| Abstract: |
In polarized MDCK cells, proteins and lipids are sorted in the trans- Golgi network/TGN) and packaged into different vesicular carriers that are delivered to the apical or basolateral cell surface. To gain insight into the sorting and trafficking machinery, we have previously isolated TGN-derived carrier vesicles from perforated MDCK cells. The composition of immuno- isolated apical and basolateral carriers was mapped by two-dimensional (2-D) gel electrophoresis. Here we describe the identification of several components of the vesicle fraction by using three different methods. 2-D gel comigration was performed with carrier vesicles isolated from metabolically labeled MDCK cells and human epidermal keratinocyte lysates. This allowed us to assign eleven known components by a comparison with the comprehensive keratinocyte 2-D gel database. These comprised two members of the 14-3-3 family of proteins that have been implicated in vesicular trafficking. Five proteins were purified from preparative 2-D gels and identified by peptide microsequencing, including the β1 and β2 subunit of trimeric G proteins and an annexin II variant. A member of the SNARE family of proteins was identified by immunoblotting. The combination of 2-D gel electrophoresis and 2-D gel databases allows the rapid assessment of the purity of subcellular fractions and to characterize components involved in vesicular transport. |
