398
[37]
IDENTIFICATION OF TRANSPORT INTERMEDIATES
[37] L o c a l i z a t i o n o f R a b F a m i l y M e m b e r s Animal Cells
in
By MARINO ZERIAL, ROBERT PARTON, PHILIPPE CHAVRIER, and RAINER FRANK Introduction Among the different classes of Ras-like low molecular weight GTPbinding proteins expressed in mammalian cells, Rab proteins are the most closely related to Yptlp and Sec4p, which are involved in control of secretion in the yeast S a c c h a r o m y c e s cerevisiae.t,2 Due to this sequence similarity they have been functionally implicated in the regulation of membrane traffic in mammalian cells. In fact, Rabl has been shown to functionally replace Yptlp in S. cerevisiae 3 and studies on Rab5 have indicated that this protein is involved in the process of early endosome fusion in vitro 4 and in control of endocytosis in vivo. 5 The localization data so far obtained have shown that Rab proteins are associated with specific subcompartments along the exocytic and endocytic pathway. Using affinity-purified antibodies in immunofluorescence and electron microscopy studies, Rab2 was found associated with an intermediate compartment between the endoplasmic reticulum (ER) and the Golgi apparatus. 6 Using similar techniques, Rab6 was found localized to the medial- and trans-Golgi cisternae. 7 Rab3a was demonstrated by subcellular fractionation to be present in synaptic vesicles in neurons and in chromaffin granules in adrenal medulla, s-~° Three other Rab proteins were found associated with compartments along the endocytic pathway: Rab5 was detected at the cytoplasmic surface of both the plasma membrane and early endo-
J A. Salminen and P. J. Novick,Ce1149, 527 (1987). 2 N. Segev, J. Mulholland, and D. Bostein, Ce1152, 915 (1988). 3H. Haubruck,R. Prange,C. Vorgias,and D. Gallwitz,EMBO J. 8, 1427 (1989). 4j..p. Gorvel,P. Chavrier,M. Zerial, and J. Gruenberg,Cell 64, 915 ( 1991). 5C. Bucci,R. G. Parton, I. Mather,H. Stunnenberg,K. Simons,B. Hoflack,and M. Zerial, Cell 70, in press (1992). 6p. Chavrier,R. G. PaTton,H. P. Hauri, K. Simons,and M. Zerial, Ce1162, 317 (1990). 7B. Goud,A. Zahraoui,A. Tavitian,and J. Saraste,Nature (London) 345, 553 (1990). s G. Fischeryon MoUard, G. A. Mignery,M. Baumert, M. S. Perin, T. J. Hanson, P. M. Burger, R. Jahn, and T. Sudhof,Prac. Natl. Acad. Sci. USA 87, 1988 (1990). 9F. Darchen, A. Zahraoui, F. Hammel, M.-P. Monteils, A. Tavitian, and D. Scherman, Proc. Natl. Acad. Sci. USA 87, 5692 (1990). ~oA. Mizoguchi,S. Kim, T. Ueda, A. Kikuchi,H. Yorifuji,N. Hirokawa,and Y. Takai, J. Biol. Chem. 265, 11872 (1990). METHODS IN ENZYMO/J3GY, VOL. 219
Copyright @ 1992 by Academ/c Press, Inc. All ri~hts of reproduction in any form reserved.
[37]
LOCALIZATIONOF Rab PROX~INS
399
somes,6 Rab4 on early endosomes, H whereas Rab7 was associated with late endosomes.6 The methods described here outline the different techniques we have used to identify and localize members of the Rab protein family. Molecular Cloning of Rab cDNAs Ras and Ras-related proteins are structurally recognizable by a high sequence conservation in the four regions forming the GTP-binding site ~2 and by the presence of one or two cysteines at their C termini (Fig. 1). Isoprenylation of these cysteines is a requirement for membrane association. The high amino acid conservation in these regions has enormously facilitated the identification of several members of the Rab protein subfamily.~3 Rab proteins could be identified by screening eDNA libraries either with oligonucleotides corresponding to highly conserved sequences~4,t5 or using Rab eDNA probes at low-stringency hybridization conditions.~6 cDNA Library Screening To identify cDNAs encoding Rab proteins we use a modification of the protocol described by Touchot et all 4 We screen an oriented 2 ZaplI (Stratagene, La Jolla, CA) MDCK cDNA library with two degenerate oligonucleotides. One corresponds to the conserved sequence WDTAGQE (single-letter amino acid code) in region 2 (olig02, Fig. 1) shared by Sec4p, Yptlp, Rab and Rho proteins: 5'-TGGGA(Cso/T~o)AC(ATo/CIo/TIo/GIo ) GC(T3o/ATo)GCr(A2JG25/C2s/T25)CA(G2o/Aso)GAA-3' (numbers in subscript refer to the relative frequency of each base at a given position). Because Ras and Rap proteins contain instead an LDTAGQE sequence, 13 this oligonucleotide preferentially hybridizes to Rab and Rho cDNAs. The second oligonucleotide corresponds to the fourth conserved domain in Ras-like proteins (olig04, Fig. 1) and has the following sequence: 5'-TT(T~/ C5o)(Tso ] Aso)T(Gso/Tso)GA(A25 / G75)(A75/ G25)C(Aso/C3o/G~o/T~o)A75 / T25(G75[C25)(T75/C25)GC-3". After a 1-hr prehybridization at 42* in 6 × ,1 p. van der Sluijs, M. Hull, A. Zahraoui, A. Tavitian, B. Goud, and I. Mellman, Proc. Natl. Acad. Sci, USA 88, 6313 (1991). ,2 E. F. Pal, W. Kabsch, U. Krengel, K. C. Holmes, J. John, and A. Wittinghofer, Nature (London) 341, 209 (1989). 13 A. Valencia, P. Chardin, A. Wittinghofer, and C. Sander, Biochemistry 30, 4637 (1991). ,4 N. Touchot, P. Chardin, and A. Tavitian, Proc. Natl. Acad. Sci. USA 84, 8210 (1987). ,5 p. Chavrier, M. Vingron, C. Sander, K. Simons, and M. Zerial, Mol. Cell. Biol. 10, 6578 (1990). ,6 A. Zahraoui, N. Touchot, P. Chardin, and A. Tavitian, J. Biol. Chem. 264, 12394 (1989).
