1 Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USA
2 Department of Pediatrics, Pulmonary Section, Baylor College of Medicine, Houston, TX 77030, USA
3 Klinik und Poliklinik für Kinderheilkunde, Westfälische Wilhelms-Universität, Albert-Schweitzer-Str. 33, D-48149 Münster, Germany
4 Department of Pharmacological and Pharmaceutical Sciences, University of Houston, College of Pharmacy, Houston, TX 77204, USA
*Author for correspondence (e-mail: bknoll{at}uh.edu)
Accepted September 13, 2001
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SUMMARY |
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Key words: Rab5a, Lysosome, Endosome, ß-hexosaminidase, ß2-adrenergic receptor
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INTRODUCTION |
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Considerable effort has been given to understanding the biogenesis of lysosomes in mammalian cells. Proteins regarded as residents of lysosomes can be directed there from the trans-Golgi network in at least two general ways (Hunziker and Geuze, 1996). Lumenal hydrolases bind mannose 6-phosphate receptors (M6PRs) in the trans-Golgi apparatus and are shuttled to the late endosome compartment for eventual transport to lysosomes (Brown et al., 1986; Duncan and Kornfeld, 1988). Alternatively, lysosome membrane proteins may first transit to the cell surface, then internalize into early endosomes and subsequently traffic to lysosomes (Lippincott-Schwartz and Fambrough, 1986). This second itinerary may also involve trafficking of proteins from the TGN to early endosomes (Ludwig et al., 1991; Nielsen et al., 2000; Press et al., 1998), then subsequent recycling to the plasma membrane (Hunziker and Geuze, 1996). Thus, while many proteins are enriched in lysosomes, this usually is a dynamic steady-state condition resulting from complex traffic amongst several cellular compartments.
Endocytic trafficking events are in part governed by ras-related GTPases of the rab family. Rab proteins are 23-25 kDa in mass and tightly bound to membranes via C-terminal geranylgeranyl modifications (Novick and Zerial, 1997). Each rab isoform appears to be associated with a specific subcellular compartment, and some rabs are known to regulate traffic in the endosome-lysosome system. Transport to late endosomes from the trans-Golgi network of lumenal lysosome proteins liganded to M6PRs is regulated by rab9 (Lombardi et al., 1993). Early events in endocytosis are regulated by rab5a (Bucci et al., 1992), while trafficking from sorting endosomes to lysosomes is governed by rab7 (Feng et al., 1995; Mukhopadhyay et al., 1997; Press et al., 1998; Vitelli et al., 1997). The delivery of transferrin receptors from sorting endosomes to perinuclear recycling endosomes appears to be regulated by both rab11 and rab5a (Ren et al., 1998; Ullrich et al., 1996; Wilcke et al., 2000).
Rab5a is a key regulator of endocytosis because it is rate limiting for homotypic endosome fusion (Bucci et al., 1992; Gorvel et al., 1991). Rab5a mutants defective in guanine-nucleotide binding (rab5a S34N or rab5a N133I) are dominant negative in action, and when expressed in transfected cells, inhibit endosome fusion and cause the accumulation of small vesicles at the cell periphery (Bucci et al., 1992). By contrast, the GTPase defective mutant rab5aQ79L is dominant active and causes the formation of greatly enlarged endosomes due to a stimulation of endosome fusion (Stenmark et al., 1994). Overexpression of rab5a wild-type (wt) or rab5aQ79L increases the steady-state accumulation of fluid phase endocytic markers (Li and Stahl, 1993); however, the rate of receptor endocytosis itself may be unaltered (Ceresa et al., 2001; Seachrist et al., 2000). Various lines of evidence suggest that while rab5a-GTP is necessary for endosome fusion, the GTPase activity of rab5a is not (Barbieri et al., 1994; Hoffenberg et al., 1995a; Rybin et al., 1996; Stenmark et al., 1994). It has been proposed that the function of the GTPase activity is to maintain a dynamic equilibrium between rab5a-GDP and rab5a-GTP (Rybin et al., 1996) that can be regulated by GTPase activators (Lanzetti et al., 2000) and proteins that stimulate guanine-nucleotide exchange (Hoffenberg et al., 2000; Horiuchi et al., 1997). Evidence from studies of rab5a (McBride et al., 1999), rab1 (Allan et al., 2000) and the yeast rab-like protein Ypt7 (Ungermann et al., 2000) suggest a functional role for these small GTPases in the recruitment of SNARE proteins to membranes to facilitate budding or fusion reactions.
