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Polarized Distribution of Endogenous Rac1 and RhoA at the Cell Surface

Michaely, Peter A., Mineo, Chieko, Ying, Yun-shu, Anderson, Richard G.W.·1999·Journal of Biological Chemistry
DOI 10.1074/jbc.274.30.21430
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FIG. 1 Rac1, RhoA are enriched in the caveolae membrane fraction. Cell lysates from serum-starved Rat-1B fibroblasts were prepared using method 1. Postnuclear supernatant ( PNS ), cytosol ( CYT ), plasma membrane ( PM ), noncaveolae membrane ( NCM ), and caveolae membrane ( CM ) were separated by gel electrophoresis (3 m g/lane), transferred to PVDF membrane, and immunoblotted with the indicated antibodies.
FIG. 1 Rac1, RhoA are enriched in the caveolae membrane fraction. Cell lysates from serum-starved Rat-1B fibroblasts were prepared using method 1. Postnuclear supernatant ( PNS ), cytosol ( CYT ), plasma membrane ( PM ), noncaveolae membrane ( NCM ), and caveolae membrane ( CM ) were separated by gel electrophoresis (3 m g/lane), transferred to PVDF membrane, and immunoblotted with the indicated antibodies.CC-BY · From the PDF
FIG. 2 Immunogold co-localization of RhoA and caveolin-1. Plasma membranes from the upper surface of normal human fibroblasts were attached to formvar-coated grids as described (11) and used in either single labeling ( A and C ) or double labeling ( B and D ) protocols. A and C , membranes were incubated for 60 min in the presence of either a RhoA mAb ( A ) or a caveolin-1 pAb ( C ) followed by gold-conjugated anti-mouse IgG (5 nm) or gold-conjugated anti-rabbit IgG (15 nm), respectively. B and D , membranes were double-labeled with a caveolin-1 pAb (15-nm gold) and a RhoA mAb (5-nm gold) ( B ) or a caveolin-1 pAb (15-nm gold) and a clathrin mAb (5 nm) ( D ) as described. Bar , 0.2 m m.
FIG. 2 Immunogold co-localization of RhoA and caveolin-1. Plasma membranes from the upper surface of normal human fibroblasts were attached to formvar-coated grids as described (11) and used in either single labeling ( A and C ) or double labeling ( B and D ) protocols. A and C , membranes were incubated for 60 min in the presence of either a RhoA mAb ( A ) or a caveolin-1 pAb ( C ) followed by gold-conjugated anti-mouse IgG (5 nm) or gold-conjugated anti-rabbit IgG (15 nm), respectively. B and D , membranes were double-labeled with a caveolin-1 pAb (15-nm gold) and a RhoA mAb (5-nm gold) ( B ) or a caveolin-1 pAb (15-nm gold) and a clathrin mAb (5 nm) ( D ) as described. Bar , 0.2 m m.CC-BY · From the PDF
FIG. 3 Effects of PDGF and LPA on the recruitment of Rac1 and RhoA to caveolae membrane of Rat-1B cells. Serum-starved Rat-1 cells were incubated in the presence of either 20 ng/ml PDGF or 100 m M LPA in Dulbecco's modified Eagle's medium at 37 °C for the indicated times. Caveolae fractions were then prepared from cell lysates made by method 1, separated by gel electrophoresis (5 m g/lane), transferred to PVDF membrane, and immunoblotted with antibodies directed against the indicated proteins. Cav1 , caveolin-1.
