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SDPR induces membrane curvature and functions in the formation of caveolae.

Hansen CG, Bright NA, Howard G, Nichols BJ - Nat. Cell Biol. (2009)

Bottom Line: Here we show that loss of SDPR (serum deprivation protein response) causes loss of caveolae.SDPR binds directly to PTRF and recruits PTRF to caveolar membranes.STB colocalizes extensively with both SDPR and caveolin 1.

View Article: PubMed Central - PubMed

Affiliation: MRC-LMB, Cambridge, UK.

ABSTRACT
Caveolae are plasma membrane invaginations with a characteristic flask-shaped morphology. They function in diverse cellular processes, including endocytosis. The mechanism by which caveolae are generated is not fully understood, but both caveolin proteins and PTRF (polymerase I and transcript release factor, also known as cavin) are important. Here we show that loss of SDPR (serum deprivation protein response) causes loss of caveolae. SDPR binds directly to PTRF and recruits PTRF to caveolar membranes. Overexpression of SDPR, unlike PTRF, induces deformation of caveolae and extensive tubulation of the plasma membrane. The B-subunit of Shiga toxin (STB) also induces membrane tubulation and these membrane tubes also originate from caveolae. STB colocalizes extensively with both SDPR and caveolin 1. Loss of caveolae reduces the propensity of STB to induce membrane tubulation. We conclude that SDPR is a membrane-curvature-inducing component of caveolae, and that STB-induced membrane tubulation is facilitated by caveolae.

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SDPR is a caveolar protein, but forms tubes at high expression levelsA. Western blots showing expression levels of SDPR, actin, caveolin 1 and PTRF in HeLa cell lines stably transfected with plasmids expressing shRNAs as shown. Arrow indicates position of 49kDa size marker (ovalbumin, not visible). Complete scans of the blots are shown in Supplementary Figure 6. B. Confocal image of a HeLa cell labeled using indirect immunofluorescence with monoclonal anti-caveolin 1 and polyclonal anti-SDPR. Yellow arrows highlight co-localization, bar 10μm. C. TIR image of caveolin 1-GFP and SDPR-mCh expressed in a HeLa cell. Yellow arrows highlight co-localization. Bar 10μm. D. Over-expression of SDPR-mCh at approximately 10x endogenous protein levels results in formation of extensive tubes. PTRF over-expressed at the same levels has a predominantly cytosolic distribution. Confocal images of live HeLa cells. Bars 10μm.
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Figure 1: SDPR is a caveolar protein, but forms tubes at high expression levelsA. Western blots showing expression levels of SDPR, actin, caveolin 1 and PTRF in HeLa cell lines stably transfected with plasmids expressing shRNAs as shown. Arrow indicates position of 49kDa size marker (ovalbumin, not visible). Complete scans of the blots are shown in Supplementary Figure 6. B. Confocal image of a HeLa cell labeled using indirect immunofluorescence with monoclonal anti-caveolin 1 and polyclonal anti-SDPR. Yellow arrows highlight co-localization, bar 10μm. C. TIR image of caveolin 1-GFP and SDPR-mCh expressed in a HeLa cell. Yellow arrows highlight co-localization. Bar 10μm. D. Over-expression of SDPR-mCh at approximately 10x endogenous protein levels results in formation of extensive tubes. PTRF over-expressed at the same levels has a predominantly cytosolic distribution. Confocal images of live HeLa cells. Bars 10μm.

Mentions: A polyclonal antibody against peptides corresponding to amino acids 9-23 and 312-325 of human SDPR was raised in rabbits. After affinity purification, the SDPR antibodies recognized a band of 49kDa on Western blots of HeLa cell extracts. This corresponds to the predicted molecular mass of SDPR. A higher molecular weight band was also observed (Figure 1A). Two HeLa cell lines stably transfected with plasmids expressing short hairpin RNAs (shRNAs) specific to different regions of the SDPR mRNA were generated. In both SDPR shRNA cell lines the 49kDa band was reduced to less than 15% of the levels observed in control cells (cells stably transfected with empty shRNA vector used or non-targeting shRNA - only the latter is shown). The SDPR shRNA cell lines therefore provide a good system to investigate SDPR function. Cell lines expressing shRNAs against PTRF were also generated (Figure 1A).