There is less information available about how rab5a activity influences the development of lysosomes. Because some fractions of lysosomal proteins reach their destinations via endosomes, experimental changes in rab5a activity might be expected to alter the morphology or function of lysosomes. Expression of the dominant-negative rab5aS34N mutant decreases the rate of endocytosis and degradation of epidermal growth factor receptors (Barbieri et al., 2000; Papini et al., 1997); however, it is unclear whether endosome to lysosome transport is affected. In addition, degradation of low-density lipoprotein (LDL) is greatly reduced by the expression of rab5aS34N, possibly due to a defect in LDL-receptor endocytosis (Vitelli et al., 1997). More direct evidence for a role of rab5a in lysosome function comes from studies of macrophage cells, where the rate of phagosome maturation is reduced by antisense inhibition of rab5a activity, while maturation is accelerated during overexpression of wild-type rab5a (Alvarez-Dominguez and Stahl, 1999). Interestingly, expression of a dominant active rab5a in MDCK cells caused the formation of enlarged rab7-positive vesicles (DArrigo et al., 1997), suggesting a role for rab5a downstream of early endosomes.
To further explore the role of rab5a in mammalian lysosome biogenesis, we expressed rab5a as a fusion with enhanced green fluorescent protein (EGFP) using an inducible expression system in cultured cells, and looked for changes in the distribution of lysosome proteins and several endocytic tracers. Surprisingly, modest expression of the GTPase-deficient EGFP-rab5aQ79L caused an extensive redistribution of lysosome proteins into large vesicles that also contain EGFP-rab5aQ79L, without an appreciable effect on the endocytosis or recycling of a cell surface receptor. These findings support the idea that rab5a GTPase activity plays a role in the formation of lysosomes.
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MATERIALS AND METHODS |
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DNA constructs
A rab5a-EGFP fusion was created by subcloning a rab5a wt cDNA fragment (Hoffenberg et al., 1995b) into pEGFP-C1 (Clontech, Palo Alto, CA). pEGFP-rab5a wt was mutagenized using the QuikChangeTM site-directed mutagenesis kit (Stratagene, La Jolla, CA) with the primer 5'-GGATACAGCTGGCCTAGAACGATACCATAG-3' to create the rab5aQ79L mutant and the primer 5'-GTCCGCTGTTGGTAAAAATAGCCTAGTGCTTC-3' to create the rab5aS34N mutant. These EGFP-rab5a fusions were then subcloned into the vector pIND-Hygro (Invitrogen) for inducible expression in EcR293 cells, and into pcDNA3.1 (Invitrogen) for transient transfections.
Inducible expression of EGFP-rab5a
The EcR293 line was derived from HEK293 and expresses the regulatory protein VgRXR, a chimeric steroid receptor that is activated by synthetic ecdysteriods such as ponasterone (No et al., 1996). The vector pIND-Hygro has a promoter element responsive to VgRXR, cloning sites for insertion of open reading frames and a hygromycin B resistance gene. pIND-Hygro/EGFP-rab5a wt, pIND-Hygro/EGFP-rab5aQ79L and pIND-Hygro/EGFP-rab5aS34N were transfected into EcR293 cells using FuGENE6, and clones resistant to hygromycin B (200 µg/ml) were screened for ponasterone-inducible fluorescence. For the induction of expression, cells were treated for up to 96 hours with 5 µM ponasterone, while control cells were treated with vehicle alone (0.125% ethanol). The fraction of cells expressing EGFP-rab5a in the presence of ponasterone was 90-95%.
Immunoblotting
Transfected cells were washed with PBS and then quickly dissolved in Laemmli sample buffer (Laemmli, 1970). Samples of 20 µg total protein were electrophoresed through SDS-PAGE and transferred to Immobilon-P membranes (Millipore, Bedford, MA). The membranes were probed with anti-rab5 monoclonal antibodies at a dilution of 1:500 and bound antibodies were detected by chemiluminescence (Pierce Chemical Co., Rockford, IL). Protein bands visualized on X-ray films were quantified by densitometry using SigmaGel 1.0 (SPSS Science, Chicago, IL).