FIG. 3 Effects of PDGF and LPA on the recruitment of Rac1 and RhoA to caveolae membrane of Rat-1B cells. Serum-starved Rat-1 cells were incubated in the presence of either 20 ng/ml PDGF or 100 m M LPA in Dulbecco's modified Eagle's medium at 37 °C for the indicated times. Caveolae fractions were then prepared from cell lysates made by method 1, separated by gel electrophoresis (5 m g/lane), transferred to PVDF membrane, and immunoblotted with antibodies directed against the indicated proteins. Cav1 , caveolin-1.CC-BY · From the PDF
FIG. 4 Caveolae fractions are enriched in ezrin, moesin and CD44. Lysates of serum-starved normal human fibroblasts were prepared by method 1 and used to isolate postnuclear supernatant ( PNS ), cytosol ( CYT ), plasma membrane ( PM ), noncaveolae membrane ( NCM ), and caveolae membrane ( CM ) as described. Samples (5 m g/lane) of each fraction were separated by gel electrophoresis, transferred to PVDF membranes, and immunoblotted with the indicated antibodies. Equivalent results were obtained with the ezrin and moesin antibodies using fractions from Rat-1B cells. The CD44 antibody, however, did not recognize any band in Rat-1 cells.
FIG. 4 Caveolae fractions are enriched in ezrin, moesin and CD44. Lysates of serum-starved normal human fibroblasts were prepared by method 1 and used to isolate postnuclear supernatant ( PNS ), cytosol ( CYT ), plasma membrane ( PM ), noncaveolae membrane ( NCM ), and caveolae membrane ( CM ) as described. Samples (5 m g/lane) of each fraction were separated by gel electrophoresis, transferred to PVDF membranes, and immunoblotted with the indicated antibodies. Equivalent results were obtained with the ezrin and moesin antibodies using fractions from Rat-1B cells. The CD44 antibody, however, did not recognize any band in Rat-1 cells.CC-BY · From the PDF
FIG. 5 GTP g S stimulates movement of RhoA from cytosol to caveolae. Cell lysates of Rat-1B cells were prepared by method 2 and divided into equal portions (1 ml). Either no additions were made to these lysates ( lanes 1 , 2 , 6 , and 7 ) or 1 mM GDP ( lanes 3 and 8 ), 1 mM GTP ( lanes 4 and 9 ), or 100 m M GTP g S ( lanes 5 and 10 ) was added, and the samples were incubated either at 4 °C ( lanes 1 and 6 ) or 30 °C ( lanes 2-5 and 7-10 ) for 30 min. Cytosol ( lanes 1-5 ) and caveolae ( lanes 6-10 ) fractions were prepared, separated by gel electrophoresis (cytosol, 50 m g/lane; caveolae, 5 m g/lane), transferred to PVDF membrane, and probed with anti-RhoA IgG.
FIG. 5 GTP g S stimulates movement of RhoA from cytosol to caveolae. Cell lysates of Rat-1B cells were prepared by method 2 and divided into equal portions (1 ml). Either no additions were made to these lysates ( lanes 1 , 2 , 6 , and 7 ) or 1 mM GDP ( lanes 3 and 8 ), 1 mM GTP ( lanes 4 and 9 ), or 100 m M GTP g S ( lanes 5 and 10 ) was added, and the samples were incubated either at 4 °C ( lanes 1 and 6 ) or 30 °C ( lanes 2-5 and 7-10 ) for 30 min. Cytosol ( lanes 1-5 ) and caveolae ( lanes 6-10 ) fractions were prepared, separated by gel electrophoresis (cytosol, 50 m g/lane; caveolae, 5 m g/lane), transferred to PVDF membrane, and probed with anti-RhoA IgG.CC-BY · From the PDF
FIG. 6 Exoenzyme C3 shifts RhoA from the caveolae to the noncaveolae membrane. Cell lysates of serum-starved Rat-1 cells were prepared by method 2. The lysates were incubated in the presence ( lanes 3 and 6 ) or absence ( lanes 1 , 2 , 4 , and 5 ) of exoenzyme C3 (5 m g/ml) for 10 min at 30 °C followed by an incubation in the absence ( lane 1 ) or presence ( lanes 2-6 ) of the indicated nucleotide (1 mM GDP, 1 mM GDP, 1 mM GTP, 100 m M GTP g S) for an additional 30 min at 30 °C. The lysate was then fractionated into cytosol (50 m g/lane), plasma membrane (20 m g/lane), noncaveolae membrane (20 m g/lane), and caveolae membrane (5 m g/lane), separated by gel electrophoresis, transferred to PVDF membrane, and immunoblotted with anti-RhoA IgG. The two RhoA bands in lane 6 correspond to the ribosylated RhoA ( upper band ) and a ribosylated degradation product of RhoA (compare with the degraded and undegraded RhoA bands in plasma membrane, lane 5 ). Exposure times were as follows: cytosol, 10 s; plasma membrane, 1 min; noncaveolae membrane, 3 min; caveolae membrane, 10 s. PNS , postnuclear supernatant; CYT , cytosol; PM , plasma membrane; NCM , noncaveolae membrane; CM , caveolae membrane.
