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A novel 14-kilodalton protein interacts with the mitogen-activated protein kinase scaffold mp1 on a late endosomal/lysosomal compartment.

Wunderlich W, Fialka I, Teis D, Alpi A, Pfeifer A, Parton RG, Lottspeich F, Huber LA - J. Cell Biol. (2001)

Bottom Line: The protein, now termed p14, is peripherally associated with the cytoplasmic face of late endosomes/lysosomes in a variety of different cell types.Moreover, expression of a plasma membrane-targeted p14 causes mislocalization of coexpressed MP1.In addition, we could reconstitute protein complexes containing the p14-MP1 complex associated with ERK and MEK in vitro.The interaction between p14 and MP1 suggests a MAPK scaffolding activity localized to the cytoplasmic surface of late endosomes/lysosomes, thereby combining catalytic scaffolding and subcellular compartmentalization as means to modulate MAPK signaling within a cell.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Molecular Pathology, A-1030 Vienna, Austria.

ABSTRACT
We have identified a novel, highly conserved protein of 14 kD copurifying with late endosomes/lysosomes on density gradients. The protein, now termed p14, is peripherally associated with the cytoplasmic face of late endosomes/lysosomes in a variety of different cell types. In a two-hybrid screen with p14 as a bait, we identified the mitogen-activated protein kinase (MAPK) scaffolding protein MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) partner 1 (MP1) as an interacting protein. We confirmed the specificity of this interaction in vitro by glutathione S-transferase pull-down assays and by coimmunoprecipitation, cosedimentation on glycerol gradients, and colocalization. Moreover, expression of a plasma membrane-targeted p14 causes mislocalization of coexpressed MP1. In addition, we could reconstitute protein complexes containing the p14-MP1 complex associated with ERK and MEK in vitro.The interaction between p14 and MP1 suggests a MAPK scaffolding activity localized to the cytoplasmic surface of late endosomes/lysosomes, thereby combining catalytic scaffolding and subcellular compartmentalization as means to modulate MAPK signaling within a cell.

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Subcellular fractionation and analysis of endosomal compartments. (A) HRP was internalized into polarized EpH4 cells for 5 min. Cells were either kept on ice (0 min chase) or incubated for another 20 min at 37°C (20 min chase). Cells were scraped, homogenized, and PNS was prepared. PNS was loaded on top of continuous sucrose gradients and separated by centrifugation as outlined in Materials and Methods. Fractions were collected, and HRP activity was measured (top) and immunoblotted for Rab4, Rab5, Rab7, Rab11, and transferrin receptor (bottom). (B–D) PNS, fractions 6+7 (LE), and fractions 18+19 (EE) were analyzed by 2DGE and compared according to enrichment of protein spots in EE and LE versus PNS, respectively. Insets show the region containing a protein (arrow) with experimental Mr ∼ 14 kD and pI ∼ 4.9, specifically enriched in LE.
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Figure 1: Subcellular fractionation and analysis of endosomal compartments. (A) HRP was internalized into polarized EpH4 cells for 5 min. Cells were either kept on ice (0 min chase) or incubated for another 20 min at 37°C (20 min chase). Cells were scraped, homogenized, and PNS was prepared. PNS was loaded on top of continuous sucrose gradients and separated by centrifugation as outlined in Materials and Methods. Fractions were collected, and HRP activity was measured (top) and immunoblotted for Rab4, Rab5, Rab7, Rab11, and transferrin receptor (bottom). (B–D) PNS, fractions 6+7 (LE), and fractions 18+19 (EE) were analyzed by 2DGE and compared according to enrichment of protein spots in EE and LE versus PNS, respectively. Insets show the region containing a protein (arrow) with experimental Mr ∼ 14 kD and pI ∼ 4.9, specifically enriched in LE.

Mentions: Fig. 1 A shows the analysis of the sucrose density gradient with respect to the activity of HRP internalized by fluid phase endocytosis for different time periods as well as by Western blotting for established compartment markers (Rab5 for the early endosome, Rab4, Rab11, and transferrin receptor for the recycling endosome, and Rab7 for the late endosome) (Bucci et al. 2000; Sonnichsen et al. 2000). Peak fractions for Rab5/HRP activity at 0 min chase (fraction 14–16) and Rab7/HRP activity at 20 min chase (fraction 6+7) were used for 2DGE analysis of early endosomal (Fig. 1 C) and late endosomal (Fig. 1 D) fractions, respectively. The second peak fraction of Rab5 (fraction 18–20) was omitted since it contained the bulk of plasma membrane and rough ER fractions.


