Mapping the protein composition of trans-Golgi network (TGN)-derived carrier vesicles from polarized MDCK cells.
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 beta1 and beta2 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.
Fiedler
K
K
Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. k-fiedler@ski.mskcc.org
Kellner
R
R
Simons
K
K
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D016764:Cell Polarity; D004285:Dogs; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006056:Golgi Apparatus / Q000737:chemistry*; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods*; D011506:Proteins / Q000032:analysis*
D011506:Proteins