40 0
[37]
IDENTIFICATION OF TRANSPORTINTERMEDIATES EFFECTOR LOOP
SYNTHE~C PEP~DES
CYST~NE MO~FS
CCXXX WDTAGQE
rab2
YEKIQEGVFDINNEANGIK
rab4b
KIQYGDASLRQLRQPRSA
tab5
PKNEPONPGANSARGR KOETEVELYNEFPEPIK KLEGNSPQGSNQ6VK
tab7 tab6
rablO
KTPVKEPNSENVDI5
FIG. 1. Schemeof a typical Rab protein showingthe variable lengths of the N and C termini, the four conservedregionsparticipating in the formationof the GTP-bindingsite (black boxes), the "effectorregion" (striped box), and the C-terminalhypervariableregion containing the diversecysteinemotifs. The syntheticpeptides used for immunizationwere derivedfrom this region. SSC (1 × SSC is 0.15 M NaC1 plus 0.015 M sodium citrate), 5X Denhardt's solution, 0.05% (w/v) sodium pyrophosphate, 0.5% (w/v) sodium dodecyl sulfate (SDS), 100 pg/ml boiled herring sperm DNA, duplicate filters are hybridized for 18 hr at 42 ° in 6× SSC, 1X Denhardt's solution, 0.05% (w/v) sodium pyrophosphate, 100 gg/ml yeast tRNA, with 25 pmol/ml of oligonucleotide 2 (32p) end-labeled using T4 polynucleotide kinase. Filters are then washed for 3 hr at 44 ° in a 6X SSC/0.05% sodium pyrophosphate solution. Positive phage DNAs are then retested by hybridization with foligo2 at 42 or 37 ° with oligo4. Washing conditions are as above except that, in the case of the latter oligonucleotide, washing is performed at 25 °. Phages hybridizing to both oligonucleotides are selected and characterized by DNA sequencing. For this purpose, oligo2 and its antisense counterpart can be efficiently used in dideoxy sequencing reactions. Using this technique the frequency of isolation of Rab-encoding cDNAs is quite low. We find an average of 1 positive clone out of 50,000 screened phages for most of the Rab cDNA clones we identify. This is due both to the relatively low abundance of Rab mRNAs and also to the use of oligonucleotides that restrict the screening to cDNA clones containing most of the coding region. Therefore, to maximize the screening efficiency it is crucial to screen a library having long cDNA inserts ( - 2 - k b average insert length). To overcome this limitation, we have employed a more efficient
[37]
LOCALIZATIONOF Rab PROTEINS
401
method with the use of oligonucleotide 2 in a rapid amplification ofcDNA ends (RACE) polymerase chain reaction ( P C R ) . 17 U s i n g this method, we could identify 11 novel Rab proteins and one new Rho protein. 18 Production of Specific Rab Proteins Antisera To determine the localization of Rab proteins we raise polyclonal antisera in rabbit either against recombinant Rab proteins produced in Escherichia coli (we use MSII polymerase-Rab fusion proteins) or against peptides derived from the C-terminal hypervariable region of Rab proteins.6 This region is not conserved among the different Rab proteins identified so far~3; therefore, the latter method circumvents the potential problem of cross-reactivity. Furthermore, peptides can be used in competition experiments to control the specificity of the antisera (see below). Using peptides, we have successfully raised antibodies against Rab2, Rab4b, Rab5, Rab7, Rab8, and Rabl0 proteins. Peptides are chosen from the C-terminal sequence 15(Fig. 1), beginning from the last amino acid residues of a helix 5, as deduced from the structure of p21r~. ~2 Before injection, synthetic peptides are covalently coupled to keyhole limpet hemocyanin (KLH; Calbiochem, San Diego, CA) as described by Kreis. 19 These peptides are 16 to 20 amino acid residues in length and contained 1 or 2 lysines required for the coupling reaction.
Injection and Bleeding of Rabbits We use the immunization procedure described by Louvard2° based on the lymph node antigen injection protocol. Typically, two rabbits are injected with each antigen, a total of 750/zg of coupled peptide or recombinant Rab protein per animal. The first injection contains 100-200 #g of antigen in 0.5 ml of Freund's complete adjuvant (Sigma, St. Louis, MO). The maximum possible is injected into the thigh lymph nodes and the remainder is injected subcutaneously in the back of the neck. After 3 weeks 50- 100 #g of antigen in 0.5 ml of Freund's incomplete adjuvant is injected, one-half subscapular and one-half in the neck region. After 6 weeks 100 #g of antigen in phosphate-buffered saline (PBS) is injected intramuscularly in the leg. At 7 weeks 50 #g is injected intramuscularly and the injection is repeated intravenously 1 and 2 days later. Animals are bled and subsequently boosted at weeks 9-12. Blood is collected into a 50-ml 77M. A. Frohman, M. K. Dush, and G. R. Martin, Proc Natl. Acad. Sci. USA 85, 8998 (1988). ~s p. Chavrier, K. Simons, and M. Zerial, Gene 112, 261 (1992). 19T. E. Kreis, E M B O J. 5, 931 (1986). 2o D. Louvard, J. Cell Biol. 92, 92 (1982).
402
IDENTIFICATION OF TRANSPORT INTERMEDIATES
[37]
Falcon (Becton Dickinson, Oxnard, CA) tube containing a long wooden stick. After a 1-hr incubation at 37 ° the blood is left at 4 ° overnight. The day after, the coagulated clot around the stick is discarded and the serum spun at 4000 g for 20 min at 4 ° to remove remaining debris. The serum is stored at 4 ° after addition of 0.02% (w/v) sodium azide or at - 7 0 °, frozen in aliquots.
