(Cell, Vol. 64, 915-925,

March

8, 1991, Copyright

rab5 Controls

0 1991 by Cell Press

Early Endosome

Jean-Pierre Gorvel, Philippe Chavrier, Marino Zerial, and Jean Gruenberg European Molecular,Biology Laboratory Postfach 10.2209 D-6900 Heidelberg f’ederal Republic of Germany

Summary The small GTP-binding protein rab5 was previously localized on early endosomes and on the cytoplasmic face of the plasma membrane. Using a cell-free assay, we have now tested whether rab5 is involved in controlling an early endocytic fusion event. Fusion could be inhibited by cytosol containing the overexpressed mutant rab511e’33, which does not bind GTP on blots, and by antibodies against rab5, but not against rab2 or rab7. In contrast, fusion was stimulated with cytosol containing overexpressed wild-type rab5. Cytosols containing high levels of rab2 or mutant rab5 with the 9 carboxy-terminal amino acids deleted, which bind GTP on blots, had no effects. Finally, the inhibition mediated by anti-rab5 antibodies could be overcome by complementing the assay with the cytosol containing wild-type rab5, but not with the same cytosol depleted of rab5, nor with cytosol containing the rab5 mutants or rab2. These in vitro findings strongly suggest that rab5 is involved in the process of early endosome fusion. Introduction Several lines of evidence indicate that small, Ras-like GTP-binding proteins are involved in the regulation of membrane traffic. In yeast, the genes YPT7 (Gallwitz et al., 1983) and SEC4 (Salminen and Novick, 1987) encode Ras-like GTP-binding proteins that are required for transport from ER to Golgi in yeast extracts (Segev et al., 1988; Baker et al., 1990) and for fusion of exocytic vesicles with the plasma membrane (Salminen and Novick, 1987; Goud et al., 1988), respectively. Two other GTP-binding proteins, SARl (Nakano and Muramatsu, 1989) and ARF (Sewell and Kahn, 1988; Stearns et al., 1990), have also been implicated in early steps of protein secretion in S. cerevisiae. Mammalian genes related to YPT7 and SEC4 have also been identified (Touchot et al., 1987; Haubruck et al., 1987; Bucci et al., 1988; Matsui et al., 1988; Didsbury et al., 1989; Polakis et al., 1989; Zahraoui et al., 1989; Chavrieret al., 1990a, 1990b). In addition, cell-free assays have shown that several steps of exocytic (MelanGon et all., 1987; Beckers and Balch, 1989; Tooze et al., 1990) and endocytic (Goda and Pfeffer, 1988; Mayorga et al., 1989; Bomsel et al., 1990) membrane traffic in mammalian cells are sensitive to low concentrations of GTPyS. Both the relatively large number of YPT7/SEC4-like genes and the GTPrS effects support the original proposal (Bourne,

Fusion In Vitro

1988) that each step of membrane transport is controlled by a distinct YPTlISECClike, GTP-binding protein. We have recently isolated 11 clones encoding YPTll SECClike proteins of the rab and rho subfamilies and localized the rab2, rab5, and rab7 proteins by immunoelectron microscopy using specific anti-peptide antibodies (Chavrier et al., 1990a, 1990b). rab2 was associated with an intermediate compartment between ER and Golgi and with the cis-Golgi, rab5 with early endosomes and the cytoplasmic face of the plasma membrane, and rab7 with late endosomes. Recent studies have shown that rab3a is present in synaptic vesicles (Fischer v. Mollard et al., 1990; Mizoguchi et al., 1990), while rab6 is localized in the Golgi complex (Goud et al., 1990). However, it has been difficult to study the direct involvement of any GTP-binding protein during any step of membrane traffic in mammalian cells. In the present paper, we have used a cell-free assay to investigate the role of rab5 in early endosome fusion. This fusion event has been reconstituted by several groups (Davey et al., 1985; Gruenberg and Howell, 1986, 1987; Braell, 1987; Diaz et al., 1988; Woodman and Warren, 1988). Our previous data indicate that this process is highly specific and reflects lateral interactions occurring between individual elements of the early endosomes (Gruenberg et al., 1989; Bomsel et al., 1990). Early endosome fusion is not facilitated by microtubules in vitro, in contrast to a later endocytic fusion event (Bomsel et al., 1990), and may be arrested during mitosis via the cellcycle control protein kinase cdc2 (Tuomikoski et al., 1989). We now have measured the fusion activity of early endosomal fractions containing the endogenous rab5 protein, which were separated from the plasma membrane and from late endosomes. To test the involvement of rab5 in the fusion reaction, we used specific antibodies as well as cytosols containing detectable amounts of overexpressed rab5 or mutants. Our results indicate that rab5 is required for the fusion of early endosomes in vitro and suggest that a cytosolic form of the protein can be utilized in this process.

