor positive, a chase of 45-min results in <20% of F-Dex–containing endosomes retaining Dor immunoreactivity on exclusively small sized endosomes observed at this time (Fig. 8 F, inset).

Quantitatively similar to Dor, Car is also associated with all 15-min F-Dex–labeled compartments (Fig. 8, C and F). However, in contrast to Dor, Car is detected on almost all (80%) endosomal compartments after a 1-h chase (Fig. 8, D and F, arrowhead); Car immunoreactivity is lost only after a chase period of 2 h (Fig. 8, E and F), wherein all the endocytosed probe is present in tubular lysosomes (Fig. 2). Together, these data indicate that Dor and Car are associated with the Rab7-positive large MVBs (Fig. S6) and small dense endosomes; the small dense endosomes rapidly lose Dor but retain Car reactivity until these structures merge with the tubular lysosomal system. This persistent presence of Car on endosomes that do not have detectable Dor suggests that Car may act independently of Dor in the fusion of small sized endosomes with tubular lysosomes.

In cells from dor mutants (including dor1car1), levels of Dor immunostaining are lower than those observed in wild-type hemocytes (unpublished data; Sevrioukov et al., 1999). However, Dor (Fig. 9, A–C and G) and Car (Fig. 9 H) immunoreactivity are persistent on the Rab7-positive large sized endosomes compared with wild-type cells. This defect is completely rescued in cells from dor4/Ydor+ animals (Fig. 9 F). These results strongly suggest that mutations in dor prevent normal release of Dor and Car proteins from endosomal membranes.

Car regulates Dor association with endosomal membranes and morphological progression of large to small sized endosomes

Endosomes in car1 cells show a wild-type loss of Car immunostaining (Fig. 9 H, gray bars) but are slowed down in the loss of Dor immunoreactivity (Fig. 9, D and E, gray bars, and G), suggesting a function for Car in the removal of Dor from endosomal membranes. To test this role of Car, we examined the kinetics of removal of Dor in cells from animals (car1/Ydor+) that overexpress Dor. Mere overexpression of Dor (in cells from +/Ydor+ animals carrying a duplication of dor) shows normal endosomal Dor dissociation kinetics (Fig. 10 C) and endosomal morphological progression (unpublished data). However, overexpression of Dor in cells with mutant Car (car1) results in a prolonged association (persistent even at 2 h) of Dor on endosomal compartments (Fig. 10 A compared with Fig. 9 E); endosomal morphological transition to small sized compartments is also blocked (Fig. 10 B compared with Fig. 6 G). Furthermore, these endosomes become fusion inaccessible at 30 min, indicating that they mature with wild-type kinetics (unpublished data). These results provide evidence that Car regulates removal of Dor from endosomal membranes; car1 mutation impairs this ability. Arrest of endosomes at the stage of large sized compartments suggests that Car and Dor are required for the morphological transition from large Rab7-positive to small sized endosomes. Endosomal degradation is unchanged with respect to cells from car1 animals (unpublished data) consistent with normal delivery of pro–cathepsin L (Fig. 10 D). Thus, blockage of morphological progression of the endosomes in cells from car1/Ydor+ animals is uncoupled from an inhibition of Golgi vesicle delivery (Fig. 10 D) and endosomal degradation.

Discussion

Here we report the characterization of the endolysosomal system in primary cultures of Drosophila hemocytes using endogenous markers (Fig. 11, schematic). The ability to visualize endosomal trafficking intermediates using quantitative fluorescence microscopy in living cells from mutant animals provides mechanistic insight into late endosomal maturation. Trafficking between late endosomes and lysosome involves three intermediates with distinct molecular identities (Fig. 11; 5 min, L; 15 min, S; and 45 min, S).

