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Rab1a regulates endocytic vesicle processing through interaction with Kifc1
Author(s) -
Quiroz Jose,
Mukhopadhyay Aparna,
Wolkoff Allan W.
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.556.4
Subject(s) - immunoprecipitation , microbiology and biotechnology , hek 293 cells , endocytic cycle , transfection , green fluorescent protein , chemistry , biology , cell , gene , biochemistry , endocytosis
Rab1a regulates endocytic vesicle processing by recruiting the microtubule‐based motor Kifc1. The mechanism by which this occurs is unknown and therefore we examined whether Kifc1 and Rab1a interact with each other either directly or through a protein complex. Human embryonic kidney‐derived HEK293 cell lines expressing super folder green fluorescent protein (SFGFP) or Rab1a‐SFGFP fusion protein were prepared. To test whether Kifc1 interacts with Rab1a, a plasmid encoding Kifc1‐flag was transfected into Rab1a‐SFGFP expressing HEK293 cells. Cell lysates were subjected to immunoprecipitation with GFP antibody. Following SDS‐PAGE, analysis revealed the presence of Kifc1 in immunoprecipitates from Rab1a‐SFGFP expressing cells. In summary, immunofluorescence microscopy verified stable cell lines expressing Rab1a‐SFGFP in HEK293. 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.