Early and Late Endosomal Fractions We first established conditions to prepare early endosomal fractions from BHK cells, which could be used to investigate the role of rab5 with our cell-free assay. To provide a general marker of the endosomal content, we used horseradish peroxidase (HRP) endocytosed in the fluid phase. HRP enters early peripheral endosomes after 5 min of internalization at 37°C and reaches later stages of the pathway after a subsequent 30 min chase in markerfreemedium(Gruenbergetal., 1989; Griffithsetal., 1989). Cells were incubated for 5 min with HRP or for 5 min with the marker followed by a 30 min chase, then homogenized, and postnuclear supernatants (PNSs) were prepared. The PNSs were adjusted to 40.6% sucrose and loaded at the bottom of a step gradient consisting of two successive

Cdl 916

Table 1. Distribution Endoaomal Fractions

Markers

Time [min]

HRP

Avidin

of the Markers

in Early and Late Distribution

Fractions

W)

5

early endosomes late endosomes

90 + 3 10 * 3

5 + 30

early endosomes late endosomes

11 +6 89 f 6

5

early endosomes late endosomes

90 f 10 f

5 + 30

early endosomes late endosomes

523 95 2 3

5 5

Cells were always incubated either for 5 min at 37OC to label the early endosomes for 5 min followed by an additional 30 min chase in marker-free medium to label late endosomes. As markers, we used either HRP internalized from the fluid phase, to follow the endosomal content, or avidin bound to the cell surface after biotinylation of the plasma membrane. In the latter case, avidin remaining at the cell surface after internalization was quenched with biotinylated insulin, and internalized avidin was quantified by immunoprecipitation, as in the cell-free assay. After internalization of either marker, the cells were homogenized and fractionated by flotation on a D20-sucrose step gradient. Early endosomes were recovered at the 16%/10% sucrose interface, whereas late endosomes were recovered in the upper region of the 10% cushion. After fractionation, both markers exhibited essentially the same distribution on the gradient. The values correspond to the means of five separate experiments, and the standard deviations are indicated.

cushions of 16% and 10% sucrose in D20. The gradient was then overlaid with homogenization buffer. The internalized HRP was recovered at the 16%/10% interface after 5 min internalization and in the uppermost part of the 10% cushion after a subsequent 30 min chase (Table 1). Either fraction contained ~20% of the total HRP

Table 2. Compared Yield and Relative Specific Internalized HRP and ?-Labeled Plasma Membrane Proteins Internalized EE fraction

Yield (%) RSA (fold)

20 f 13 f

LE fraction

Yield (O/o) RSA (fold)