Uptake of endocytic tracers
For labeling with transferrin Texas Red, the cells were washed and incubated in serum-free medium for 30 minutes, pulsed with 20 µg/ml of the tracer for 5 minutes, then rapidly chilled and fixed. For pulse-chase analysis of fluid phase uptakes, dextran Texas Red (1 mg/ml) was added directly to the medium for 1 hour, then the cells were washed and incubated in complete medium for an additional 6 hours before washing and fixation. To assess lysosome to endosome traffic, cells were incubated with dextran Texas Red for 1 hour, then washed and chased for 6 hours, and finally incubated with ponasterone for 72 hours prior to fixation. For LDL uptakes, the cells were cultured in the presence of ponasterone or vehicle for 72 hours in medium containing 10% NuSerum (Life Technologies) in place of fetal bovine serum, then Di-LDL was added to 5 µg/ml for 5 minutes. The labeled medium was removed, the cells washed and then incubated in complete medium for a further 1 hour prior to fixation.
Immunofluorescence microscopy
Cells growing on glass coverslips in 6-well clusters were washed in PBS with 1.2% sucrose (PBSS), fixed with 4% paraformaldehyde in PBSS at 4°C for 10 minutes, and then washed again with PBSS. The following steps were done at room temperature, with PBSS used for washes. The coverslips were incubated in 0.34% L-lysine, 0.05% Na-m-periodate for 20 minutes and permeabilized with 0.2% Triton X-100. The cells were then blocked with 10% normal goat serum (NGS) for 15 minutes. Primary antibodies, diluted in PBSS with 0.2% NGS and 0.05% Triton X-100, were added to the cells and left for 1 hour. The coverslips were washed four times before incubation with secondary antibodies using the same procedure as for the primary antibodies. We used the following concentrations of antibodies: anti-cathepsin D, 20 µg/ml; anti-LAMP-1, 5 µg/ml; anti-LAMP-2, 5 µg/ml; anti-CI-M6PR, 1:200; secondary antibodies, 1:100 dilution. The coverslips were mounted in Mowiol and viewed using a DeltaVision deconvolution microscopy system (Applied Precision Inc., Issaquah, WA) in the Baylor College of Medicine Integrated Microscopy Core. Optical sections of 150 nm (10-15 in number) were obtained and deconvolved, and then five sections through the cell center were combined to produce the images shown.
Immunoelectron microscopy
Sectioning and labeling of ultrathin frozen sections (50 nm) of EGFP-rab5aQ79L expressing cells using the technique of Tokuyasu were performed as described in detail elsewhere (Zimmer et al., 1998). Small specimens were cryoprotected by polyvinylpyrrolidone/sucrose, frozen in liquid nitrogen and sectioned with a cryoultramicrotome (LEICA EM Ultracut R FCS) at 100 to 110°C. Thawed sections were incubated at room temperature with the monoclonal antibody against GFP (Scompany, dilution of 1:10) for 45 minutes and goat anti-mouse IgG conjugated with 6 nm gold (Dianova, D-Hamburg, dilution of 1:10). Double-labeling was performed by incubating the sections with a polyclonal antibody against LAMP-2 (a gift of M. Fukuda, San Diego, dilution of 1:50) and goat anti-mouse IgG conjugated with 12 nm gold (Dianova, dilution of 1:50). After labeling, the grids were contrasted, embedded in 2% methylcellulose and examined in a Philips 400 electron microscope (Kassel, Germany).