Hemocytes from all mutants of the eye color gene, dor, show defects in degradation of the dSR ligand because of the lack of delivery of Golgi-derived hydrolytic enzymes to Rab7-positive MVBs. As observed by immunofluorescence microscopy, aberrant large ring-like structures containing pro–cathepsin L accumulate in cells from mutant dor alleles, inaccessible to endocytosed probes. These structures are likely to represent vesicles involved in Golgi to late endosome traffic. This is similar to the vesicles containing Golgi-derived alkaline phosphatase and carboxypeptidase Y that accumulate at the restrictive temperature in a temperature-sensitive allele of yeast VPS18 (Rieder and Emr, 1997). These results provide evidence that, similar to Vps18p (Rieder and Emr, 1997), Dor functions in the fusion of Golgi-derived vesicles with the MVB endosomes in a metazoan system.

In contrast to dor, only a single mutant allele of another eye color gene, the Drosophila homologue of VPS33, car1 (Lindsley and Zimm, 1992), has been characterized. Cells from this mutant background have a small but significant endosomal degradation defect. In this respect, car1 behaves like a “weak” allele of a mutation in the class C complex genes. Distinct from dor4 cells that exhibit only a similar defect in endosomal degradation but clearly mislocalize pro–cathepsin L into aberrant structures, pro–cathepsin L is delivered normally to endocytic organelles in cells from car1. Normal endosomal delivery of pro–cathepsin L in car1 mutant suggests that there may be a redundant role for Car in this process. In conjunction with genes of the class C complex, VPS11, VPS16, and VPS18, recent studies (Webb et al., 1997; Nichols et al., 1998; Gerrard et al., 2000) have implicated VPS45 as another sec1 gene involved in Golgi to endosome traffic in S. cerevisiae. It is likely that similar to yeast, in addition to car, a VPS45 homologue may participate in this step (Littleton, 2000). Likely candidates for this gene exist at 85D-E (Lloyd et al., 2000). The reason for a degradation defect may be due to the failure of endosomal contents in car1 to be well mixed in a large stable pool of degradative enzymes normally found in tubular lysosomes of wild-type cells.

A suggestion for a role for Car in endosomal delivery of Golgi-derived cargo comes from analyses of the endocytic phenotype of cells from the synthetic lethal double mutant dor1car1. A small but significant enhancement of the dor1 endosomal degradation defect and an exaggeration of the aberration of endosomal morphology (compared with the defects observed in dor1 alone) implicate car in the same step; endosomal degradation may be a more sensitive read out of the impairment of delivery of cargo from the Golgi.

Large sized endosomes that accumulate in mutant dor alleles and dor1car1 mature with normal kinetics into fusion-inaccessible organelles but fail to lose Dor and Car. This suggests that, unlike morphological progression, persistent localization of Dor and Car does not impair maturation. Both the mutations in the dor alleles map to the COOH-terminal cysteine-rich RING domain of the protein; dor4 has a frame-shift mutation, which adds 30 new amino acids, whereas dor1 has a similar lesion as the VPS18tsf allele in terms of a mutation in a RING finger cysteine (Rieder and Emr, 1997). The inability of dor mutants to lose Dor immunoreactivity from endosomes may be a consequence of the specific molecular lesion in the RING domain in mutant alleles of dor. This is likely to affect the localization dynamics of Car. Car remains persistently localized on the membrane (Fig. 9 H), consistent with a possible physical interaction between Dor and Car (Sevrioukov et al., 1999).

These experiments clearly indicate sequential use of Dor (possibly with other players of the class C complex), first in forming large sized endosomes (fusion between Rab7-positive endosomes and Golgi-derived vesicles) and later in progression to small sized Dor-negative structures. Removal of Dor is implicated in this progression; Dor is retained on the Rab7-positive large endosomes in all dor mutants. At the same time, in wild-type and car1 cells morphological progression of the large sized to small sized endosomes precedes the loss of Dor from the small sized endosomes. This may be explained by a mechanism where Dor function is necessary for this progression and is then followed by inactivation of the membrane-associated Dor leading to its subsequent removal.

