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First published online 4 April 2006
doi: 10.1242/jcs.02885

Journal of Cell Science 119, 1703-1714 (2006)
Published by The Company of Biologists 2006
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Evidence for a role of transmembrane protein p25 in localization of protein tyrosine phosphatase TC48 to the ER

Vijay Gupta and Ghanshyam Swarup*

Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India


Figure 1
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  		Fig. 1. Yeast two-hybrid analysis reveals an interaction between TC48 and p23/p25. (A) The PJ69-4A yeast strain was co-transformed with TC-PTP (TC48 or TC45) GAL4-DNA-binding-domain fusion constructs and p23 or p25 GAL4-activation-domain fusion constructs obtained in the yeast two hybrid screen. Transformants were grown on plates with (Ade+) or without (Ade-) adenine and also on plates supplemented with ß-galactosidase substrate X-gal (X-gal+). Ade- is the yeast-medium-plate lacking Leu, Trp and Ade, in which activation of the adenine reporter gene is being tested. Ade+ is the yeast-medium-plate lacking Leu and Trp, selecting only for the presence of plasmids to be tested for interaction. (B) Interaction of the C-terminal 66 aa (C-66) of TC-48 with p23 and p25. (C) Schematic representation of TC48 and TC45. The C66-TC48 construct encodes the C-terminal 66 aa of TC48 (from 350-415); aa 1-381 are identical in TC48 and TC45. (C) Control plasmid pGBKT-7; TC45-IP is a positive control for the TC45-interacting protein.

 

Figure 2
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  		Fig. 2. Interaction of C-terminal sequences in p23 and p25 with TC48. (A) Schematic representation of p23 and p25 and various deletion mutants cloned in yeast vector pAct-II. LD, lumenal domain; CC, coiled-coil domain; TM, transmembrane domain; CT, cytoplasmic tail. (B) Yeast strain PJ69-4A was co-transformed with TC48 (cloned in pGBKT-7) and various mutants of p23 or p25. Transformants were grown on plates with (Ade+) or without adenine (Ade-).

 

Figure 3
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  		Fig. 3. Western blots of cell lysates and immunoprecipitates (IP). Interaction of TC48 with p23 and p25 in mammalian cells. Cos-1 cells were co-transfected with GFP-TC48 and myc-p25 (A), or GFP-TC48 and myc-p23 (B) expression constructs. After 36 hours of transfection, cell lysates were prepared and immunoprecipitation with anti-myc or control antibody was performed as described in Materials and Methods. Input is 5% of the total cell lysate; IgG(H) is the band generated by the heavy chain of antibodies used for immunoprecipitation. G11 is the mouse monoclonal antibody that recognizes overexpressed TCPTP. (C) Cos-1 cells were transfected with GFP-TC48 and, after 36 hours, cell lysates were subjected to immunoprecipitation with control antibody (C) or antisera that recognize native p23 or p25 proteins. Western blot of immunoprecipitates was done with GFP monoclonal antibody. Lysates from untransfected cells were also used as controls for immunoprecipitation.

 

Figure 4
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  		Fig. 4. Time course of TC48 localization in Cos-1 cells. (A) Cos-1 cells transfected with GFP-TC48 were fixed at 12 hours, 24 hours and 48 hours after transfection and stained with anti-giantin mouse monoclonal antibody (red) to stain the Golgi complex, and observed by confocal microscopy. Yellow colour generated in the merged images indicates colocalization of TC48 and Golgi-localized giantin. Notice the progressive increase of GFP-TC48 localization in ER-like reticular structures with increasing time. (B) Cos-1 cells overexpressing GFP-TC48 were fixed after 12 hours of transfection and stained with mouse monoclonal anti-ERGIC-53 antibody. Cy3-conjugated secondary antibodies were used to visualize ERGIC (red). Yellow colour generated in the merged image indicates colocalization of TC48 and ERGIC-localized ERGIC-53. (C) Cos-1 cells were transfected with plasmids expressing VSVG-GFP and HA-TC48. After 15 hours at 40°C, the cells were shifted to 15°C for 3 hours. Cells were fixed and stained with HA-tag antibody (red) and analyzed by confocal microscopy. Bars, 10 µm.