Measurement of ß2AR endocytosis and recycling kinetics
Cells growing in 24-well clusters were treated with 5 µM ponasterone or vehicle alone (0.125% ethanol) for 72 hours. Isoproterenol (10 µM) was added to the cells in triplicate wells for varying times up to 20 minutes, then the wells were aspirated and washed with ice-cold serum-free medium containing 20 mM Hepes pH 7.4 (DMEM-H). Cell surface receptors were quantified by incubation at 4°C for 90 minutes with 6 nM [3H]CGP12177, a hydrophilic radioligand that selectively binds surface ß2ARs (Staehelin and Hertel, 1992). The cells were washed twice with cold DMEM-H, then lysed in the wells with 0.1% SDS, 0.1% NP-40 and the lysates counted by scintillation spectroscopy. Nonspecific binding was determined by incubations with 3 µM propranol, and was always less than 5%. The fraction of receptors left on the cell surface was plotted versus time of agonist exposure, and the curves fitted by nonlinear regression using the program GraphPad Prism (v. 3). The rate of approach to a steady-state level of surface and internal receptors is determined by the first-order rate constants for receptor endocytosis (ke) and recycling (kr). Unique values for these rate constants were estimated by curve-fitting to equation 4 in Morrison et al. (Morrison et al., 1996).
Percoll density gradients
Stably transfected cells inducibly expressing EGFP-rab5aQ79L were grown in the presence of 5 µM ponasterone or vehicle alone for 72 hours. The cells were washed with PBS and then suspended in 1x SHE, which contained 0.25 M sucrose, 10 mM Hepes, pH 7.42, 2 mM EDTA, 1x Complete® proteinase inhibitor (Roche Biomolecular). The cell suspension was homogenized by repeated passage through a 25G needle, and then centrifuged at 500 g to obtain a post-nuclear supernatant. This was overlayed onto a solution of 25% Percoll in 1x SHE with 25 mM ATP and centrifuged at 36,000 g for 65 minutes in a 70.1 Ti rotor at 4°C. Immediately after collection of fractions, 25 µl aliquots were assayed for ß-hexosaminidase activity by incubation with 0.3 mM methylumbelliferone in acetate buffer (100 mM sodium acetate and 0.1% Triton X-100) at 37°C for 1 hour in the dark. Trichloroacetic acid was added to a final concentration of 10% to stop the reactions. Samples were diluted 1:20 with 0.5 M glycine and 0.5 M sodium carbonate buffer, added in triplicate to white U-bottom 96-well trays, then read at an excitation of 365 nm and emission of 450 nm on a Dynatech Fluorolite 100.
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RESULTS |
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Endocytic tracers accumulate in EGFP rab5aQ79L containing endosomes
The proteins so far examined traffic to lysosomes by various pathways that are believed to originate at the trans-Golgi network. To determine specifically whether traffic between the plasma membrane and lysosomes is affected by expression of EGFP-rab5aQ79L, cells were labeled with fluorescent endocytic tracers that are known to traffic from endosomes to lysosomes, then subjected to imaging. Dextran Texas Red was fed to cells for 1 hour, then washed and chased for 6 hours prior to fixation and imaging. In cells expressing EGFP-rab5a wt, dextran Texas Red was chased from endosomes into punctate vesicles that showed no overlap with EGFP-rab5a wt (Fig. 6A). By contrast, a considerable proportion of dextran Texas Red remained within the lumen of enlarged EGFP-rab5aQ79L containing vesicles after chase (Fig. 6B). Similar results were obtained using BSA Texas Red (not shown). To determine whether trapping of endocytic tracer within endosomes by expression of EGFP-rab5aQ79L was limited to fluid-phase proteins, DiI-LDL was added to cells for 5 minutes, then removed and chased for 1 hour. In cells expressing EGFP-rab5a wt, internalized label sorted from endosomes into discrete, punctate vesicles (Fig. 6C). However, in cells expressing EGFP-rab5aQ79L, the majority of Di-LDL remained within endosomes after a 1 hour chase (Fig. 6D).