Our results suggest that Car is responsible for the inactivation of Dor. This is because in car1 cells there is delayed endosomal dissociation of Dor from the small sized endosomes, a phenotype that is greatly enhanced by overexpression of Dor (car1/Ydor+ cells; Fig. 10). This overexpression also results in blockage of the morphological transition of the Rab7-positive endosomes (but not its maturation). These observations together suggest that Dor must undergo a Car-dependent modulation necessary for morphological progression to the small sized endosomes, before its removal from the membrane.

Our results implicate Car in a step that is likely to be independent of Dor. First, in wild-type cells, although Dor and Car associate with Rab7-positive MVBs, only Car remains associated with small dense Rab7-positive organelles before fusion with the tubular-vesicular compartments. Second, in the car1 mutant, Dor-negative and Car-positive small dense endosomal structures formed by maturation of large MVBs that fail to fuse with tubular lysosomes accumulate. This shows that Car remains functionally associated with the endosomal system in the absence of Dor. Consistent with this hypothesis, although Car has been shown to physically interact with Dor, not all membrane-bound Car is in a complex with Dor (Sevrioukov et al., 1999). Furthermore, in yeast Vps33p is obtained in a membrane-associated complex with Vps16p in a deletion strain of VPS18 (Sato et al., 2000).

Data presented here strongly suggest that although Dor may function similar to VPS18 in its role in Golgi to endosome traffic, the Sec1p homologue Car may be involved in the Dor-dependent and -independent steps, potentially modulating interaction with endosome stage-specific SNAREs. One of the two point mutations in car1 (Val to Gly at position 249) is located in the region conserved in all Vps33 family members (Sevrioukov et al., 1999), implicating this region in the regulation of SNARE function. Analyses of the endosomal phenotype of syntaxin 7 mutant fly cells will be necessary to address this question. Finally, the role of Rab7 in the context of this late endosomal pathway needs to be elucidated, since different from yeast, Dor and Car dissect the Rab7-positive endosomal compartments into subsets based on their fusion with either Golgi-derived compartments or with the lysosome.

In conclusion, these studies provide evidence for interplay of Dor and Car in ordering the sequence of endosomal biogenesis. Functional analyses of genes involved in this pathway in a metazoan cell is extremely relevant because these and other studies suggest that the endolysosomal system in higher eukaryotes is likely to be more complex than that present in yeast; some genes involved in cellular functions are present only in higher eukaryotes (Dell'Angelica et al., 2000).

Materials And Methods

Materials

All chemicals and media reagents were obtained from Sigma-Aldrich or GIBCO BRL, respectively, unless otherwise specified. Fluorescent dyes for conjugation and LysotrackerTM Red DND-99 were obtained from Molecular Probes. F-Dex and LR-Dex were made as described previously (Sabharanjak et al., 2002). Cy3-mBSA and A488-mBSA were prepared as described (Guha et al., 2003). Secondary antibodies (Jackson ImmunoResearch Laboratories) were conjugated to fluorophores as recommended by the manufacturer.

Fly stocks

All Drosophila stocks obtained from the Bloomington Stock Center were grown at 20°C in cornmeal agar bottles. dor1car1 was provided by Mani Ramaswami (University of Arizona, Tucson, AZ). Squash-GAL4 was provided by Dan Kiehart (Duke University, Durham, NC).

Cell culture

Cells from larvae were obtained as described previously (Braun et al., 1998). Briefly, third instar larvae were surface sterilized, and hemolymph was collected by puncturing the integument using dissection forceps into 150 μl of complete medium (CM; Schneider's insect medium supplemented with 10% nonheat inactivated FBS, 1 μg/ml bovine pancreatic insulin, 150 μg/ml penicillin, 250 μg/ml streptomycin, 750 μg/ml glutamine) in 35-mm coverslip-bottom dishes (Mayor et al., 1998). Labeling incubations were performed on adherent hemocytes ∼2 h postdissection.

