Gaining insight into a complex organelle, the phagosome, using two-dimensional gel electrophoresis.
Phagosomes are the organelles formed de novo in a variety of cells by the internalization of large particulate materials, including a wide range of pathogenic microorganisms. We present here a systematic approach that can be used to study the polypeptide composition of phagosomes/phagolysosomes and to yield analytical information on the characteristics of their proteins. A density shift approach was used to isolate pure preparations of phagosomes filled with low density latex beads from mouse J774 and human U937 macrophages. High resolution two-dimensional (2-D) gel electrophoresis was performed to generate a map of the overall [35S]methionine-labeled protein profile of the isolated phagosomes. The resulting map showed the minimal presence of over 200 polypeptides, indicating the complexity of this organelle. Comigration experiments showed that several phagosome polypeptides, among them several known proteins, are shared by the two species. Extraction with Triton X-114 and sodium carbonate was performed to distinguish between membrane and soluble proteins, and sensitivity to a panel of proteases was measured to identify proteins exposed on the cytoplasmic face of the phagosome membrane. The general value of the 2-D gel approach in the mapping of organelle proteins is discussed.
Burkhardt
J
J
European Molecular Biology Laboratory, Heidelberg.
Huber
L A
LA
Dieplinger
H
H
Blocker
A
A
Griffiths
G
G
Desjardins
M
M
D000818:Animals; D002460:Cell Line; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D008264:Macrophages / Q000737:chemistry* / Q000166:cytology; D051379:Mice; D015388:Organelles; D010449:Peptide Mapping; D010455:Peptides / Q000032:analysis; D010588:Phagosomes / Q000737:chemistry*; D011506:Proteins / Q000032:analysis*
D010455:Peptides; D011506:Proteins