23 f 5 17 2 2

5 2

HRP

Activity

of

?-pm

Proteins

0.8 + 0.5 0.8 + 0.2 1.0 f 1.4 f

0.3 0.1

The experiments were carried out as described in Table 1. The early endosomal fractions (EE fraction) were prepared from cells with HRP internalized for 5 min at 37OC and were collected at the 16%-10% sucrose interface on the gradient. The late endosomal fractions (LE fraction) were obtained after a subsequent 30 min chase in marker-free medium and were collected from the upper region of the 10% cushion. To label plasma membrane proteins with 1251,the cells were maintained on ice, homogenized, and a PNS was prepared. The distribution of ‘Z5-labeled plasma membrane proteins ]‘?-pm proteins] was analyzed on the gradients. The yield of each marker in the desired fraction was calculated as a percentage of the amount present in the corresponding homogenate. We used gentle homogenization conditions to limit damage to endosomal elements. As a result, ~30% of the cells remained intact, and ~50% of internalized HRP as well as *70% of ‘nal-pm proteins were lost to the nuclear pellet. The relative specific activity (RSA) of each marker was calculated from its specific activity (OD units per mg of protein) in the fraction divided by its specific activity in the homogenate.

present in the corresponding homogenate (Table 2). (We used gentle homogenization conditions, which resulted in a loss of ~50% of the total HRP activity to the nuclear pellet.) In both fractions, the specific activity of HRP (OD units per mg of protein) was enriched ~18fold over the corresponding homogenate. When the 16%/l 0% interface was examined in the electron microscope, HRP was observed after 5 min internalization within cisternal and tubular structures (Figure 1A) resembling closely the appearance of early endosomes observed in intact cells (Gruenberget al., 1969; Griffithset al.., 1969). Inagreement with the biochemical data, these structures were depleted of HRP after the chase (data not shown). Conversely, the upper region of the 10% cushion contained little if any labeled structures 5 min after internalization (not shown) but numerous HRP-positive structures after the chase (Figure 16). These latter structures exhibited the typical morphology of late endosomes and lysosomes in intact cells (Gruenberg et al., 1969; Griffithset al., 1990). We will refer to the fractions recovered at the 16%/l 0% interface and in the upper region of the 10% cushion as early and late endosomal fractions, respectively. Next, we quantified the amount of plasma membrane present in the fractions. The cell surface was iodinated on ice, using the glucose oxidase-lactoperoxidase reaction (Hubbard and Cohn, 1972; Gruenberg and Howell, 1966). The cells were homogenized, and a PNS was prepared that contained ~30% of the 1Z51-labeled plasma membrane proteins; the rest sedimented in the nuclear pellet. The bound 1251present in the PNS (60%) was rapidly separated from the free ‘*Y (20%) using a PD-10 column, adjusted to 40.6% sucrose, and loaded at the bottom of the gradient. On the gradient, ~70% of the label remained in the heavy sucrose, and only ~3% was collected in each endosomal fraction. As a result, plasma membrane contamination of either endosomal fraction corresponded to 65% (measured as in Bomsel et al., 1990). The PNS was then brought to 40.6% sucrose using a stock solution of 62% sucrose, 3 mM imidazole (pH 7.4) (final volume = 1 ml) and loaded at the bottom of a SW 60 centrifugation tube. A gradient consisting of three steps was then poured (2 ml of 16% sucrose in DzO,

r(ab+Mediated 923

Control

of Fusion

3 mM imidazole [pH 7.41; 1.5 ml of 10% sucrose in DzO, 3 mM imidazole [pH 7.4); 0.5 ml of homogenization buffer). The gradient was centrifuged at 35,000 rpm for 60 min at 4OC in a Beckman L5-65 ultracentrifuge using a SW 60 rotor. The early and late endosomal fractions were collected at the 16%/10% sucrose interface and in the uppermost portion of the 10% sucrose cushion, respectively.

Early Endosomal

Fractions

Labeled

with

bHRP

The conditions used for HRP biotinylation did not modify its enzymatic activity (Gruenberg et al., 1969). bHRP was endocytosed in the fluid phase at 1.7 mglml, and then the cells were fractionated as described for HRP. The endosomal fractions were frozen in 20 pg aliquots in liquid nitrogen and stored at - 70DC.