Affinity Purification of Antisera Both for biochemical and morphological studies anti-Rab protein antisera are first affinity purified. In the ease of antiserum raised against MSII polymerase-Rab fusion proteins, affinity purification is performed by adsorbing the antiserum onto nitrocellulose filters containing the corresponding proteins. Anti-peptide antibodies are affinity purified on the same filters or on Sepharose 4B-peptide columns. For this purpose, 1 g of CNBr-activated Sepharose 4B (Pharmacia, Piscataway, N J) is swollen in 50 ml of 1 m M HCI for 20 rain at room temperature and washed on a sintered glass filter (No. 3, Schott Duran, Mainz, Germany) with 500 ml of the same buffer. Beads are then transferred to a 15-ml Falcon tube and washed twice with coupling buffer (0.2 M NaHCO3, pH 8.5, 0.5 M NaCI). After centrifugation at 1000 g for 1 rain the liquid is removed and the Sepharose 4B incubated with 10 mg of coupled peptide dissolved in 7.5 ml of coupling buffer, first for 2 hr at room temperature and then overnight at 4 ° on a rotating wheel. Beads are washed with 30 ml of coupling buffer and the reacting groups blocked by incubation in 40 ml of 0.2 M glycine, pH 8.5, for 4 hr at 4 °. After three cycles of washes consisting of 40 ml sodium acetate (0.1 M)/NaC1 (0.5 M), pH 4.0, and then coupling buffer, beads are washed twice in PBS and stored in PBS containing 0.02% sodium azide a t 4 °. For affinity purification, 1 - 2 ml of antiserum is diluted to I 0 ml with PBS and bound at 4 ° overnight on their corresponding MSII polymeraseRab fusion proteins immobilized on nitrocellulose filter or peptide columns. Antibodies are eluted in 100 m M glycine hydrochloride, pH 2.8, and neutralized by addition of 3 M Tris-HC1, pH 8.8. Affinity-purified antibodies are supplemented with 0.5% (w/v) IgG-free bovine serum albumin (BSA) (Sigma) and 0.02% sodium azide and stored at 4 °. Despite the affinity purification, antisera occasionally gave some background in immunofluorescence or electron microscopy studies. To avoid this, antisera are first cleared by adsorption at 4 ° on a Sepharose 4B matrix coupled to a bacterial lysate prior to affinity purification (we used K537 cells transformed with plasmid pEX34b and temperature induced to express the MSII polymerase polypeptide6; other bacterial extracts can be used instead). This step removed antibodies unspecifieally bound to the
[37]
LOCALIZATIONOF Rab PROTEINS
403
peptide column. To prepare the lysate, bacteria from a 40-ml overnight culture are spun and lysed in 5 ml of 50 m M Tris-HC1, pH 8.0, 5.0 m M ethylenediaminetetraacetic acid (EDTA), 0.2 mg/ml lysozyme for 30 min at 37 °. After addition of Triton X-100 to a concentration of 1% (v/v) the extract is ultrasonified. Insoluble aggregates are pelleted in a mierofuge. One milliliter of the supernatant ( - 3 0 mg/ml) is used in the coupling reaction performed as described above. Coupling efficiency is monitored by analyzing samples of the lysate before and after the coupling reaction by polyacrylamide gel electrophoresis. The affinity-purified antibodies are characterized by immunoblot analysis and by immunofluorescence staining. Different amounts ( 1 rig- 1 #g) of recombinant Rab proteins produced in E. coli are used to titer the affinity-purified antisera by Western blot analysis. Immunofluorescence Localization Analysis For this type of analysis we permeabilize cells using saponin as detergent prior to fixation. This procedure removes most of the cytosolic proteins and allows us to better visualize the membrane staining pattern. This method is particularly useful in the case oftransfected cells expressing high levels of Rab proteins (see below). In these cells the excess of cytosolic Rab protein can be washed off during the permeabilization step. Cells are grown on 10-ram round cover slips for 24 hr prior to treatment. Cells are washed once with PBS and permeabilized with 0.05% (w/v) saponin in 80 m M K+-piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), pH 6.8, 5 mMethylene glycol-bis(fl-aminoethylether)-N,N,N',N'tetraacetic acid (EGTA), 1 m M MgC12 for 5 min. After a 15-min fixation in 3% (w/v) formaldehyde in PBS, pH 7.4, cells are washed with 0.05% saponin in PBS for 5 min and free aldehyde groups quenched with 50 m M NH4C1 in PBS for 10 min. Cells are washed with 0.05% saponin in PBS for 5 min and then incubated with the first antibody in PBS-0.05% saponin for 20 min. Depending on the antiserum, antibodies are diluted at volume ratios of 1 : 10- 1:300. After rinsing the cells three times (15 min in total) primary antibody binding is visualized with goat anti-rabbit fluorescein isothiocyanate (FITC) or goat anti-rabbit RITC diluted in 0.05% saponinPBS for 20 min. After one wash in PBS-0.05% saponin and three washes in PBS (20 min in total), the cover slips can be mounted on glass sides in Mowiol (Hoechst, Frankfurt, Germany) and viewed.
Overexpression of Rab Proteins Using T7 RNA Polymerase Recombinant Vaccinia Virus System To facilitate the localization studies of Rab proteins we make use of a transient expression system based on the T7 RNA polymerase recombi-
404
IDENTIFICATION OF TRANSPORT INTERMEDIATES
[3 7]
FIG. 2. Immunofluorescence localization of Rab2 in untransfected and transfected cells. (A) BHK cells labeled with affinity-purified anti-Rab2 antibodies. The staining pattern of vesicular structures restricted to the perinuclear area corresponds to an intermediate compartment between the ER and the Golgi apparatus. ~ (B) BHK cells infected with the T7 RNA polymerase-recombinant vaccinia virus and transfected with Rab2 construct. The exogenous protein gives an intense signal close to the nucleus, a staining pattern similar to that of the endogenous protein.
nant vaccinia virus. 21 Cells are infected with this vaccinia virus and transfected with a plasmid containing the Rab eDNA under the control of the T7 promoter. We currently use plasmid pGEM 1 (Promega, Madison, WI) for these studies. This system has been useful in proving the specificity of the affinity-purified antibodies. We often obtain affinity-purified antibodies reacting well in Western blot experiments but giving artifactual staining by immunofluorescence analysis. When antisera are specific, in cells expressing the exogenous Rab protein, the staining pattern is similar and of higher signal intensity compared to that of nontransfected cells. Figure 2 shows an immunofluorescence analysis using anti-Rab2 antibodies of untransfected cells and cells overexpressing Rab2. For these experiments cells are split 18- 24 hr before transfection, so that on the day of transfection they are about 80% confluent. The cells are washed twice with serum-free medium and infected with T7 RNA polymerase-recombinant vaccinia virus. Infections are carried out with 3 - 5 pfu/cell at room temperature for 30 min with intermittent agitation. The cells are then washed twice with serum-free medium and transfected by lipofection using the DOTAP reagent according to the instructions of 2~ T. T. Fuerst, E. G. Niles, F. W. Studier, and B. Moss, Proc. Natl. Acad. Sci. USA 83, 8122 (1986).
