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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: We have identified a novel, highly conserved protein of 14 kD copurifying with late endosomes/lysosomes on density gradients.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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Membrane topology of p14. (A) PNS from polarized EpH4 cells was loaded on top of a continuous sucrose gradient and sedimented organelle fractions enriched in p14 were extracted with Na2CO3, pH 11.0. Input (left), carbonate insoluble (middle), and carbonate soluble (right) proteins were analyzed by 2DGE. Respective areas of high resolution two-dimensional gels are shown; arrows indicate p14. (B) PNS from polarized EpH4 cells was treated with increasing concentrations of proteinase K. Digested samples were analyzed by immunoblot, quantified, and the percentage of intact protein compared with undigested controls was plotted (numbers are an average of three independent experiments ± SD). A lumenal protein (calreticulin) and Rab5 as a protein associated with the cytoplasmic leaflet of membranes were taken as controls.
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Figure 3: Membrane topology of p14. (A) PNS from polarized EpH4 cells was loaded on top of a continuous sucrose gradient and sedimented organelle fractions enriched in p14 were extracted with Na2CO3, pH 11.0. Input (left), carbonate insoluble (middle), and carbonate soluble (right) proteins were analyzed by 2DGE. Respective areas of high resolution two-dimensional gels are shown; arrows indicate p14. (B) PNS from polarized EpH4 cells was treated with increasing concentrations of proteinase K. Digested samples were analyzed by immunoblot, quantified, and the percentage of intact protein compared with undigested controls was plotted (numbers are an average of three independent experiments ± SD). A lumenal protein (calreticulin) and Rab5 as a protein associated with the cytoplasmic leaflet of membranes were taken as controls.

Mentions: In a secondary analysis, we investigated the membrane topology of p14. The protein displayed features of a peripheral membrane protein since it was partially extractable by carbonate at high pH (Fig. 3 A). Furthermore, we prepared PNSs under conditions that maintain the integrity of vesicles and subjected them to proteinase K digestion. Proteinase K is not membrane permeable and thus, digests at limited concentrations only proteins accessible at the cytoplasmic face of membranes. Under these conditions, p14 was much more sensitive to digestion than known lumenal marker proteins, such as calreticulin (Krause and Michalak 1997), or even proteins on the cytoplasmic side, such as Rab5 (Somsel Rodman and Wandinger-Ness 2000) (Fig. 3 B). Calreticulin was chosen since it represented a well-characterized lumenal marker protein of the ER, the most abundant vesicle fraction in PNSs. Taken together, these results suggested a peripheral association of p14 with the cytoplasmic face of membranes.


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)

Membrane topology of p14. (A) PNS from polarized EpH4 cells was loaded on top of a continuous sucrose gradient and sedimented organelle fractions enriched in p14 were extracted with Na2CO3, pH 11.0. Input (left), carbonate insoluble (middle), and carbonate soluble (right) proteins were analyzed by 2DGE. Respective areas of high resolution two-dimensional gels are shown; arrows indicate p14. (B) PNS from polarized EpH4 cells was treated with increasing concentrations of proteinase K. Digested samples were analyzed by immunoblot, quantified, and the percentage of intact protein compared with undigested controls was plotted (numbers are an average of three independent experiments ± SD). A lumenal protein (calreticulin) and Rab5 as a protein associated with the cytoplasmic leaflet of membranes were taken as controls.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195784&req=5

Figure 3: Membrane topology of p14. (A) PNS from polarized EpH4 cells was loaded on top of a continuous sucrose gradient and sedimented organelle fractions enriched in p14 were extracted with Na2CO3, pH 11.0. Input (left), carbonate insoluble (middle), and carbonate soluble (right) proteins were analyzed by 2DGE. Respective areas of high resolution two-dimensional gels are shown; arrows indicate p14. (B) PNS from polarized EpH4 cells was treated with increasing concentrations of proteinase K. Digested samples were analyzed by immunoblot, quantified, and the percentage of intact protein compared with undigested controls was plotted (numbers are an average of three independent experiments ± SD). A lumenal protein (calreticulin) and Rab5 as a protein associated with the cytoplasmic leaflet of membranes were taken as controls.
Mentions: In a secondary analysis, we investigated the membrane topology of p14. The protein displayed features of a peripheral membrane protein since it was partially extractable by carbonate at high pH (Fig. 3 A). Furthermore, we prepared PNSs under conditions that maintain the integrity of vesicles and subjected them to proteinase K digestion. Proteinase K is not membrane permeable and thus, digests at limited concentrations only proteins accessible at the cytoplasmic face of membranes. Under these conditions, p14 was much more sensitive to digestion than known lumenal marker proteins, such as calreticulin (Krause and Michalak 1997), or even proteins on the cytoplasmic side, such as Rab5 (Somsel Rodman and Wandinger-Ness 2000) (Fig. 3 B). Calreticulin was chosen since it represented a well-characterized lumenal marker protein of the ER, the most abundant vesicle fraction in PNSs. Taken together, these results suggested a peripheral association of p14 with the cytoplasmic face of membranes.

Bottom Line: We have identified a novel, highly conserved protein of 14 kD copurifying with late endosomes/lysosomes on density gradients.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