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DISCUSSION |
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The remarkable redistribution of lysosome proteins to EGFP-rab5aQ79L-containing vesicles is reminiscent of what occurs when cells are treated with chloroquine to block endosome-lysosome transport (Lippincott-Schwartz and Fambrough, 1987). This similarity suggests that EGFP-rab5aQ79L inhibits such transport events, causing the accumulation within endosomes of lysosome proteins that normally traverse the endosome compartment before reaching lysosomes. This interpretation would be consistent with findings that expression of rab5aQ79L inhibits transport (of transferrin) from early endosomes to perinuclear recycling endosomes (Ullrich et al., 1996), inhibits degradation of LDL and epidermal growth factor (McCaffrey et al., 2001) and inhibits to some degree the degradation of ricin in MDCK cells (DArrigo et al., 1997). In that study, abnormalities in late endosomes/lysosomes was further suggested by the observation of enlarged rab7-positive vesicles in cells expressing myc-tagged rab5aQ79L, and a partial colocalization of these two proteins. We observed a similar morphology in HEK293 cells expressing GFP-rab7 and myc-tagged rab5aQ79L (data not shown). However, the inhibition of transport from endosomes to lysosomes per se may not be sufficient to explain our findings. Expression of a dominant-negative syntaxin-7 blocks traffic from early to late endosomes in NIH3T3 cells, yet does not appear to cause the accumulation of LAMP-2 or cathepsin D in early endosomes (Nakamura et al., 2000). Also, while our data suggest that the expression of EGFP-rab5aQ79L may inhibit trafficking from early endosomes to lysosomes, there also appears to be a stimulation of traffic in the opposite direction (Fig. 8). However, our results do not allow us to distinguish between a stimulation of direct fusion between lysosome and endosomes versus a stimulation of vesicular trafficking from lysosome to endosomes.
Recent evidence suggests that rab7 also plays an important role in lysosome biogenesis. Expression of GTPase-defective rab7 in HeLa cells increases the size of lysosomes and the extent of perinuclear aggregation, while expression of dominant negative rab7 causes dispersion of lysosomes throughout the cytoplasm. These aberrant organelles are not accessible to endocytic tracers; however, there is no apparent change in the early or late endosome compartments (Bucci et al., 2000). In BHK-21 cells, dominant negative rab7 expression causes an increase in the proportion of CI-MPR and cathepsin D in early endosome compartments. By contrast, lgp 120, a homologue of LAMP-1, shows a normal distribution under these conditions (Press et al., 1998). These results suggest that, in BHK-21 cells, CI-M6PR/cathepsin D is delivered to lysosomes through the early endosome compartment, whereas lgp 120 is delivered via late endosomes. The localization of LAMP-1 and LAMP-2 with EGFP-rab5aQ79L observed in our study suggests that, in contrast with BHK-21 cells, these lysosomal proteins traffic to lysosomes via early endosomes in HEK293 cells. Our results are consistent with what was found in rat hepatocytes, where significant fractions of both LAMP-1 and LAMP-2 are sorted to the plasma membrane and early endosomes before eventual transport to lysosomes (Akasaki et al., 1995).
In our experimental system, we do not detect significant changes in ß2AR recycling during EGFP-rab5aQ79L expression (Fig. 7), suggesting a relatively specific effect on trafficking of proteins between endosomes and lysosomes. Previous studies where rab5aQ79L caused changes in transferrin recycling and endocytosis employed acute 5-10-fold overexpression mediated by recombinant vaccinia viruses over a 4-5-hour period after infection (Stenmark et al., 1994), rather than the approximately threefold expression relative to endogenous rab5a after 48-72 hours reported here. Stenmarks study also used HeLa or BHK-21 cells, so that differences in cell type could be significant. Further, subsequent studies of rab5aQ79L overexpression using recombinant adenoviruses failed to detect changes in the rates of transferrin recycling or endocytosis (Ceresa et al., 2001). Our findings suggest that alteration of intracellular sorting events may be a more significant consequence of inactivating the rab5a GTPase.
The accumulation of cathepsin D within enlarged rab5a-containing endosomes was recently observed in pyramidal neurons from patients with sporadic Alzheimers disease (Cataldo et al., 1997). These abnormal endosomes appeared to be positive for rabaptin 5 and EEA-1, proteins that bind rab5a-GTP specifically, suggesting enhanced rab5a activation (Cataldo et al., 2000). Since these changes occurred in preclinical stages of Alzheimers disease, inappropriate activation of rab5a may be proposed as an important factor in the pathogenesis of this disease. The enrichment of proteases within early endosomes could conceivably facilitate the proteolytic processing of amyloid precursor protein, and the secretion of amyloid fragments, perhaps by a form of recycling, could be especially rapid from this peripheral compartment.
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ACKNOWLEDGMENTS |
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