[37]
LOCALIZATION OF R a b PROTEINS
405
the manufacturer (Boehringer Mannheim, Indianapolis, IN). To obtain high transfection efficiencies (up to 95%) 5 we use plasmids purified twice on CsC1 density gradients and dialyzed against 0.1 × TE (1.0 m M Tris, pH 7.5, 0.1 m M EDTA). Cells are incubated for 3 - 4 hr at 37 ° in a 5% CO2 incubator and processed for immunofluorescence microscopy (Fig. 2) or immunoelectron microscopy. Longer incubation times lead to accumulation of Rab protein, posttranslationally unmodified in the cytosol. This does not affect the immunotluorescence analysis owing to the permeabilization step. However, accumulation of cytosolic Rab protein should be avoided in the case of immunoelectron microscopic analysis (see below). I m m u n o e l e c t r o n Microscopic Localization of Rab Proteins
Practical Details We examined the intracellular location of Rab2, 5, and 7 in cultured cells. For this, affinity-purified anti-peptide antibodies were applied to thawed cryosections according to published procedures. 22-25 Pellets of BHK, NRK, or HeLa cells were prepared for cryosectioning as follows.26 Cultured cells on 6-cm culture dishes were washed with icecold PBS and then were removed from the culture dish by incubation on ice with 1 ml of 20- to 50-/zg/ml proteinase K in PBS. This is a relatively gentle procedure for removal of the cells from the dish. The cells start to detach from the dish in sheets after about 2 min; the cells do not noticeably round up and most surface antigens are unaffected by this treatment (G. Griffiths, personal communication, 1991). The detached cells were layered onto 200#1 of 8% (w/v) paraformaldehyde in 250 m M N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.4, and were pelleted in a microfuge at 1000 rpm for 1 rain at room temperature. The supernatant is then removed and replaced with fresh fixative as above without disturbing the pellet. After 30 rain at room temperature the cells were spun at maximum speed in the microfuge before replacing the supernatant once more with fresh fixative. To allow freezing of the cells without destructive ice crystal formation the cells must be cryoprotected. This is achieved by incubation of small pieces of the pellet with sucrose (2.1 M in PBS) for 15 - 30 min. The pieces 22 K. T. Tokuyasu, J. Cell Biol. 57, 551 (1973). 23 K. T. Tokuyasu, J. Ultrastruct. Res. 63, 287 (1978). 24 G. Grittiths, K. Simons, G. Warren, and K. T. Tokuyasu, this series, Vol. 96, p. 435. 2s G. Griitiths, A. MeDowell, R. Back, and J. Dubochet, J. Ultrastruct. Res. 89, 65 (1984). 26 j. Green, G. Grittiths, D. Louvard, P. Quinn, and G. Warren, J. Mol. Biol. 152, 663 (1981).
406
IDENTIFICATION OF TRANSPORT INTERMEDIATES
[37]
of pellet were then mounted on copper stubs and plunged into liquid nitrogen, the specimens were then ready for cryosectioning. This is performed at - 100 ° using glass or diamond knives. Sections were transferred from the knife to copper grids using a loop with a drop of 2.3 M sucrose in PBS. All labeling is carried out at room temperature with antibodies followed by protein A-gold (6- or 9-nm diameter) diluted in PBS containing 5% (w/v) fetal calf serum and 0.12% (w/v) glycine. After washing the grids with distilled water they are embedded in a methylcellulose/uranyl acetate (MC/UA) mixture. Excess MC/UA is then removed, leaving a thin film over the sections. After drying (a few minutes at room temperature) the grids are viewed at an accelerating voltage of 80 kV.
Specificity of Labeling The antibodies used for all electron microscopy (EM) analyses were first checked by Western blotting and by immunofluorescence. However, there is still the possibility that artifactual "sticking" of the antibody to intraceUular structures occurs. Normally the specificity is monitored by checking the amount of labeling on structures known to be negative for the antigen, for example, the nucleus. For anti-peptide antibodies, however, the specificity can often be unequivocably determined by incubating the antibody prior to applying it to sections with the specific peptide to which it was raised. In principle, this should abolish the labeling whereas incubation with the same concentration of a nonspecific peptide should have no effect. In the case of the anti-Rab antibodies, relatively high concentrations (0.1 mg/ml) of the specific peptide were required to inhibit binding. This presumably reflects the fact that the affinity-purified antibody recognizes a limited number of conformational forms that the peptide can attain in solution.27,28 This control alone cannot rule out that the antibody recognizes different proteins sharing the same epitopes, but this can be checked by experiments on overexpressing cells and Western blotting analyses as described above.
Colocalization Studies The unequivocal localization of any antigen to a particular compartment by morphology alone is often extremely difficult. Well-defined markers are then required for colocalization studies to identify the corn27 H. J. Dyson, M. Rance, R. A. Houghten, R. A. I_¢rner, and P. E. Wright, J. MoL BioL 201, 161 (1988). 28 H. J. Dyson, M. Ranee, R. A. Houghten, P. E. Wright, and R. A. Lerner, J. Mol. Biol. 201, 201 (1988).
[37]
LOCALIZATIONOF Rab PROTEINS
407
partment of interest. Both antibodies and endocytic tracers were used as markers for endocytic and exocytic compartments. Rab2 was found to colocalize with labeling for a 53-kDa antigen previously shown to be present in the putative intermediate compartment located between the rough endoplasmic reticulum (RER) and Golgi. 29 Double labeling was achieved by one of two methods. In the protein A method the sections were treated with 1 mg/ml protein A in PBS after the first antibody and protein A-gold incubation steps. This quenches any free antibody before the incubations with the second antibody followed by a different size of protein A-gold. In the second method, 3° the sections were instead incubated with 1% glutaraldehyde in PBS at the same stage in the labeling scheme to destroy the ability of the first antibody to bind protein A-gold. For the endocytic pathway well-characterized particulate markers were used to label previously defined compartments of the cells of interest? ~a2 Bovine serum albumin-gold was internalized for either a short time (5 min) to label early compartments or for longer times to label late endosomes and lysosomes.
Experiments on Vaccinia Virus-Infected/Transfected Cells Rab proteins are produced as soluble proteins that then appear to associate with the membrane of specific compartments. On overexpression of these proteins an increase of labeling associated with a specific compartment occurs before the cytoplasmic pool of soluble protein starts to increase. For immunoelectron microscopy a large fraction of the cell population should express the protein at a high level, as the sample size for electron microscopy is relatively small when compared to, for example, immunofluorescence. However, the time after transfection should not be too long, as a high level of cytosolic protein will obscure the signal associated with a specific membrane. One way to overcome this is to extract the cells with saponin before fixation as for immunofluorescence (see above). However, in our experience a simpler solution, which was more compatible with fine structure preservation, was to choose a transfection time when the membrane-associated signal of protein is increased but the cytosolic pool of the protein is still below detectable levels.
~9 A. Schweizer, J. A, M. Fransen, T. Bachi, L. Ginsel, and H. P. Hauri, J. Cell Biol. 107, 1643 (1988). 3o j. W. Slot, H. J. Geuze, S. Gigenack, G. E. Lienhard, and D. E. James, J. Cell Biol. 113, 123 (1991). 31 G. Griffiths, B. Hoflack, K. Simons, I. Mellman, and S. Kornfeld, Cell 52, 329 0988). 32 G. Gdfliths, R. Back, and M. Marsh, J. Cell Biol. 109, 2703 (1989).