Early Endosomal

Fractions

Labeled

wlth Avldln

The cell surface was first biotinylated, using essentially the protocol described by Le Bivic et al. (1969). Briefly, three petri dishes were washed three times with PBS containing 0.05 mM CaCI, and 0.5 mM MgCll (PBS+). The cells were then incubated for 30 min on ice in 2.5 ml per dish of PBS+ containing 2.5 mg of biotin-X-SS-NHS (Pierce) that had been freshly disolved in 12.5 PI of dimethyl sulfoxide. The ceils were washed once in PBS+, and the treatment was repeated. The reagent was then quenched for 15 min at 4OC with 50 mM glycine in PBS+. The cells were rinsed one time with PBS+, washed for 10 min with internalization medium, rinsed three times with PBS+, and finally one time with internalization medium. Each dish was then incubated with 900 pg of avidin in 3 ml of internalization medium for 45 min at 4OC. Since saturating amountsof avidin were used, the avidin bound to biotinylated cell surface proteins retained free binding sites (not shown). After washing the cells three times for 10 min in PBS-BSA and one time in PBS+, the cells were incubated for 5 min at 37OC in inlernalization medium containing 2 mg/ml BSA to label early endosomes. When late endosomes were labeled, the cells were returned to ice temperature, and the avidin binding sites remaining on the cell surface were quenched with 30 pg/ml biotinylated insulin in PBS+ for 30 min at 4OC. The cells were then washed three times in PBS+-ESA and one time in PBS+, and reincubated in internalization medium for 30 min at 37OC. Cells were then harvested and homogenized as above. The fractions used in the cell-free assay were collected, frozen in 20 pg aliquots in liquid nitrogen, and stored at - 70°C.

Analysis

of the Fractions

after

Fractionation

The distribution of endocytosed HAP or bHRP on the gradient was assayed as in Gruenberg and Howell (1966). The gradient was also analyzed for the distribution of endocytosed avidin bound to biotinylated proteins. A 200 pl aliquot of each fraction supplemented with the cocktail of protease inhibitors was extracted with 2% Triton X-100 for 30 min at 4OC in the presence of 250 ng of bHRP to label the free avidin-binding sites. As a control, an excess of 10 pg of biotin-insulin was used. After centrifugation of the detergent extract at 100,000 x g, lhe avidin-bHRP complex present in the supernatant was immunoprecipitated overnight at 4OC in 1 ml of PBS-BSA, 0.5% Triton X-100 using protein A-Sepharose with coupled affinity-purified antibodies against avidin. The enzymatic activity of bHRP present in the complex was then quantified as in Bomsel et al. (1990). When the distribution of the plasma membrane was analyzed on the gradient, the same protocol was used, except that the internalization step was omitted. We also analyzed the distribution of the plasma membrane after cell surface iodination. One petri dish was prewashed three times for 5 min with PBS-BSA and three times with PBS at 4OC and then iodinated for 10 min at 4OC with 0.1 mCi of carrier-free Y (Amersham) using the glucose oxidase-lactoperoxidase reaction (as in Gruenberg and Howell, 1966). The cells were then washed eight times for 10 min in PBS-BSA containing 5 mM KI and two times in PBS+. After homogenization, the PNS was mixed with another PNS prepared from two dishes that had not been labeled. The bound ‘%I present in the PNS (~60%) was rapidly separated from the free l-1 (~20%) with a PD-l0 column equlibrated at 4OC with homogenization buffer. After flotation on the gradient, the amount of lZ51 was counted in each fraction.

Cytosol

and Mlcrosomes

Cytosol and microsomes from transfected or nontransfected cells were obtained from supernatant and pellet, respectively, after centrifugation

of PNS at 60,000 rpm for 30 min at 4OC in a TL 100 Beckman centrifuge using a TLA 100 rotor. For the cell-free analysis, 50 PI aliquots of the supernatant (cytosol) were frozen in liquid nitrogen and stored at - 7ooc.

