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First published online 12 August 2008
doi: 10.1242/jcs.026153

Journal of Cell Science 121, 2921-2929 (2008)
Published by The Company of Biologists 2008
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Multiple functions encoded by the N-terminal PAT domain of adipophilin

David J. Orlicky1, Greg DeGala2, Carrie Greenwood2, Elise S. Bales2, Tanya D. Russell2,3 and James L. McManaman2,3,4,5,*

1 Department of Pathology, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO 80045, USA
2 Department of Obstetrics and Gynecology, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO 80045, USA
3 Graduate Program in Molecular Biology, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO 80045, USA
4 Graduate Program in Reproductive Sciences, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO 80045, USA
5 Graduate Program in Cell Biology, Stem Cells and Development, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO 80045, USA


Figure 1
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  		Fig. 1. Effects of oleic acid on the stability of ADPH variants in stably transfected HEK 293 cells. (A) Immunoblot analysis of parental 293 cultures and cultures stably expressing ADPH variants following a 20-hour incubation in the absence (C) or presence of 300 µM oleic acid (OA). Parental 293 culture extracts were immunoblotted with chicken anti-human TIP47, extracts of cultures expressing ADPH were immunoblotted with guinea pig anti-mouse ADPH, and extracts of cultures expressing VSV-tagged forms of ADPH were immunoblotted with mouse anti-VSV. Immunoblots of whole gels are shown to demonstrate specificity and the absence of breakdown products. The boxed regions beneath show the results of immunoblots that were stripped and re-probed for β-actin to control for sample loading. (B) Representative anti-VSV immunoblots of cultures expressing ADPH[fl]-VSV or {Delta} 2,3 ADPH-VSV following a 20-hour incubation in the indicated concentrations of OA. Anti-β-actin immunoblots are shown beneath the anti-VSV immunoblots. (C) Quantitation of the immunoblots in B. The data show relative steady-state levels of ADPH[fl]-VSV (white bars) and {Delta} 2,3 ADPH-VSV (black bars) normalized to β-actin. The values are means ± s.d. for triplicate cultures. (D) Effects of OA and/or triacsin C on ADPH[fl]-VSV and {Delta} 2,3 ADPH-VSV levels. Representative anti-VSV immunoblots of extracts of ADPH[fl]-VSV or {Delta} 2,3 ADPH-VSV cell lines following a 20-hour incubation in unsupplemented basal culture media (Cont), or in basal media supplemented with 300 µM OA, 5 µM triacsin C (Triac-C), or with 300 µM OA plus 5 µM triacsin C (Triac-C + OA). β-actin loading controls for each sample are shown beneath the anti-VSV blots. Representative immunoblots are from a single well of triplicate cultures treated as indicated. All experiments were repeated at least twice, with similar results.

 

Figure 2
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  		Fig. 2. N-terminally modified ADPH is resistant to proteolytic degradation. (A) Effects of MG132 concentration on steady-state levels of ADPH variants and TIP47. Representative immunoblots of parental 293 cultures and ADPH variant cultures following a 20-hour incubation in unsupplemented basal culture media containing increasing concentrations of MG132. Anti-β-actin immunoblots are shown beneath the anti-VSV immunoblots. The relative effects of increasing MG132 concentrations on steady-state levels of each ADPH variant and endogenous TIP47 are shown alongside each immunoblot. The values are averages ± s.d. for six samples, each normalized to β-actin. (B) Effects of MG132 and OA on steady-state levels of ADPH[fl]-VSV, {Delta} 2,3 ADPH-VSV and TIP47. Representative immunoblots of parental 293 cultures and ADPH[fl]-VSV and {Delta} 2,3 ADPH-VSV cultures following a 20-hour incubation in basal culture media (control), or in basal culture media supplemented with 3 µM MG132 (MG), 300 µM OA, or 3 µM MG132 plus 300 µM OA (MG/OA). The relative effects of these treatments on steady-state levels of each protein are shown alongside each immunoblot. The values are averages ± s.d. for six samples, each normalized to β-actin. (C) Effects of lysosomal protease inhibitors on steady-state levels of ADPH[fl]-VSV, {Delta} 2,3 ADPH-VSV and endogenousTIP47. Representative immunoblots of parental 293 cultures and ADPH[fl]-VSV and {Delta} 2,3 ADPH-VSV cultures after a 20-hour incubation with the indicated inhibitors of lysosomal proteases. The relative effects of these inhibitors on steady-state cellular levels of each protein are shown alongside each blot. The values are averages ± s.d. for six samples, each normalized to β-actin. All experiments were repeated twice, with similar results. In-depth statistical analyses of these results are given in the text.