[37]
IDENTIFICATION OF TRANSPORT INTERMEDIATES
[37] L o c a l i z a t i o n o f R a b F a m i l y M e m b e r s Animal Cells
in
By MARINO ZERIAL, ROBERT PARTON, PHILIPPE CHAVRIER, and RAINER FRANK Introduction Among the different classes of Ras-like low molecular weight GTPbinding proteins expressed in mammalian cells, Rab proteins are the most closely related to Yptlp and Sec4p, which are involved in control of secretion in the yeast S a c c h a r o m y c e s cerevisiae.t,2 Due to this sequence similarity they have been functionally implicated in the regulation of membrane traffic in mammalian cells. In fact, Rabl has been shown to functionally replace Yptlp in S. cerevisiae 3 and studies on Rab5 have indicated that this protein is involved in the process of early endosome fusion in vitro 4 and in control of endocytosis in vivo. 5 The localization data so far obtained have shown that Rab proteins are associated with specific subcompartments along the exocytic and endocytic pathway. Using affinity-purified antibodies in immunofluorescence and electron microscopy studies, Rab2 was found associated with an intermediate compartment between the endoplasmic reticulum (ER) and the Golgi apparatus. 6 Using similar techniques, Rab6 was found localized to the medial- and trans-Golgi cisternae. 7 Rab3a was demonstrated by subcellular fractionation to be present in synaptic vesicles in neurons and in chromaffin granules in adrenal medulla, s-~° Three other Rab proteins were found associated with compartments along the endocytic pathway: Rab5 was detected at the cytoplasmic surface of both the plasma membrane and early endo-
J A. Salminen and P. J. Novick,Ce1149, 527 (1987). 2 N. Segev, J. Mulholland, and D. Bostein, Ce1152, 915 (1988). 3H. Haubruck,R. Prange,C. Vorgias,and D. Gallwitz,EMBO J. 8, 1427 (1989). 4j..p. Gorvel,P. Chavrier,M. Zerial, and J. Gruenberg,Cell 64, 915 ( 1991). 5C. Bucci,R. G. Parton, I. Mather,H. Stunnenberg,K. Simons,B. Hoflack,and M. Zerial, Cell 70, in press (1992). 6p. Chavrier,R. G. PaTton,H. P. Hauri, K. Simons,and M. Zerial, Ce1162, 317 (1990). 7B. Goud,A. Zahraoui,A. Tavitian,and J. Saraste,Nature (London) 345, 553 (1990). s G. Fischeryon MoUard, G. A. Mignery,M. Baumert, M. S. Perin, T. J. Hanson, P. M. Burger, R. Jahn, and T. Sudhof,Prac. Natl. Acad. Sci. USA 87, 1988 (1990). 9F. Darchen, A. Zahraoui, F. Hammel, M.-P. Monteils, A. Tavitian, and D. Scherman, Proc. Natl. Acad. Sci. USA 87, 5692 (1990). ~oA. Mizoguchi,S. Kim, T. Ueda, A. Kikuchi,H. Yorifuji,N. Hirokawa,and Y. Takai, J. Biol. Chem. 265, 11872 (1990). METHODS IN ENZYMO/J3GY, VOL. 219
Copyright @ 1992 by Academ/c Press, Inc. All ri~hts of reproduction in any form reserved.
[37]
LOCALIZATIONOF Rab PROX~INS
399
somes,6 Rab4 on early endosomes, H whereas Rab7 was associated with late endosomes.6 The methods described here outline the different techniques we have used to identify and localize members of the Rab protein family. Molecular Cloning of Rab cDNAs Ras and Ras-related proteins are structurally recognizable by a high sequence conservation in the four regions forming the GTP-binding site ~2 and by the presence of one or two cysteines at their C termini (Fig. 1). Isoprenylation of these cysteines is a requirement for membrane association. The high amino acid conservation in these regions has enormously facilitated the identification of several members of the Rab protein subfamily.~3 Rab proteins could be identified by screening eDNA libraries either with oligonucleotides corresponding to highly conserved sequences~4,t5 or using Rab eDNA probes at low-stringency hybridization conditions.~6 cDNA Library Screening To identify cDNAs encoding Rab proteins we use a modification of the protocol described by Touchot et all 4 We screen an oriented 2 ZaplI (Stratagene, La Jolla, CA) MDCK cDNA library with two degenerate oligonucleotides. One corresponds to the conserved sequence WDTAGQE (single-letter amino acid code) in region 2 (olig02, Fig. 1) shared by Sec4p, Yptlp, Rab and Rho proteins: 5'-TGGGA(Cso/T~o)AC(ATo/CIo/TIo/GIo ) GC(T3o/ATo)GCr(A2JG25/C2s/T25)CA(G2o/Aso)GAA-3' (numbers in subscript refer to the relative frequency of each base at a given position). Because Ras and Rap proteins contain instead an LDTAGQE sequence, 13 this oligonucleotide preferentially hybridizes to Rab and Rho cDNAs. The second oligonucleotide corresponds to the fourth conserved domain in Ras-like proteins (olig04, Fig. 1) and has the following sequence: 5'-TT(T~/ C5o)(Tso ] Aso)T(Gso/Tso)GA(A25 / G75)(A75/ G25)C(Aso/C3o/G~o/T~o)A75 / T25(G75[C25)(T75/C25)GC-3". After a 1-hr prehybridization at 42* in 6 × ,1 p. van der Sluijs, M. Hull, A. Zahraoui, A. Tavitian, B. Goud, and I. Mellman, Proc. Natl. Acad. Sci, USA 88, 6313 (1991). ,2 E. F. Pal, W. Kabsch, U. Krengel, K. C. Holmes, J. John, and A. Wittinghofer, Nature (London) 341, 209 (1989). 13 A. Valencia, P. Chardin, A. Wittinghofer, and C. Sander, Biochemistry 30, 4637 (1991). ,4 N. Touchot, P. Chardin, and A. Tavitian, Proc. Natl. Acad. Sci. USA 84, 8210 (1987). ,5 p. Chavrier, M. Vingron, C. Sander, K. Simons, and M. Zerial, Mol. Cell. Biol. 10, 6578 (1990). ,6 A. Zahraoui, N. Touchot, P. Chardin, and A. Tavitian, J. Biol. Chem. 264, 12394 (1989).
