An internalized transmembrane protein resides in a fusion-competent endosome for less than 5 minutes.
We have used our assay for the cell-free reconstitution of vesicle fusion occurring in endocytosis to investigate the fusion competence of defined endosomal fractions containing the G protein of vesicular stomatitis virus, G protein was first implanted into the plasma membrane, and endocytosis was then allowed to proceed for defined periods of time. Endosomal fractions were prepared by "immuno-isolation" on a solid support with a monoclonal antibody against the cytoplasmic domain of the G protein. Maximal internalization of the G protein occurred within 5 min at 37 degrees C. From this early endosome the G molecules follow a branched pathway: 50% recycles to the cell surface, while 50% is transported along the endocytic route to the lysosomal compartment. The proportion of G protein following each pathway can be modulated. When the amount of implanted G protein was increased, the fraction of G molecules recycling to the cell surface was reduced. When the G molecules were cross-linked with an antibody prior to the internalization step, recycling to the cell surface was abolished. The cell-free analysis of vesicle fusion was carried out with endosomal fractions immuno-isolated after 5, 10, 15, and 30 min of G-protein internalization at 37 degrees C. Fusion competence was at a maximum with the fraction isolated 5 min after internalization and then decreased with a half-life of approximately equal to 3 min with fractions isolated at later time points. The fusion-competent compartment is the early endosome where sorting of the transmembrane G protein to recycling or degradation occurs.
D000818:Animals; D006224:Cricetinae; D004705:Endocytosis; D007700:Kinetics; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000032:analysis*; D008715:Methionine / Q000378:metabolism; D009940:Organoids / Q000648:ultrastructure*; D013997:Time Factors; D014721:Vesicular stomatitis Indiana virus; D014759:Viral Envelope Proteins; D014764:Viral Proteins / Q000378:metabolism
C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D014759:Viral Envelope Proteins; D014764:Viral Proteins; D008715:Methionine