A novel 14-kilodalton protein interacts with the mitogen-activated protein kinase scaffold mp1 on a late endosomal/lysosomal compartment.

Wunderlich W, Fialka I, Teis D, Alpi A, Pfeifer A, Parton RG, Lottspeich F, Huber LA - J. Cell Biol. (2001)

Subcellular fractionation and analysis of endosomal compartments. (A) HRP was internalized into polarized EpH4 cells for 5 min. Cells were either kept on ice (0 min chase) or incubated for another 20 min at 37°C (20 min chase). Cells were scraped, homogenized, and PNS was prepared. PNS was loaded on top of continuous sucrose gradients and separated by centrifugation as outlined in Materials and Methods. Fractions were collected, and HRP activity was measured (top) and immunoblotted for Rab4, Rab5, Rab7, Rab11, and transferrin receptor (bottom). (B–D) PNS, fractions 6+7 (LE), and fractions 18+19 (EE) were analyzed by 2DGE and compared according to enrichment of protein spots in EE and LE versus PNS, respectively. Insets show the region containing a protein (arrow) with experimental Mr ∼ 14 kD and pI ∼ 4.9, specifically enriched in LE.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195784&req=5

Figure 1: Subcellular fractionation and analysis of endosomal compartments. (A) HRP was internalized into polarized EpH4 cells for 5 min. Cells were either kept on ice (0 min chase) or incubated for another 20 min at 37°C (20 min chase). Cells were scraped, homogenized, and PNS was prepared. PNS was loaded on top of continuous sucrose gradients and separated by centrifugation as outlined in Materials and Methods. Fractions were collected, and HRP activity was measured (top) and immunoblotted for Rab4, Rab5, Rab7, Rab11, and transferrin receptor (bottom). (B–D) PNS, fractions 6+7 (LE), and fractions 18+19 (EE) were analyzed by 2DGE and compared according to enrichment of protein spots in EE and LE versus PNS, respectively. Insets show the region containing a protein (arrow) with experimental Mr ∼ 14 kD and pI ∼ 4.9, specifically enriched in LE.
Mentions: Fig. 1 A shows the analysis of the sucrose density gradient with respect to the activity of HRP internalized by fluid phase endocytosis for different time periods as well as by Western blotting for established compartment markers (Rab5 for the early endosome, Rab4, Rab11, and transferrin receptor for the recycling endosome, and Rab7 for the late endosome) (Bucci et al. 2000; Sonnichsen et al. 2000). Peak fractions for Rab5/HRP activity at 0 min chase (fraction 14–16) and Rab7/HRP activity at 20 min chase (fraction 6+7) were used for 2DGE analysis of early endosomal (Fig. 1 C) and late endosomal (Fig. 1 D) fractions, respectively. The second peak fraction of Rab5 (fraction 18–20) was omitted since it contained the bulk of plasma membrane and rough ER fractions.

Bottom Line: The protein, now termed p14, is peripherally associated with the cytoplasmic face of late endosomes/lysosomes in a variety of different cell types.Moreover, expression of a plasma membrane-targeted p14 causes mislocalization of coexpressed MP1.In addition, we could reconstitute protein complexes containing the p14-MP1 complex associated with ERK and MEK in vitro.The interaction between p14 and MP1 suggests a MAPK scaffolding activity localized to the cytoplasmic surface of late endosomes/lysosomes, thereby combining catalytic scaffolding and subcellular compartmentalization as means to modulate MAPK signaling within a cell.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Molecular Pathology, A-1030 Vienna, Austria.

ABSTRACT
We have identified a novel, highly conserved protein of 14 kD copurifying with late endosomes/lysosomes on density gradients. The protein, now termed p14, is peripherally associated with the cytoplasmic face of late endosomes/lysosomes in a variety of different cell types. In a two-hybrid screen with p14 as a bait, we identified the mitogen-activated protein kinase (MAPK) scaffolding protein MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) partner 1 (MP1) as an interacting protein. We confirmed the specificity of this interaction in vitro by glutathione S-transferase pull-down assays and by coimmunoprecipitation, cosedimentation on glycerol gradients, and colocalization. Moreover, expression of a plasma membrane-targeted p14 causes mislocalization of coexpressed MP1. In addition, we could reconstitute protein complexes containing the p14-MP1 complex associated with ERK and MEK in vitro.The interaction between p14 and MP1 suggests a MAPK scaffolding activity localized to the cytoplasmic surface of late endosomes/lysosomes, thereby combining catalytic scaffolding and subcellular compartmentalization as means to modulate MAPK signaling within a cell.

Show MeSH
Related in: MedlinePlus