 

Figure 3
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  		Fig. 3. Immunolocalization of endogenous TIP47, ADPH[fl]-VSV and {Delta} 2,3 ADPH-VSV. (A) Immunostaining of endogenous TIP47 (green) and ADPH[fl]-VSV (red) or {Delta} 2,3 ADPH-VSV (red) following a 24-hour incubation of 293 cells (a,d,g,j), {Delta} 2,3 ADPH-VSV cells (b,e,h,k) and ADPH[fl]-VSV cells (c,f,i,l) in basal media (Cont, a-c), 300 µM OA (d-f), 5 µM MG132 (MG, g-i) or 300 µM OA plus 5 µM MG132 (OA + MG, j-l). Panels a,d,g,j were immunostained with chicken anti-human TIP47; b,e,h,k and c,f,i,l were immunostained with mouse anti-VSV and chicken anti-human TIP47. (B) Enlargement of regions in Ad-f are shown (a-c) to better illustrate CLD staining properties of endogenous TIP47, {Delta} 2,3 ADPH-VSV and ADPH[fl]-VSV. Nuclei were stained with DAPI (blue). All experiments were performed at least three times, with similar results. Multiple images were captured in each experiment; representative images are shown. Images were originally taken at 600x magnification. Scale bar: 20 µm.

 

Figure 4
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  		Fig. 4. Specificity of N-terminal control of ADPH stability. (A,B) Schematics of ADPH-TIP47-VSV and TIP47-ADPH-VSV chimeras. (C,D) Representative immunoblots of ADPH-TIP47-VSV (C) and TIP47-ADPH-VSV (D) chimeras following a 20-hour incubation of their respective cultures in basal culture media (control) or in basal culture media supplemented with 3 µM MG132 (MG), 300 µM OA, or 3 µM MG132 plus 300 µM OA (MG/OA). Chimeras were detected by probing with mouse anti-VSV antibodies; blots were stripped and reprobed for β-actin to control for loading. (E,F) Quantitation of the respective immunoblots in C and D. The values are averages ± s.d. for three samples, each normalized to β-actin. The experiment was repeated twice, with similar results. Statistical analyses of these results are given in the text.

 

Figure 5
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  		Fig. 5. Immunolocalization of chimeric proteins. Cultures stably expressing ADPH-TIP47-VSV (A-D) or TIP47-ADPH-VSV (E-H) were incubated for 20 hours in unsupplemented basal media (Cont; A,E), or in basal media supplemented with 300 µM OA (B,F), 3 µM MG132 (MG, C,G), or 3 µM MG132 plus 300 µM OA (MG+OA, D,H). Chimeric proteins were localized in cells by staining with mouse anti-VSV (green); nuclei were stained with DAPI (blue). All experiments were performed at least three times, with similar results. Multiple images were captured in each experiment; representative images are shown. Images were originally taken at 600x magnification. Scale bar: 20 µm.

 

Figure 6
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  		Fig. 6. Specificity of N-terminal control of TIP47 binding to CLDs. (A) Specificity of TIP47 antibodies. Extracts of cultures stably expressing the TIP47-ADPH-VSV or ADPH-TIP47-VSV chimeric protein were immunoblotted with chicken anti-human TIP47 antibody ({alpha}hTIP47), rabbit antibodies to the N-terminus of mouse TIP47 ({alpha} N-term mTIP47) or mouse anti-VSV antibody ({alpha}VSV). {alpha}hTIP47 detected a single species at ~47 kDa in cultures of both chimeric cell lines (calculated molecular weight of hTIP47=47032.88; ProSite, http://ca.expasy.org/tools/dna.html). {alpha} N-term mTIP47 and {alpha}VSV antibodies detected a single species of ~52 kDa in TIP-ADPH-VSV cultures (calculated molecular weight=50907.48) and a single species of ~49 kDa (calculated molecular weight=48019.52) in ADPH-TIP47-VSV cultures. (B) Immunolocalization of endogenous TIP47 (green, a-d) and chimeric proteins in ADPH-TIP-VSV (red, a,b) and TIP-ADPH-VSV (red, c,d) in cells after a 20-hour incubation in the absence (Cont, a,c) or presence of 300 µM OA (b,d). TIP47 was localized by staining with {alpha}hTIP47; chimeric proteins were localized by staining with mouse anti-VSV; nuclei were stained with DAPI (blue). All experiments were performed at least three times, with similar results. Multiple images were captured in each experiment; representative images are shown. Images were originally taken at 600x magnification. Scale bar: 20 µm.

 

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© The Company of Biologists Ltd 2008