Cell labeling

Adherent hemocytes were washed (three times with medium 1 [Mayor et al., 1998]) before addition of endocytic probes. Fl-mBSA and dextran were used at 800 ng/ml and 1 mg/ml, respectively, in labeling medium (LM; Schneider's insect medium supplemented with 1.5 mg/ml BSA). Hemocytes were incubated with the endocytic probes for the indicated times (pulse period) and washed extensively with medium 1 before further incubation of cells for different intervals of time (chase periods) in CM. Fluorescently labeled mBSA probes were completely competed by incubation of cells with unlabeled mBSA (0.8 mg/ml). Cells were imaged live in imaging medium (IM; medium 1 supplemented with 1 mg/ml BSA and 2 mg/ml D-glucose), or fixed in 2.5% PFA in medium 1 for 20 min before imaging.

Immunofluorescence microscopy

Labeled and fixed cells were permeabilized using 0.4% Igepal for 13 min and incubated in blocking solution (BS; medium 1 with 2 mg/ml BSA), before incubation with primary antiserum. The primary antisera were obtained and used at the indicated dilutions in BS as described in Table I. Cells were incubated with labeled secondary antibodies diluted in BS for 45 min.

Endosomal degradation assays

Cells were incubated with 800 ng/ml Cy3-mBSA for 5 min, washed with medium 1, fixed either immediately or after chase times in CM, and imaged on a wide field microscope. Endosomal degradation was inhibited by using a protease inhibitor cocktail (Set III; Calbiochem). Total fluorescence of cells was determined by marking out a cell outline from the bright field image, and the fluorescence per cell was obtained using MetamorphTM software (Universal Imaging Corp.). Integrated values of cell fluorescence were corrected for background autofluorescence. Extent of degradation was estimated by normalizing the amount of cell-associated fluorescence remaining at the indicated chase time to the amount internalized in 5 min. An experiment consisted of two dishes in which >20 cells per dish were quantified.

Ratiometric pH estimation

To measure endosomal acidification, we used the pH sensitivity of F-Dex (Ohkuma and Poole, 1978). Cells were incubated with F-Dex for 5 min and either imaged immediately or after a 2-h chase. Two images were collected for each field, one before and the other after endosomal pH was neutralized using 10 μM nigericin (Sabharanjak et al., 2002). FITC fluorescence associated with the endosomes per cell before and after addition of nigericin was quantified using MetamorphTM software (Universal Imaging Corp.) and corrected for photobleaching during consecutive exposures. The extent of photobleaching was determined by exposing cells for exactly the same time without any treatment. Ratio of FITC fluorescence associated with the endosomes before addition of nigericin to that after its addition is a measure of the extent of endosomal acidification. Each data point was obtained from >10 cells per allele.

Imaging and image processing

Confocal and wide field imaging was performed exactly as described (Sabharanjak et al., 2002). All images were processed for output purposes using Adobe Photoshop® software.

Quantitative analyses of colocalization

Quantification of colocalization for measuring maturation kinetics was performed as described (Dunn and Maxfield, 1992; Sabharanjak et al., 2002). All processing including determination of colocalization was performed using similar parameters regardless of the type of endocytic tracer used. Colocalization index was calculated as the ratio of the colocalized intensity to the total intensity of the probe in endosomes in each cell. Maximum extent of colocalization obtained by this method is 94% for the colocalization of cointernalized F-Dex and Cy3-mBSA in the same cell. Ratios of fluorophore intensities in individual endosomes were obtained by determining the individual fluorophore intensities in endosome that exhibited colocalization. For the quantification of endocytic probe-containing endosomes colocalized with immunodetected proteins, the total number of endosomes labeled by the endocytic tracer was identified and counted manually using MetamorphTM software (Universal Imaging Corp.). Endosomes positive for the immunodetected protein were recorded and expressed as the percentage of the total number of endosomes. Each experiment consisted of two dishes with >15 cells per dish.

