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The small GTPase Rab22 interacts with EEA1 and controls endosomal membrane trafficking

¹ Department of Molecular Medicine, National Public Health Institute (KTL), Biomedicum, PO Box 104, FIN-00251 Helsinki, Finland
² Department of Biochemistry, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway

View larger version (57K): [in a new window]   Fig. 1. Localization of Rab22a and its mutant forms in BHK-21 cells. Rab22a or the Q64L and S19N mutants were expressed by transfection of constructs in pcDNA3.1, followed by immunofluorescent double stainings to visualize the expressed protein and compartmental markers by confocal microscopy analysis. Wild-type Rab22a (A,D,G) localized on the surface of large vacuole-appearing structures, as well as on the plasma membrane. The vacuolar elements were positive for EEA1 (B) but did not contain LAMP-1 (E) or Rab11 (H). The Q64L mutant (J) showed a similar distribution and colocalized with EEA1 (K); the S19N mutant (M) displayed in many cells a diffuse cytosolic appearing staining often with additional brightly stained structures of variable size and shape in the perinuclear region. This mutant did not affect the intracellular distribution of EEA1 or show specific colocalization with it (N). Overlays are shown in panels C,F,I,L and O. Bar=10 µm.

View larger version (18K): [in a new window]   Fig. 2. In vitro verification of the Rab22a-EEA1 interaction. GST-Rab5a and Rab22a fusion proteins, or GST as a negative control, were bound to glutathione sepharose beads, loaded with either GDP or GTPS and incubated with a MBP-EEA1 (aa 1-209) fusion protein as descibed in the Materials and Methods. The binding of MBP-EEA1 was visualized by SDS-PAGE and western blotting with anti-EEA1 antibodies (the top panels). Specific binding of MBP-EEA1 to the GTPS-bound forms of both Rab5a and Rab22a was observed; the signal for the GDP-loaded proteins was very weak. Background binding of MBP-EEA1 to plain GST was undetectable. Visualization of the fusion proteins on the filters by Ponceau staining (the bottom panels) reveals that the amount of Rab22a required for a given signal intensity is markedly lower than that for Rab5a.

View larger version (48K): [in a new window]   Fig. 3. Rab22a overexpression makes EEA1 resistant to Wortmannin treatment. Wild-type myc-Rab22a was expressed in BHK-21 cells for 14 hours using a recombinant SFV, the cells were then incubated for 30 minutes in the absence (-Wortmannin) or the presence (+Wortmannin) of 100 nM Wortmannin and prepared for double immunofluorescence microscopy with anti-myc and human anti-EEA1 antibodies. In untransfected cells (arrows), EEA1 showed in the absence of Wortmannin (A-B) a characteristic staining of small early endosomal structures and localized in transfected cells on the large vacuole-like Rab22a-positive endosomes. Upon Wortmannin treatment (C,D), a majority of EEA1 was lost from the endosomes of untransfected cells (arrowheads), but intense EEA1 staining remained detectable on the large Rab22a-positive endosomes of the tranfected cells. Bar=10 µm.

View larger version (84K): [in a new window]   Fig. 4. Effects of Rab22a overexpression on the uptake and intracellular distibution of Alexa-transferrin (Tfn). HeLa cells were infected with the Ankara strain Vaccinia T7 virus, followed by transfection of myc-tagged Rab22a wt, Q64L or S19N constructs in pGEM-1. Cell-surface binding and uptake (30 min) of Alexa-Tfn were then carried out as described in Materials and Methods, and the cells were processed for confocal immunofluorescence microscopy. Immunostaining for Rab22a is shown in A,C,F,I; Alexa-Tfn fluorescence in panels B,D,G,J; and overlays in E,H,K. In non-transfected cells (A,B), the Tfn was observed in characteristic endosomal structures. The volume or distribution of these structures was not significantly affected by expression of wt Rab22a (C-E). However, in cells expressing Rab22a Q64L (F-H) the Alexa-Tfn containing endosomes were redistributed to clusters near the leading edge. The Rab22a S19N mutant (I-K) had no detectable effect on the volume or distribution of internalized Alexa-Tfn. The wt Rab22a (E, arrowheads), but not the mutant proteins (H,K), showed a partial colocalization with Alexa-Tfn. Bar=10 µm.

View larger version (59K): [in a new window]   Fig. 5. Effects of Rab22a on the uptake and degradation of rhodamine-conjugated epidermal growth factor (rho-EGF). Hep2 cells were infected with the Ankara strain Vaccinia T7 virus, followed by transfection of myc-tagged Rab22a wt, Q64L or S19N constructs in pGEM-1. Internalization (1 hour) and chase (3 hours) of rho-EGF were then carried out as described in Materials and Methods, and the cells were processed for confocal immunofluorescence microscopy. The rho-EGF fluorescence is shown in panels A,D,G,J,M,P, immunostaining for Rab22a in B,E,H,K,N,Q, and overlays in C,F,I,L,O,R. After the 1 hour internalization period, the rho-EGF was found in intracellular endocytic structures, the volume and distribution of which were highly similar in untransfected cells (arrows) and those expressing wt Rab22a (A-C), Rab22 Q64L (G-I) or Rab22 S19N (M-O). After 3 hours chase, the rho-EGF fluorescence had disappeared from untransfected cells and those expressing Rab22 S19N (P-R), although it remained detectable in cells expressing Rab22a wt (D-F) or the Q64L mutant (J-L). Colocalization of Rab22a wt (C,F) but not Q64L (I,L) was detected with the rho-EGF positive structures. Bar=10 µm.

