RAB1A REGULATES ENDOCYTIC VESICLE PROCESSING THROUGH INTERACTION WITH KIFC1

picture of Jose Quiroz presenting his/her poster: RAB1A REGULATES ENDOCYTIC VESICLE PROCESSING THROUGH INTERACTION WITH KIFC1

Jose Quiroz , Aparna mukhopadhya, Allan W. Wolkoff

RAB1A REGULATES ENDOCYTIC VESICLE PROCESSING THROUGH INTERACTION WITH KIFC1

Receptor-mediated endocytosis is a mechanism by which cells take up and process macromolecules via a series of endocytic vesicles. We recently reported a novel pathway by which Rab1a regulates endocytic vesicle processing by recruiting the microtubule-based motor Kifc1. The mechanism by which this occurs is unknown and was the subject of the current investigation in which we examined whether Kifc1 and Rab1a interact with each other either directly or through a protein complex. 

Human hepatoma-derived HuH7 and human embryonic kidney-derived HEK293 cell lines expressing super folder green fluorescent protein (SFGFP) or Rab1a-SFGFP fusion protein were prepared. Fluorescence microscopy of Rab1a-SFGFP expressing cells revealed punctate distribution of fluorescence throughout the cytoplasm. This distribution was similar to that of endogenous Raba1a in untransfected cells as determined by immunofluorescence using specific antibody. To test whether Kifc1 interacts with Rab1a, a plasmid encoding Kifc1-flag or empty vector (pFlag-LMV-5c) was transfected into Rab1a-SFGFP or SFGFP expressing HEK293 cells. Cell lysates were subjected to immunoprecipitation with GFP antibody linked to agarose beads. Following SDS-PAGE, immunoblot analysis revealed the presence of Kifc1 in immunoprecipitates from Rab1a-SFGFP expressing cells but not from cells expressing SFGFP alone.

In summary, immunofluorescence microscopy verified the successful preparation of stable cell lines expressing Rab1a-SFGFP in HEK293 and HuH7 cells. Immunoprecipitation of Rab1a-SFGFP cells transiently transfected with Kifc1-Flag showed that Rab1a and Kifc1 were in the immunoprecipitate. We conclude that Rab1a and Kifc1 are associated with each other in a protein complex and do not just independently bind to endocytic vesicles. Whether other proteins are in this complex remains to be discovered. 

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