 

Figure 5
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  		Fig. 5. Effects of drugs on the localization of TC48. (A) Cycloheximide-chase causes depletion of the Golgi pool of TC48. Cos-1 cells transfected with GFP-TC48 were allowed to express the exogenous cDNA for 12 hours at 37°C after which incubation was continued in the presence of cycloheximide (10 µg/ml) for 6 hours (Cyc+) and in the absence of cycloheximide (UT). Cells were fixed and stained with anti-giantin mouse monoclonal antibody (red) to stain the Golgi complex and observed by confocal microscopy (upper panels). Yellow colour in merged images indicates colocalization of TC48 and Golgi-localized giantin, and is evident in untreated cells (UT) but not in cells chased for 6 hours with cycloheximide (Cyc+). Notice, after 6 hours of cycloheximide-chase the Golgi pool of TC48 appears depleted. GFP-TC48 transfected and cycloheximide-treated or untreated cells were also stained with calnexin (ER marker) antibody (lower panels). (B) Nocodazole and BFA disturb the Golgi localization of TC48. Cos-1 cells expressing GFP-TC48 were treated, after 12 hours of transfection, with 5 µg/ml nocodazole at 37°C for 2 hours (Noc+) or with 5 µg/ml BFA for 30 minutes (BFA+). After fixation, cells were stained with giantin antibody and visualized by confocal microscopy. Bars, 10 µm.

 

Figure 6
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  		Fig. 6. Overexpression of p23 and p25 affect localization of TC48. GFP-TC48 was transfected in Cos-1 cells; after 24 hours cells were stained with antibodies that recognize endogenous p23 (A) or p25 (D). Stained cells were examined by confocal microscopy. Cos-1 cells were co-transfected with GFP-TC48 and myc-p23 (B) or myc-p25 (E) and fixed after 12 hours of transfection. Fixed cells were stained with anti-myc mouse monoclonal antibody and Texas-Red-conjugated secondary antibody to visualize p23 and p25 proteins. The images shown are the middle optical sections taken, using the apotome fluorescence microscope. (C) GFP-p23- and rat TC48-expression plasmids were co-transfected in Cos-1 cells. After 24 hours cells were fixed, stained with G11 monoclonal antibody (red) and visualized by confocal microscopy. (F) Cells were transfected with myc-p25SS and GFP-TC48 and processed as in (E) after 12 hours of transfection. Bars, 10 µm.

 

Figure 7
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  		Fig. 7. Interaction of C-terminal sequences of TC48 with p23 and p25. (A) Yeast two-hybrid analysis was performed between various N-terminal deletion mutants of TC48 and p23 (pAct-II) or p25 (pAct-II). TC48 is the full-length TC48, whereas C40aa and C66aa are the C-terminal 40 aa and 66 aa of TC48 fused with the DNA-binding domain of Gal4 in pGBKT-7 (bait vector). pGBKT-7 and pAct-II are the empty Gal4-DNA-binding domain and Gal4-activation-domain-containing yeast two-hybrid expression vectors, respectively. (B) Cos-1 cells were co-transfected with GFP-C40 and myc-p23 (upper panels) or GFP-C66 and myc-p25 (lower panels) expression plasmids. After 36 hours cell lysates were prepared and immunoprecipitated with myc antibody or control antibody (C). Western blots of cell lysates and immunoprecipitates (IP) with GFP and myc antibodies are shown. Input is 5% total cell lysate.

 

Figure 8
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  		Fig. 8. Localization of GFP-C40 and GFP-C66 fusion proteins. Cos-1 cells transfected with vectors expressing GFP-C40 (A) or GFP-C66 (B) were allowed to express exogenous cDNA for 24 hours. Cells were fixed and stained with anti-giantin mouse monoclonal antibody (red) to stain the Golgi complex, and observed by confocal microscopy. Yellow colour indicates colocalization of GFP-C40 or GFP-C66 and Golgi-localized giantin. (C) GFP-C66 and myc-p25 expression plasmids were co-transfected in Cos-1 cells; after 12 hours of transfection the cells were fixed, stained with anti-myc antibody (red) and observed by confocal microscopy. (D) GFP-C40 and myc-p23 expression plasmids were co-transfected in Cos-1 cells; after 12 hours of transfection the cells were fixed, stained with myc antibody (red) and observed by confocal microscopy. Bars, 10 µm.

 

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© The Company of Biologists Ltd 2006