FIG. 6 Exoenzyme C3 shifts RhoA from the caveolae to the noncaveolae membrane. Cell lysates of serum-starved Rat-1 cells were prepared by method 2. The lysates were incubated in the presence ( lanes 3 and 6 ) or absence ( lanes 1 , 2 , 4 , and 5 ) of exoenzyme C3 (5 m g/ml) for 10 min at 30 °C followed by an incubation in the absence ( lane 1 ) or presence ( lanes 2-6 ) of the indicated nucleotide (1 mM GDP, 1 mM GDP, 1 mM GTP, 100 m M GTP g S) for an additional 30 min at 30 °C. The lysate was then fractionated into cytosol (50 m g/lane), plasma membrane (20 m g/lane), noncaveolae membrane (20 m g/lane), and caveolae membrane (5 m g/lane), separated by gel electrophoresis, transferred to PVDF membrane, and immunoblotted with anti-RhoA IgG. The two RhoA bands in lane 6 correspond to the ribosylated RhoA ( upper band ) and a ribosylated degradation product of RhoA (compare with the degraded and undegraded RhoA bands in plasma membrane, lane 5 ). Exposure times were as follows: cytosol, 10 s; plasma membrane, 1 min; noncaveolae membrane, 3 min; caveolae membrane, 10 s. PNS , postnuclear supernatant; CYT , cytosol; PM , plasma membrane; NCM , noncaveolae membrane; CM , caveolae membrane.CC-BY · From the PDF
FIG. 7 Recruitment of RhoA to purified caveolae membrane. A , cell lysates from serum-starved Rat-1B cells were prepared by method 2. Cytosol, noncaveolae membrane, and caveolae membrane were isolated as described. Cytosol (250 m g) was mixed with 10 m g of either noncaveolae or caveolae membrane in buffer C and incubated for the indicated time at 30 °C. At the end of the incubation, membranes were separated from cytosol by centrifugation. The entire membrane pellet and a 40m l sample of the supernatant fraction were separated by gel electrophoresis, transferred to PVDF, and immunoblotted with antiRhoA IgG. B , cytosol (250 m g) and membranes (10 m g) were mixed and incubated in the presence ( lanes 3 , 6 , and 9 ) or absence ( lanes 1 , 2 , 4 , 5 , 7 , and 8 ) of exoenzyme C3 (5 m g) for 10 min at 30 °C before the addition of either 1 mM GTP or 100 m M GTP g S and further incubation for 30 min at 30 °C. The plasma membrane ( PM ), noncaveolae ( NCM ), and caveolae ( CM ) were isolated, separated by gel electrophoresis, transferred to PVDF membrane, and immunoblotted with anti-RhoA IgG.