Cell-Free

Assay

of Endosome

Fusion

The cell-free assay was modified from our previous assay (Gruenberg et al., 1969; Tuomikoski et al., 1969; Bomsel et al., 1990). Two early endosomal fractions were prepared on the flotation gradient after separate internalization of bHRP from the medium or avidin prebound to cell surface biotinylated proteins. A 50 ~1 aliquot (20 pg of protein) of each fraction was gently mixed with 50 ~1 of cytosol(2.5-10 mglml, as indicated) and adjusted to 12.5 mM HEPES (pH 7.4), 1.5 mM MgOAc, 3 mM imidazole, 1 mM DTT, 50 mM KOAc, and complemented with 6 ~1 of an ATP-regenerating system (1:l :l mixture of 100 mM ATP brought to pH 7.0 with KOH, 600 mM creatine phosphate, and 4 mg/ ml creatine phosphokinase) and with 8 ~1 of biotin-insulin (to quench the avidin-binding sites that may remain accessible). To deplete ATP, 1.6 ~1 of apyrase (1200 U/ml) replaced the ATP-regenerating system. The reaction mixture was first incubated 10 min at 4OC, and then the fusion reaction was initiated by raising the temperature to 37°C. After 45 min, the reaction was stopped by returning the mixture to ice temperature. The avidin-bHRP complex, which may have formed upon fusion, was extracted in 2% Triton X-100 for 30 min at 4OC, and the extract was clarified by a 100,000 x g centrifugation. The supernatant was brought to 1 ml with PBS-BSA containing 1% Triton X-100 and incubated overnight at 4V with protein A-Sepharose beads coupled to affinity-purified anti-avidin antibodies. The beads were then washed, and the enzymatic activity of bHRP was quantified (Bomsel et al., 1990). In experiments in which the effects of antibodies oh the fusion reaction were tested, the PNSs were complemented with a 1:20 dilution of the desired antiserum and incubated for 60 min at 4°C. The PNS was then fractionated on the gradient to remove the free antibody, and the corresponding early endosomal fractions were used in the cell-free assay. Alternatively, a 1:50 dilution of the desired antiserum was directly added to the reaction mixture, which was then incubated for 60 min at 4OC, before raising the temperature to 37OC. lmmunodepletion of rab5 was carried out by mixing cytosol-containing overexpressed rab5 with protein A-Sepharose beads that had been pretreated with anti-rab5 antiserum or with a control antiserum. The mixtures were incubated on ice for 30 min, then the beads were removed by centrifugation, and the cytosols were used in the assay.

Electron

Microscopy

The fractions were fixed in 1% glutaraldehyde in 200 mM cacodylate buffer (pH 7.4) and centrifuged in a Beckman airfuge for 15 min. The pellet was then treated with osmium tetroxide and embedded in Epon. Internalized HRP was visualized as described by Marsh et al. (1988).

Analytlcal

Methods

HRP biotinylation was as in Gruenberg et al. (1989). Protein was determined according to Bradford (1978). SDS-PAGE was performed with the system of Maize1 (1971). Protein transfer onto nitrocellulose was by the Western procedure (Burnette, 1981).

Acknowledgments We are particularly grateful to Dr. Gareth Griffiths for carrying out the electron microscopy analysis. We are also grateful to Drs. Bernard Hoflack, Kai Simons, and John Tooze for critically reading the manuscript. We also wish to thank Ruth Jelinek and Carmen Walter for expert technical assistance. We thank Dr. Bernard Hoflack for the generous gift of antibodies against the Cl-MPR. J.-P. G. was supported by a fellowship from the Alexander-von-Humbolt Stiftung (FRG) and P. C. by a fellowship from the European Molecular Biology Organization. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

September

11, 1990; revised

November

19, 1990

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rab5 controls early endosome fusion in vitro.

The small GTP-binding protein rab5 was previously localized on early endosomes and on the cytoplasmic face of the plasma membrane. Using a cell-free a...
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