HRP quenching assay

To ascertain lumenal connectivity (Mayor et al., 1998) of optically colocalized Cy3-mBSA and F-Dex in endosomes, HRP was cointernalized as a fluid phase probe along with F-Dex at 1 mg/ml in the presence of 0.5 mg/ml mannan. To prevent the internalization of HRP by mannose receptor expressed on hemocytes, cells were also preincubated with Mannan. To estimate the extent of fusion of two discrete pulses of endocytosed probes, hemocytes were incubated with Cy3-mBSA for 5 min in LM and chased for either 5 or 45 min in CM before a second pulse of 5 min of F-Dex and HRP. HRP-mediated quenching was performed at 4°C for 45 min (Mayor et al., 1998). Fluorescence of Cy3-mBSA and F-Dex was quantified using the MetamorphTM software (Universal Imaging Corp.). The extent of HRP-mediated quenching was expressed as the percentage of Cy3-mBSA fluorescence that was quenched by exposure to H2O2. Efficacy of HRP quenching was independently confirmed by ensuring that cointernalized F-Dex fluorescence was completely quenched for each condition.

EM

Hemocytes incubated with 1.5 mg/ml HRP in LM were fixed using 2.5% PFA for 3 min, and HRP enzymatic activity was developed with DAB and 0.003% H2O2 diluted in medium 1 at 4°C for 45 min. The cells were washed and postfixed using a mixture of 1.5% gluteraldehyde and 2.5% PFA for 1 h, treated with osmium tetroxide, dehydrated, and embedded in araldite (TAAB). Sections (50–150 nm) were viewed using a Jeol CXII 100 transmission electron microscope. Images were captured on photographic emulsion and scanned at 1,200 dpi for output purposes.

Online supplemental material

Figs. S1–S6 are available. Fig. S1 provides information regarding the specificity of the Rab7 antisera used and the nature of the compartments containg this marker. Fig. S2 shows that Hrs, Deep-orange, Carnation, and Hook are colocalized in the late endolysosomal system in larval hemocytes. Fig. S3 shows that mutant alleles of dor and car do not affect the maturation kinetics of large sized endosomes. Fig. S4 shows that the block in endosomal delivery of Golgi-derived hydrolase is completely rescued in hemocytes from dor4/Ydor+. Fig. S5 provides evidence that Golgi morphology is not affected in hemocytes from the eye color mutants. Fig. S6 shows that both Deep-orange and Carnation label GFP-Rab7–positive late endosomes accessed by 5-min pulse of Fl-mBSA.

Acknowledgments

We thank H. Kramer (University of Texas Southwestern Medical Center, Dallas, TX), S. Natori (RIKEN, Japan), M. Zerial (Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany), and J.C. Sisson and W. Sullivan (University of California, Santa Cruz, CA) for antibodies, M. Ramaswami and R. Narayanan (University of Arizona, Tucson, AZ), M. Gonzalez-Gaitan (Max Planck Institute of Molecular Cell Biology and Genetics), and Dan Kiehart (Duke University, Durham, NC) for fly strains, and C. Sagar and Y. Ramamohan (National Institute of Mental Health and Sciences, Bangalore, India) for EM, and members of the Mayor laboratory for critically reading the manuscript. S. Mayor thanks K. Belur and F.F. Bosphorus for inspiration.

This work was supported by a grant from the Department of Biotechnology, India, and from intramural funds from the National Centre for Biological Sciences. V. Sriram is supported by a Kanwal Rekhi fellowship from the Tata Institute of Fundamental Research endowment fund and Appam. S. Mayor is a Senior Research fellow of the Wellcome Trust.

The online version of this article includes supplemental material.

*

Abbreviations used in this paper: car, carnation; DAB, diaminobenzidine; dor, deep-orange; dSR, Drosophila scavenger receptor; F-Dex, FITC-dextran; Fl-mBSA, fluorescently conjugated mBSA; LR-Dex, lissamine rhodamine–labeled dextran; mBSA, maleylated BSA; MVB, multivesicular body; vps, vacuolar protein sorting; Ydor+, Dp(Y:1)1E.

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Supplementary data