40 0
[37]
IDENTIFICATION OF TRANSPORTINTERMEDIATES EFFECTOR LOOP
SYNTHE~C PEP~DES
CYST~NE MO~FS
CCXXX WDTAGQE
rab2
YEKIQEGVFDINNEANGIK
rab4b
KIQYGDASLRQLRQPRSA
tab5
PKNEPONPGANSARGR KOETEVELYNEFPEPIK KLEGNSPQGSNQ6VK
tab7 tab6
rablO
KTPVKEPNSENVDI5
FIG. 1. Schemeof a typical Rab protein showingthe variable lengths of the N and C termini, the four conservedregionsparticipating in the formationof the GTP-bindingsite (black boxes), the "effectorregion" (striped box), and the C-terminalhypervariableregion containing the diversecysteinemotifs. The syntheticpeptides used for immunizationwere derivedfrom this region. SSC (1 × SSC is 0.15 M NaC1 plus 0.015 M sodium citrate), 5X Denhardt's solution, 0.05% (w/v) sodium pyrophosphate, 0.5% (w/v) sodium dodecyl sulfate (SDS), 100 pg/ml boiled herring sperm DNA, duplicate filters are hybridized for 18 hr at 42 ° in 6× SSC, 1X Denhardt's solution, 0.05% (w/v) sodium pyrophosphate, 100 gg/ml yeast tRNA, with 25 pmol/ml of oligonucleotide 2 (32p) end-labeled using T4 polynucleotide kinase. Filters are then washed for 3 hr at 44 ° in a 6X SSC/0.05% sodium pyrophosphate solution. Positive phage DNAs are then retested by hybridization with foligo2 at 42 or 37 ° with oligo4. Washing conditions are as above except that, in the case of the latter oligonucleotide, washing is performed at 25 °. Phages hybridizing to both oligonucleotides are selected and characterized by DNA sequencing. For this purpose, oligo2 and its antisense counterpart can be efficiently used in dideoxy sequencing reactions. Using this technique the frequency of isolation of Rab-encoding cDNAs is quite low. We find an average of 1 positive clone out of 50,000 screened phages for most of the Rab cDNA clones we identify. This is due both to the relatively low abundance of Rab mRNAs and also to the use of oligonucleotides that restrict the screening to cDNA clones containing most of the coding region. Therefore, to maximize the screening efficiency it is crucial to screen a library having long cDNA inserts ( - 2 - k b average insert length). To overcome this limitation, we have employed a more efficient
[37]
LOCALIZATIONOF Rab PROTEINS
401
method with the use of oligonucleotide 2 in a rapid amplification ofcDNA ends (RACE) polymerase chain reaction ( P C R ) . 17 U s i n g this method, we could identify 11 novel Rab proteins and one new Rho protein. 18 Production of Specific Rab Proteins Antisera To determine the localization of Rab proteins we raise polyclonal antisera in rabbit either against recombinant Rab proteins produced in Escherichia coli (we use MSII polymerase-Rab fusion proteins) or against peptides derived from the C-terminal hypervariable region of Rab proteins.6 This region is not conserved among the different Rab proteins identified so far~3; therefore, the latter method circumvents the potential problem of cross-reactivity. Furthermore, peptides can be used in competition experiments to control the specificity of the antisera (see below). Using peptides, we have successfully raised antibodies against Rab2, Rab4b, Rab5, Rab7, Rab8, and Rabl0 proteins. Peptides are chosen from the C-terminal sequence 15(Fig. 1), beginning from the last amino acid residues of a helix 5, as deduced from the structure of p21r~. ~2 Before injection, synthetic peptides are covalently coupled to keyhole limpet hemocyanin (KLH; Calbiochem, San Diego, CA) as described by Kreis. 19 These peptides are 16 to 20 amino acid residues in length and contained 1 or 2 lysines required for the coupling reaction.
Injection and Bleeding of Rabbits We use the immunization procedure described by Louvard2° based on the lymph node antigen injection protocol. Typically, two rabbits are injected with each antigen, a total of 750/zg of coupled peptide or recombinant Rab protein per animal. The first injection contains 100-200 #g of antigen in 0.5 ml of Freund's complete adjuvant (Sigma, St. Louis, MO). The maximum possible is injected into the thigh lymph nodes and the remainder is injected subcutaneously in the back of the neck. After 3 weeks 50- 100 #g of antigen in 0.5 ml of Freund's incomplete adjuvant is injected, one-half subscapular and one-half in the neck region. After 6 weeks 100 #g of antigen in phosphate-buffered saline (PBS) is injected intramuscularly in the leg. At 7 weeks 50 #g is injected intramuscularly and the injection is repeated intravenously 1 and 2 days later. Animals are bled and subsequently boosted at weeks 9-12. Blood is collected into a 50-ml 77M. A. Frohman, M. K. Dush, and G. R. Martin, Proc Natl. Acad. Sci. USA 85, 8998 (1988). ~s p. Chavrier, K. Simons, and M. Zerial, Gene 112, 261 (1992). 19T. E. Kreis, E M B O J. 5, 931 (1986). 2o D. Louvard, J. Cell Biol. 92, 92 (1982).
402
IDENTIFICATION OF TRANSPORT INTERMEDIATES
[37]
Falcon (Becton Dickinson, Oxnard, CA) tube containing a long wooden stick. After a 1-hr incubation at 37 ° the blood is left at 4 ° overnight. The day after, the coagulated clot around the stick is discarded and the serum spun at 4000 g for 20 min at 4 ° to remove remaining debris. The serum is stored at 4 ° after addition of 0.02% (w/v) sodium azide or at - 7 0 °, frozen in aliquots.
