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Immunocytochemical techniques reveal multiple, distinct cellular pools of PtdIns4P and PtdIns(4,5)P(2).

Hammond GR, Schiavo G, Irvine RF - Biochem. J. (2009)

Bottom Line: PtdIns4P is the major precursor for the synthesis of the multifunctional plasma membrane lipid, PtdIns(4,5)P(2).Yet PtdIns4P also functions as a regulatory lipid in its own right, particularly at the Golgi apparatus.In addition, we present evidence that the majority of PtdIns4P resides in the plasma membrane, where it is metabolically distinct from the steady-state plasma membrane pool of PtdIns(4,5)P(2).

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Cambridge, Cambridge, UK. gruh2@cam.ac.uk

ABSTRACT
PtdIns4P is the major precursor for the synthesis of the multifunctional plasma membrane lipid, PtdIns(4,5)P(2). Yet PtdIns4P also functions as a regulatory lipid in its own right, particularly at the Golgi apparatus. In the present study we define specific conditions that enable preservation of several organellar membranes for the immunocytochemical detection of PtdIns4P. We report distinct pools of this lipid in both Golgi and plasma membranes, which are synthesized by different PI4K (phosphatidylinositol 4-kinase) activities, and also the presence of PtdIns4P in cytoplasmic vesicles, which are not readily identifiable as PI4K containing trafficking intermediates. In addition, we present evidence that the majority of PtdIns4P resides in the plasma membrane, where it is metabolically distinct from the steady-state plasma membrane pool of PtdIns(4,5)P(2).

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Competition for Golgi PtdIns4P staining by PH-FAPP1(a) The indicated GST-tagged fusion proteins (10 μM) were included with the primary anti-PtdIns4P antibody and anti-giantin antibodies applied to COS-7 cells. Staining conditions are as Figure 3. Single confocal optical sections are shown. Scale bar=20 μm. (b) Quantification of anti-PtdIns4P antibody labelling (n=207). See the Experimental section for details. ***P<0.001.
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Figure 5: Competition for Golgi PtdIns4P staining by PH-FAPP1(a) The indicated GST-tagged fusion proteins (10 μM) were included with the primary anti-PtdIns4P antibody and anti-giantin antibodies applied to COS-7 cells. Staining conditions are as Figure 3. Single confocal optical sections are shown. Scale bar=20 μm. (b) Quantification of anti-PtdIns4P antibody labelling (n=207). See the Experimental section for details. ***P<0.001.

Mentions: To further assess the specificity of Golgi staining with anti-PtdIns4P, we used GST-tagged PH-FAPP1 at high concentration (10 μM) to sequester the endogenous PtdIns4P. This protein indeed abolishes staining, whereas GST alone had no affect (Figures 5a and 5b). GST–PH-PLCδ1, which should be more specific for PtdIns(4,5)P2 compared with PtdIns4P [47,48] did not abolish Golgi staining (Figure 5a), but quantitative analysis revealed an approx. 50% decrease in staining intensity (Figure 5b). This might suggest that some of the observed signal with the anti-PtdIns4P antibody is due to the presence of PtdIns(4,5)P2, as PH-FAPP1 can bind both lipids in vitro [23]. This, however, seems unlikely given the specificity of the antibody for exogenous competing lipid (Figure 1), as well as the fact that anti-PtdIns(4,5)P2 does not label the Golgi (see Supplemental Figure S3), which is known to anyway contain relatively little PtdIns(4,5)P2 [31]. Rather, we suspect the partial competition is due to some weak PtdIns4P-binding by PH-PLCδ1, which may be exacerbated by the high concentration of protein used, and the presence of the GST tag, which effectively increases affinity [49].


Immunocytochemical techniques reveal multiple, distinct cellular pools of PtdIns4P and PtdIns(4,5)P(2).

Hammond GR, Schiavo G, Irvine RF - Biochem. J. (2009)

Competition for Golgi PtdIns4P staining by PH-FAPP1(a) The indicated GST-tagged fusion proteins (10 μM) were included with the primary anti-PtdIns4P antibody and anti-giantin antibodies applied to COS-7 cells. Staining conditions are as Figure 3. Single confocal optical sections are shown. Scale bar=20 μm. (b) Quantification of anti-PtdIns4P antibody labelling (n=207). See the Experimental section for details. ***P<0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2722159&req=5

Figure 5: Competition for Golgi PtdIns4P staining by PH-FAPP1(a) The indicated GST-tagged fusion proteins (10 μM) were included with the primary anti-PtdIns4P antibody and anti-giantin antibodies applied to COS-7 cells. Staining conditions are as Figure 3. Single confocal optical sections are shown. Scale bar=20 μm. (b) Quantification of anti-PtdIns4P antibody labelling (n=207). See the Experimental section for details. ***P<0.001.
Mentions: To further assess the specificity of Golgi staining with anti-PtdIns4P, we used GST-tagged PH-FAPP1 at high concentration (10 μM) to sequester the endogenous PtdIns4P. This protein indeed abolishes staining, whereas GST alone had no affect (Figures 5a and 5b). GST–PH-PLCδ1, which should be more specific for PtdIns(4,5)P2 compared with PtdIns4P [47,48] did not abolish Golgi staining (Figure 5a), but quantitative analysis revealed an approx. 50% decrease in staining intensity (Figure 5b). This might suggest that some of the observed signal with the anti-PtdIns4P antibody is due to the presence of PtdIns(4,5)P2, as PH-FAPP1 can bind both lipids in vitro [23]. This, however, seems unlikely given the specificity of the antibody for exogenous competing lipid (Figure 1), as well as the fact that anti-PtdIns(4,5)P2 does not label the Golgi (see Supplemental Figure S3), which is known to anyway contain relatively little PtdIns(4,5)P2 [31]. Rather, we suspect the partial competition is due to some weak PtdIns4P-binding by PH-PLCδ1, which may be exacerbated by the high concentration of protein used, and the presence of the GST tag, which effectively increases affinity [49].

Bottom Line: PtdIns4P is the major precursor for the synthesis of the multifunctional plasma membrane lipid, PtdIns(4,5)P(2).Yet PtdIns4P also functions as a regulatory lipid in its own right, particularly at the Golgi apparatus.In addition, we present evidence that the majority of PtdIns4P resides in the plasma membrane, where it is metabolically distinct from the steady-state plasma membrane pool of PtdIns(4,5)P(2).

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Cambridge, Cambridge, UK. gruh2@cam.ac.uk

ABSTRACT
PtdIns4P is the major precursor for the synthesis of the multifunctional plasma membrane lipid, PtdIns(4,5)P(2). Yet PtdIns4P also functions as a regulatory lipid in its own right, particularly at the Golgi apparatus. In the present study we define specific conditions that enable preservation of several organellar membranes for the immunocytochemical detection of PtdIns4P. We report distinct pools of this lipid in both Golgi and plasma membranes, which are synthesized by different PI4K (phosphatidylinositol 4-kinase) activities, and also the presence of PtdIns4P in cytoplasmic vesicles, which are not readily identifiable as PI4K containing trafficking intermediates. In addition, we present evidence that the majority of PtdIns4P resides in the plasma membrane, where it is metabolically distinct from the steady-state plasma membrane pool of PtdIns(4,5)P(2).

Show MeSH
Related in: MedlinePlus