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.

Members of the epidermal growth factor (EGF) receptor family are known to be specifically involved in mammary carcinogenesis. As a nuclear target of activated receptors, we examined c-Jun in mammary epithelial cells. For this, we used a c-JunER fusion protein which was tightly controlled by estrogen. Activation of the JunER by hormone resulted in the transcriptional regulation of a variety of AP-1 target genes. Hormone-activated JunER induced the loss of epithelial polarity, a disruption of intercellular junctions and normal barrier function and the formation of irregular multilayers. These changes were completely reversible upon hormone withdrawal. Loss of epithelial polarity involved redistribution of both apical and basolateral proteins to the entire plasma membrane. The redistribution of E-cadherin and beta-catenin was accompanied by a destabilization of complexes formed between these two proteins, leading to an enrichment of beta-catenin in the detergent-soluble fraction. Uninduced cells were able to form three-dimensional tubular structures in collagen I gels which were disrupted upon JunER activation, leading to irregular cell aggregates. The JunER-induced disruption of tubular structures was dependent on active signaling by growth factors. Moreover, the effects of JunER could be mimicked in normal cells by the addition of acidic fibroblast growth factor (aFGF). These data suggest that a possible function of c-Jun in epithelial cells is to modulate epithelial polarity and regulate tissue organization, processes which may be equally important for both normal breast development and as initiating steps in carcinogenesis.

Electrophoretic techniques, and especially two-dimensional gel electrophoresis (2-DE), have provided an indispensable set of tools for the separation of complex protein mixtures as well as for the identification of protein-protein interactions. Nevertheless, after its introduction more than twenty years ago and even with recent technical developments, the separation of integral and peripheral membrane proteins, in amounts sufficient for microsequencing, is still a difficult task. Lipids present in the membrane as well as the low solubility of hydrophobic membrane proteins result in protein aggregation both on the sample application point and on isoelectric focusing. As a consequence many proteins do not enter the first or second dimension of 2-DE. Here we describe the modification of a protocol using a combination of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS), chaotropic agents (thiourea, urea), Tris base and reducing agents (1,4-dithioerythritol) to improve solubilization of integral and peripheral membrane proteins. Preparative amounts of membrane proteins (up to 2 mg) were loaded during reswelling of dry immobilized pH gradients and the resulting Coomassie staining patterns were largely superimposable with silver-stained gels obtained from identical samples (4 microg). This indicates that the recovery of proteins from the sample is not significantly compromised by the scale-up procedure. A direct application of this method for the characterization and identification of membrane proteins from cellular organelles is described in another paper in this issue (I. Fialka et al., Electrophoresis 1997, 18, 2582-2590).

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Protein targeting and sorting is accomplished by complex vesicular transport processes that are tightly regulated within a cell. This is especially important for epithelial cells because correct delivery of newly synthesized proteins as well as recycling and sorting of internalized membrane proteins is essential for the establishment and preservation of cellular polarity. Many transport events, linking various subcellular compartments, have been analyzed, but many transport mechanisms still remain unresolved. In this study we attempted to identify proteins specifically associated with distinct organelles in murine mammary epithelial cells (EpH4). We isolated subcellular compartments by continuous sucrose gradient centrifugation in order to further analyze their protein composition by high-resolution two-dimensional gel electrophoresis (2-DE). The successful separation of late endosomes (LE), early endosomes (EE) and most of the rough endoplasmic reticulum (RER) was confirmed by subsequent analysis of gradient fractions for compartment-specific enzymes and marker proteins. Both Golgi and plasma membrane (PM) were found to partially co-purify with EE in such gradients. Characteristic polypeptide patterns were revealed on 2-DE gels for fractions enriched in membranes of different origin. Based on improved sample preparation and loading techniques (this issue, C. Pasquali et al., Electrophoresis, 1997, 18, 2573-2581), we were able to identify several proteins by immunoblotting or microsequencing of Coomassie-stained spots. This will be the basis for a further characterization of organelle-specific molecules in epithelial cells as well as for the establishment of a 2-DE reference map of membrane proteins from murine mammary epithelium.