Affinity Purification of Antisera Both for biochemical and morphological studies anti-Rab protein antisera are first affinity purified. In the ease of antiserum raised against MSII polymerase-Rab fusion proteins, affinity purification is performed by adsorbing the antiserum onto nitrocellulose filters containing the corresponding proteins. Anti-peptide antibodies are affinity purified on the same filters or on Sepharose 4B-peptide columns. For this purpose, 1 g of CNBr-activated Sepharose 4B (Pharmacia, Piscataway, N J) is swollen in 50 ml of 1 m M HCI for 20 rain at room temperature and washed on a sintered glass filter (No. 3, Schott Duran, Mainz, Germany) with 500 ml of the same buffer. Beads are then transferred to a 15-ml Falcon tube and washed twice with coupling buffer (0.2 M NaHCO3, pH 8.5, 0.5 M NaCI). After centrifugation at 1000 g for 1 rain the liquid is removed and the Sepharose 4B incubated with 10 mg of coupled peptide dissolved in 7.5 ml of coupling buffer, first for 2 hr at room temperature and then overnight at 4 ° on a rotating wheel. Beads are washed with 30 ml of coupling buffer and the reacting groups blocked by incubation in 40 ml of 0.2 M glycine, pH 8.5, for 4 hr at 4 °. After three cycles of washes consisting of 40 ml sodium acetate (0.1 M)/NaC1 (0.5 M), pH 4.0, and then coupling buffer, beads are washed twice in PBS and stored in PBS containing 0.02% sodium azide a t 4 °. For affinity purification, 1 - 2 ml of antiserum is diluted to I 0 ml with PBS and bound at 4 ° overnight on their corresponding MSII polymeraseRab fusion proteins immobilized on nitrocellulose filter or peptide columns. Antibodies are eluted in 100 m M glycine hydrochloride, pH 2.8, and neutralized by addition of 3 M Tris-HC1, pH 8.8. Affinity-purified antibodies are supplemented with 0.5% (w/v) IgG-free bovine serum albumin (BSA) (Sigma) and 0.02% sodium azide and stored at 4 °. Despite the affinity purification, antisera occasionally gave some background in immunofluorescence or electron microscopy studies. To avoid this, antisera are first cleared by adsorption at 4 ° on a Sepharose 4B matrix coupled to a bacterial lysate prior to affinity purification (we used K537 cells transformed with plasmid pEX34b and temperature induced to express the MSII polymerase polypeptide6; other bacterial extracts can be used instead). This step removed antibodies unspecifieally bound to the
[37]
LOCALIZATIONOF Rab PROTEINS
403
peptide column. To prepare the lysate, bacteria from a 40-ml overnight culture are spun and lysed in 5 ml of 50 m M Tris-HC1, pH 8.0, 5.0 m M ethylenediaminetetraacetic acid (EDTA), 0.2 mg/ml lysozyme for 30 min at 37 °. After addition of Triton X-100 to a concentration of 1% (v/v) the extract is ultrasonified. Insoluble aggregates are pelleted in a mierofuge. One milliliter of the supernatant ( - 3 0 mg/ml) is used in the coupling reaction performed as described above. Coupling efficiency is monitored by analyzing samples of the lysate before and after the coupling reaction by polyacrylamide gel electrophoresis. The affinity-purified antibodies are characterized by immunoblot analysis and by immunofluorescence staining. Different amounts ( 1 rig- 1 #g) of recombinant Rab proteins produced in E. coli are used to titer the affinity-purified antisera by Western blot analysis. Immunofluorescence Localization Analysis For this type of analysis we permeabilize cells using saponin as detergent prior to fixation. This procedure removes most of the cytosolic proteins and allows us to better visualize the membrane staining pattern. This method is particularly useful in the case oftransfected cells expressing high levels of Rab proteins (see below). In these cells the excess of cytosolic Rab protein can be washed off during the permeabilization step. Cells are grown on 10-ram round cover slips for 24 hr prior to treatment. Cells are washed once with PBS and permeabilized with 0.05% (w/v) saponin in 80 m M K+-piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), pH 6.8, 5 mMethylene glycol-bis(fl-aminoethylether)-N,N,N',N'tetraacetic acid (EGTA), 1 m M MgC12 for 5 min. After a 15-min fixation in 3% (w/v) formaldehyde in PBS, pH 7.4, cells are washed with 0.05% saponin in PBS for 5 min and free aldehyde groups quenched with 50 m M NH4C1 in PBS for 10 min. Cells are washed with 0.05% saponin in PBS for 5 min and then incubated with the first antibody in PBS-0.05% saponin for 20 min. Depending on the antiserum, antibodies are diluted at volume ratios of 1 : 10- 1:300. After rinsing the cells three times (15 min in total) primary antibody binding is visualized with goat anti-rabbit fluorescein isothiocyanate (FITC) or goat anti-rabbit RITC diluted in 0.05% saponinPBS for 20 min. After one wash in PBS-0.05% saponin and three washes in PBS (20 min in total), the cover slips can be mounted on glass sides in Mowiol (Hoechst, Frankfurt, Germany) and viewed.
Overexpression of Rab Proteins Using T7 RNA Polymerase Recombinant Vaccinia Virus System To facilitate the localization studies of Rab proteins we make use of a transient expression system based on the T7 RNA polymerase recombi-
404
IDENTIFICATION OF TRANSPORT INTERMEDIATES
[3 7]
FIG. 2. Immunofluorescence localization of Rab2 in untransfected and transfected cells. (A) BHK cells labeled with affinity-purified anti-Rab2 antibodies. The staining pattern of vesicular structures restricted to the perinuclear area corresponds to an intermediate compartment between the ER and the Golgi apparatus. ~ (B) BHK cells infected with the T7 RNA polymerase-recombinant vaccinia virus and transfected with Rab2 construct. The exogenous protein gives an intense signal close to the nucleus, a staining pattern similar to that of the endogenous protein.
nant vaccinia virus. 21 Cells are infected with this vaccinia virus and transfected with a plasmid containing the Rab eDNA under the control of the T7 promoter. We currently use plasmid pGEM 1 (Promega, Madison, WI) for these studies. This system has been useful in proving the specificity of the affinity-purified antibodies. We often obtain affinity-purified antibodies reacting well in Western blot experiments but giving artifactual staining by immunofluorescence analysis. When antisera are specific, in cells expressing the exogenous Rab protein, the staining pattern is similar and of higher signal intensity compared to that of nontransfected cells. Figure 2 shows an immunofluorescence analysis using anti-Rab2 antibodies of untransfected cells and cells overexpressing Rab2. For these experiments cells are split 18- 24 hr before transfection, so that on the day of transfection they are about 80% confluent. The cells are washed twice with serum-free medium and infected with T7 RNA polymerase-recombinant vaccinia virus. Infections are carried out with 3 - 5 pfu/cell at room temperature for 30 min with intermittent agitation. The cells are then washed twice with serum-free medium and transfected by lipofection using the DOTAP reagent according to the instructions of 2~ T. T. Fuerst, E. G. Niles, F. W. Studier, and B. Moss, Proc. Natl. Acad. Sci. USA 83, 8122 (1986).