View larger version (8K): [in a new window]   Fig. 6. Quantitation of the effects of Rab22a on rho-EGF degradation in Hep2 cells. Hep2 cells were transfected with pGEM-myc, -mycRab22a wt, -mycRab22a Q64L or -mycRab22a S19N using the Vaccinia system. The cells were incubated with rhodamine-labelled EGF for 1 hour at 37°C, followed by a 3 hour chase at 37°C before fixation and staining with an anti-myc antibody. Transfected cells were visualized by confocal immunofluorescence microscopy, and 150 cells on separate coverslips were counted for the presence of rho-EGF signal after a 3 hour chase. The data are presented as the % (±s.e.m.) of transfected cells having a visible rho-EGF signal after the chase.

View larger version (68K): [in a new window]   Fig. 7. The effect of Rab22a on the endocytic trafficking of internalized TRITC-dextran. BHK-21 cells were transfected (for 24 hours) with the mycRab22a wt, Q64L or S19N cDNAs (indicated on the left) in pcDNA3.1 or the plain vector plasmid (Mock), followed by 1 hour internalization of TRITC-dextran and a 3 hour chase in the absence of the compound. The cells were then immunostained for EEA1 and LAMP-1 and viewed under a confocal microscope. TRITC-dextran fluorescence is shown in panels A,F,K,P, staining for EEA1 in B,G,L, LAMP-1 staining in C,H,M,R, and Rab22a staining in Q. Pairwise overlays of the channels are shown in D,E,I,J,N,O,S and T. In mock-transfected cells the TRITC-dextran was transported to structures that contained LAMP-1 and showed no overlap with the early endosome marker EEA1 during the chase (A-E). In cells expressing Rab22a wt, the transport of TRITC-dextran proceeded in a similar fashion, despite the presence of abnormally large EEA1-positive vacuolar endosomes induced by the GTPase (F-J). In a majority of cells expressing Rab22a Q64L, however, the fluorescent dextran ended up in large vacuolar endosomes which now contained markers of both early (EEA1) and late (LAMP-1) endocytic compartments (K-O). Expression of the S19N mutant had no detectable effect on the trafficking of TRITC-dextran (P-T). In Q, staining for the Rab is shown instead of EEA1, as S19N-expressing cells cannot be identified on the basis of their endosome morphology. Bar=10 µm.

View larger version (73K): [in a new window]   Fig. 8. The effect of Rab22a on the transport of aspartylglucosaminidase (AGA) to lysosomes. BHK-21 cells were double transfected for 24 hours with Rab22a wt or mutant cDNAs (indicated on the left) in pcDNA3.1 or the plain vector plasmid (Mock) and human AGA cDNA in pSVPoly, followed by a 3 hour chase in the presence of cycloheximide (25 µg/ml). Thereafter the cells were triple immunostained for AGA, EEA1 and LBPA, and viewed under a confocal microscope. Staining for AGA is shown in panels A,F,K,P, for EEA1 in B,G,L,Q, and for LBPA in C,H,M,R. Pairwise overlays of the channels are shown in D,E,I,J,N,O,S and T. In mock-transfected cells (A-E), the AGA was chased into punctate structures that colocalized with the late endosome/lysosome marker LBPA but not with EEA1. In a majority of cells expressing the wt Rab22a, the trafficking of AGA to late endocytic compartments proceeded in a similar fashion, despite the presence of enlarged EEA1-positive endosomes induced by the GTPase (F-J). In cells expressing the Q64L mutant, however, AGA accumulated in large vacuolar endosomes, which contained both early (EEA1) and late (LBPA) endosomal markers (K-O). Expression of the Rab22a S19N mutant had no significant effect on the transport of AGA to late endocytic compartments (P-T). Bar=10 µm.

View larger version (63K): [in a new window]   Fig. 9. The effect of Rab22a expression on Golgi apparatus morphology in HeLa cells. Rab22a or its mutant forms were expressed with the Vaccinia T7 system, and the cells were processed for immunofluorescence microscopy; triple stainings with antibodies against GM130, EEA1 and the expressed Rab were carried out. A-E: cells expressing wt Rab22a; F-J: cells expressing the Q64L mutant; K-O, cells expressing the S19N mutant. A,F,K, GM130 staining; B,G,L, EEA1 staining; C,H,M, Rab22a staining. Overlays are shown in D,E,I,J,N and O. The wt Rab22a and the Q64L mutant cause a complete fragmentation of the Golgi apparatus and colocalize significantly with both GM130 and EEA1. No significant mixing of the Golgi and early endosome markers is detected. An untransfected cell with a compact Golgi is seen, for example, in the upper right-hand corner of panel A. The S19N mutant shows colocalization with GM130 but does not induce any redistribution of the Golgi apparatus. Bar=10 µm.

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