FIG. 7 Recruitment of RhoA to purified caveolae membrane. A , cell lysates from serum-starved Rat-1B cells were prepared by method 2. Cytosol, noncaveolae membrane, and caveolae membrane were isolated as described. Cytosol (250 m g) was mixed with 10 m g of either noncaveolae or caveolae membrane in buffer C and incubated for the indicated time at 30 °C. At the end of the incubation, membranes were separated from cytosol by centrifugation. The entire membrane pellet and a 40m l sample of the supernatant fraction were separated by gel electrophoresis, transferred to PVDF, and immunoblotted with antiRhoA IgG. B , cytosol (250 m g) and membranes (10 m g) were mixed and incubated in the presence ( lanes 3 , 6 , and 9 ) or absence ( lanes 1 , 2 , 4 , 5 , 7 , and 8 ) of exoenzyme C3 (5 m g) for 10 min at 30 °C before the addition of either 1 mM GTP or 100 m M GTP g S and further incubation for 30 min at 30 °C. The plasma membrane ( PM ), noncaveolae ( NCM ), and caveolae ( CM ) were isolated, separated by gel electrophoresis, transferred to PVDF membrane, and immunoblotted with anti-RhoA IgG.CC-BY · From the PDF
FIG. 8 Localization of RhoA and Rac1 to caveolae does not depend on effector interactions. A , Rat-1 cells were transiently transfected either with Myc-tagged RhoA (V14/A39) or Rac1 (L61/A37) cDNA. Cells were grown to confluence and fractionated into postnuclear supernatant ( PNS ), cytosol ( CYT ), plasma membrane ( PM ), noncaveolae membrane ( NCM ), or caveolae membrane ( CM ). Samples were separated and immunoblotted with a Myc mAb. B , cell lysates were prepared from Rat-1 cells and incubated in the presence of either buffer, GTP g S or C3 plus GTP g S, using the same conditions as described in the legend to Fig. 4. The lysates were fractionated, and each fraction was processed for immunoblotting with either a ROK a mAb ( lanes 4-6 ) or a RhoA mAb ( lanes 1-3 ).
FIG. 8 Localization of RhoA and Rac1 to caveolae does not depend on effector interactions. A , Rat-1 cells were transiently transfected either with Myc-tagged RhoA (V14/A39) or Rac1 (L61/A37) cDNA. Cells were grown to confluence and fractionated into postnuclear supernatant ( PNS ), cytosol ( CYT ), plasma membrane ( PM ), noncaveolae membrane ( NCM ), or caveolae membrane ( CM ). Samples were separated and immunoblotted with a Myc mAb. B , cell lysates were prepared from Rat-1 cells and incubated in the presence of either buffer, GTP g S or C3 plus GTP g S, using the same conditions as described in the legend to Fig. 4. The lysates were fractionated, and each fraction was processed for immunoblotting with either a ROK a mAb ( lanes 4-6 ) or a RhoA mAb ( lanes 1-3 ).CC-BY · From the PDF
FIG. 9 Prenylation is sufficient to localize a protein to caveolae. Cells, transiently expressing EGFP, EGFP-CLVL, EGFP-CLLL, EGFP-RRGKKKCLVL, or EGFP-KKRKRKCLLL, were harvested and fractionated as described. Samples (5 m g) of postnuclear supernatant ( PNS ), cytosol ( CYT ), plasma membrane ( PM ), noncaveolae membrane ( NCM ), and caveolae membrane ( CM ) were processed for immunoblotting using a green fluorescent protein mAb.
FIG. 9 Prenylation is sufficient to localize a protein to caveolae. Cells, transiently expressing EGFP, EGFP-CLVL, EGFP-CLLL, EGFP-RRGKKKCLVL, or EGFP-KKRKRKCLLL, were harvested and fractionated as described. Samples (5 m g) of postnuclear supernatant ( PNS ), cytosol ( CYT ), plasma membrane ( PM ), noncaveolae membrane ( NCM ), and caveolae membrane ( CM ) were processed for immunoblotting using a green fluorescent protein mAb.CC-BY · From the PDF

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Metadata

Authors
Michaely, P. A., Mineo, C., Ying, Y.-S., & Anderson, R. G. W.
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
1999-07
Volume
274
Issue
30
Pages
21430-21436
ISSN
0021-9258
DOI
10.1074/jbc.274.30.21430
Cited by
139
References
0
Language
en
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Michaely, P. A., Mineo, C., Ying, Y.-S., & Anderson, R. G. W. (1999). Polarized Distribution of Endogenous Rac1 and RhoA at the Cell Surface. Journal of Biological Chemistry. https://doi.org/10.1074/jbc.274.30.21430