SDPR induces membrane curvature and functions in the formation of caveolae.

Hansen CG, Bright NA, Howard G, Nichols BJ - Nat. Cell Biol. (2009)

SDPR is a caveolar protein, but forms tubes at high expression levelsA. Western blots showing expression levels of SDPR, actin, caveolin 1 and PTRF in HeLa cell lines stably transfected with plasmids expressing shRNAs as shown. Arrow indicates position of 49kDa size marker (ovalbumin, not visible). Complete scans of the blots are shown in Supplementary Figure 6. B. Confocal image of a HeLa cell labeled using indirect immunofluorescence with monoclonal anti-caveolin 1 and polyclonal anti-SDPR. Yellow arrows highlight co-localization, bar 10μm. C. TIR image of caveolin 1-GFP and SDPR-mCh expressed in a HeLa cell. Yellow arrows highlight co-localization. Bar 10μm. D. Over-expression of SDPR-mCh at approximately 10x endogenous protein levels results in formation of extensive tubes. PTRF over-expressed at the same levels has a predominantly cytosolic distribution. Confocal images of live HeLa cells. Bars 10μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2712677&req=5

Figure 1: SDPR is a caveolar protein, but forms tubes at high expression levelsA. Western blots showing expression levels of SDPR, actin, caveolin 1 and PTRF in HeLa cell lines stably transfected with plasmids expressing shRNAs as shown. Arrow indicates position of 49kDa size marker (ovalbumin, not visible). Complete scans of the blots are shown in Supplementary Figure 6. B. Confocal image of a HeLa cell labeled using indirect immunofluorescence with monoclonal anti-caveolin 1 and polyclonal anti-SDPR. Yellow arrows highlight co-localization, bar 10μm. C. TIR image of caveolin 1-GFP and SDPR-mCh expressed in a HeLa cell. Yellow arrows highlight co-localization. Bar 10μm. D. Over-expression of SDPR-mCh at approximately 10x endogenous protein levels results in formation of extensive tubes. PTRF over-expressed at the same levels has a predominantly cytosolic distribution. Confocal images of live HeLa cells. Bars 10μm.
Mentions: A polyclonal antibody against peptides corresponding to amino acids 9-23 and 312-325 of human SDPR was raised in rabbits. After affinity purification, the SDPR antibodies recognized a band of 49kDa on Western blots of HeLa cell extracts. This corresponds to the predicted molecular mass of SDPR. A higher molecular weight band was also observed (Figure 1A). Two HeLa cell lines stably transfected with plasmids expressing short hairpin RNAs (shRNAs) specific to different regions of the SDPR mRNA were generated. In both SDPR shRNA cell lines the 49kDa band was reduced to less than 15% of the levels observed in control cells (cells stably transfected with empty shRNA vector used or non-targeting shRNA - only the latter is shown). The SDPR shRNA cell lines therefore provide a good system to investigate SDPR function. Cell lines expressing shRNAs against PTRF were also generated (Figure 1A).

Bottom Line: Here we show that loss of SDPR (serum deprivation protein response) causes loss of caveolae.SDPR binds directly to PTRF and recruits PTRF to caveolar membranes.STB colocalizes extensively with both SDPR and caveolin 1.

View Article: PubMed Central - PubMed

Affiliation: MRC-LMB, Cambridge, UK.

ABSTRACT
Caveolae are plasma membrane invaginations with a characteristic flask-shaped morphology. They function in diverse cellular processes, including endocytosis. The mechanism by which caveolae are generated is not fully understood, but both caveolin proteins and PTRF (polymerase I and transcript release factor, also known as cavin) are important. Here we show that loss of SDPR (serum deprivation protein response) causes loss of caveolae. SDPR binds directly to PTRF and recruits PTRF to caveolar membranes. Overexpression of SDPR, unlike PTRF, induces deformation of caveolae and extensive tubulation of the plasma membrane. The B-subunit of Shiga toxin (STB) also induces membrane tubulation and these membrane tubes also originate from caveolae. STB colocalizes extensively with both SDPR and caveolin 1. Loss of caveolae reduces the propensity of STB to induce membrane tubulation. We conclude that SDPR is a membrane-curvature-inducing component of caveolae, and that STB-induced membrane tubulation is facilitated by caveolae.

Show MeSH
Related in: MedlinePlus