SUBCELLULAR FRACTIONATION OF POLARIZED EPITHELIAL-CELLS AND IDENTIFICATION OF ORGANELLE-SPECIFIC PROTEINS BY 2-DIMENSIONAL GEL-ELECTROPHORESIS

Protein targeting and sorting is accomplished by complex vesicular tra nsport processes that are tightly regulated within a cell. This is esp ecially important for epithelial cells because correct delivery of new ly synthesized proteins as well as recycling and sorting of internaliz ed membrane proteins is essential for the establishment and preservati on of cellular polarity. Many transport events, linking various subcel lular compartments, have been analyzed, but many transport mechanisms still remain unresolved. In this study we attempted to identify protei ns specifically associated with distinct organelles in murine mammary epithelial cells (EpH4). We isolated subcellular compartments by conti nuous sucrose gradient centrifugation in order to further analyze thei r protein composition by high-resolution two-dimensional gel electroph oresis (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. Char acteristic polypeptide patterns were revealed on 2-DE gels for fractio ns enriched in membranes of different origin. Based on improved sample preparation and loading techniques (this issue. C. Pasquali et al., E lectrophoresis, 1997, 18, 2573-2581), we were able to identify several proteins by immunoblotting or microsequencing of Coomassie-stained sp ots. This will be the basis for a further characterization of organell e-specific molecules in epithelial cells as well as for the establishm ent of a 2-DE reference map of membrane proteins from murine mammary e pithelium.