[37]
LOCALIZATION OF R a b PROTEINS
405
the manufacturer (Boehringer Mannheim, Indianapolis, IN). To obtain high transfection efficiencies (up to 95%) 5 we use plasmids purified twice on CsC1 density gradients and dialyzed against 0.1 × TE (1.0 m M Tris, pH 7.5, 0.1 m M EDTA). Cells are incubated for 3 - 4 hr at 37 ° in a 5% CO2 incubator and processed for immunofluorescence microscopy (Fig. 2) or immunoelectron microscopy. Longer incubation times lead to accumulation of Rab protein, posttranslationally unmodified in the cytosol. This does not affect the immunotluorescence analysis owing to the permeabilization step. However, accumulation of cytosolic Rab protein should be avoided in the case of immunoelectron microscopic analysis (see below). I m m u n o e l e c t r o n Microscopic Localization of Rab Proteins
Practical Details We examined the intracellular location of Rab2, 5, and 7 in cultured cells. For this, affinity-purified anti-peptide antibodies were applied to thawed cryosections according to published procedures. 22-25 Pellets of BHK, NRK, or HeLa cells were prepared for cryosectioning as follows.26 Cultured cells on 6-cm culture dishes were washed with icecold PBS and then were removed from the culture dish by incubation on ice with 1 ml of 20- to 50-/zg/ml proteinase K in PBS. This is a relatively gentle procedure for removal of the cells from the dish. The cells start to detach from the dish in sheets after about 2 min; the cells do not noticeably round up and most surface antigens are unaffected by this treatment (G. Griffiths, personal communication, 1991). The detached cells were layered onto 200#1 of 8% (w/v) paraformaldehyde in 250 m M N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.4, and were pelleted in a microfuge at 1000 rpm for 1 rain at room temperature. The supernatant is then removed and replaced with fresh fixative as above without disturbing the pellet. After 30 rain at room temperature the cells were spun at maximum speed in the microfuge before replacing the supernatant once more with fresh fixative. To allow freezing of the cells without destructive ice crystal formation the cells must be cryoprotected. This is achieved by incubation of small pieces of the pellet with sucrose (2.1 M in PBS) for 15 - 30 min. The pieces 22 K. T. Tokuyasu, J. Cell Biol. 57, 551 (1973). 23 K. T. Tokuyasu, J. Ultrastruct. Res. 63, 287 (1978). 24 G. Grittiths, K. Simons, G. Warren, and K. T. Tokuyasu, this series, Vol. 96, p. 435. 2s G. Griitiths, A. MeDowell, R. Back, and J. Dubochet, J. Ultrastruct. Res. 89, 65 (1984). 26 j. Green, G. Grittiths, D. Louvard, P. Quinn, and G. Warren, J. Mol. Biol. 152, 663 (1981).
406
IDENTIFICATION OF TRANSPORT INTERMEDIATES
[37]
of pellet were then mounted on copper stubs and plunged into liquid nitrogen, the specimens were then ready for cryosectioning. This is performed at - 100 ° using glass or diamond knives. Sections were transferred from the knife to copper grids using a loop with a drop of 2.3 M sucrose in PBS. All labeling is carried out at room temperature with antibodies followed by protein A-gold (6- or 9-nm diameter) diluted in PBS containing 5% (w/v) fetal calf serum and 0.12% (w/v) glycine. After washing the grids with distilled water they are embedded in a methylcellulose/uranyl acetate (MC/UA) mixture. Excess MC/UA is then removed, leaving a thin film over the sections. After drying (a few minutes at room temperature) the grids are viewed at an accelerating voltage of 80 kV.
Specificity of Labeling The antibodies used for all electron microscopy (EM) analyses were first checked by Western blotting and by immunofluorescence. However, there is still the possibility that artifactual "sticking" of the antibody to intraceUular structures occurs. Normally the specificity is monitored by checking the amount of labeling on structures known to be negative for the antigen, for example, the nucleus. For anti-peptide antibodies, however, the specificity can often be unequivocably determined by incubating the antibody prior to applying it to sections with the specific peptide to which it was raised. In principle, this should abolish the labeling whereas incubation with the same concentration of a nonspecific peptide should have no effect. In the case of the anti-Rab antibodies, relatively high concentrations (0.1 mg/ml) of the specific peptide were required to inhibit binding. This presumably reflects the fact that the affinity-purified antibody recognizes a limited number of conformational forms that the peptide can attain in solution.27,28 This control alone cannot rule out that the antibody recognizes different proteins sharing the same epitopes, but this can be checked by experiments on overexpressing cells and Western blotting analyses as described above.
Colocalization Studies The unequivocal localization of any antigen to a particular compartment by morphology alone is often extremely difficult. Well-defined markers are then required for colocalization studies to identify the corn27 H. J. Dyson, M. Rance, R. A. Houghten, R. A. I_¢rner, and P. E. Wright, J. MoL BioL 201, 161 (1988). 28 H. J. Dyson, M. Ranee, R. A. Houghten, P. E. Wright, and R. A. Lerner, J. Mol. Biol. 201, 201 (1988).
[37]
LOCALIZATIONOF Rab PROTEINS
407
partment of interest. Both antibodies and endocytic tracers were used as markers for endocytic and exocytic compartments. Rab2 was found to colocalize with labeling for a 53-kDa antigen previously shown to be present in the putative intermediate compartment located between the rough endoplasmic reticulum (RER) and Golgi. 29 Double labeling was achieved by one of two methods. In the protein A method the sections were treated with 1 mg/ml protein A in PBS after the first antibody and protein A-gold incubation steps. This quenches any free antibody before the incubations with the second antibody followed by a different size of protein A-gold. In the second method, 3° the sections were instead incubated with 1% glutaraldehyde in PBS at the same stage in the labeling scheme to destroy the ability of the first antibody to bind protein A-gold. For the endocytic pathway well-characterized particulate markers were used to label previously defined compartments of the cells of interest? ~a2 Bovine serum albumin-gold was internalized for either a short time (5 min) to label early compartments or for longer times to label late endosomes and lysosomes.
Experiments on Vaccinia Virus-Infected/Transfected Cells Rab proteins are produced as soluble proteins that then appear to associate with the membrane of specific compartments. On overexpression of these proteins an increase of labeling associated with a specific compartment occurs before the cytoplasmic pool of soluble protein starts to increase. For immunoelectron microscopy a large fraction of the cell population should express the protein at a high level, as the sample size for electron microscopy is relatively small when compared to, for example, immunofluorescence. However, the time after transfection should not be too long, as a high level of cytosolic protein will obscure the signal associated with a specific membrane. One way to overcome this is to extract the cells with saponin before fixation as for immunofluorescence (see above). However, in our experience a simpler solution, which was more compatible with fine structure preservation, was to choose a transfection time when the membrane-associated signal of protein is increased but the cytosolic pool of the protein is still below detectable levels.
~9 A. Schweizer, J. A, M. Fransen, T. Bachi, L. Ginsel, and H. P. Hauri, J. Cell Biol. 107, 1643 (1988). 3o j. W. Slot, H. J. Geuze, S. Gigenack, G. E. Lienhard, and D. E. James, J. Cell Biol. 113, 123 (1991). 31 G. Griffiths, B. Hoflack, K. Simons, I. Mellman, and S. Kornfeld, Cell 52, 329 0988). 32 G. Gdfliths, R. Back, and M. Marsh, J. Cell Biol. 109, 2703 (1989).
