title: Rabenosyn-5 and EHD1 interact and sequentially regulate protein recycling to the plasma membrane.
abstract: EHD1 has been implicated in the recycling of internalized proteins to the plasma membrane. However, the mechanism by which EHD1 mediates recycling and its relationship to Rab-family-controlled events has yet to be established. To investigate further the mode of EHD1 action, we sought to identify novel interacting partners. GST-EHD1 was used as bait to isolate a approximately 120-kDa species from bovine and murine brain cytosol, which was identified by mass spectrometry as the divalent Rab4/Rab5 effector Rabenosyn-5. We mapped the sites of interaction to the EH domain of EHD1, and the first two of five NPF motifs of Rabenosyn-5. Immunofluorescence microscopy studies revealed that EHD1 and Rabenosyn-5 partially colocalize to vesicular and tubular structures in vivo. To address the functional roles of EHD1 and Rabenosyn-5, we first demonstrated that RNA interference (RNAi) dramatically reduced the level of expression of each protein, either individually or in combination. Depletion of either EHD1 or Rabenosyn-5 delayed the recycling of transferrin and major histocompatibility complex class I to the plasma membrane. However, whereas depletion of EHD1 caused the accumulation of internalized cargo in a compact juxtanuclear compartment, Rabenosyn-5-RNAi caused its retention within a dispersed peripheral compartment. Simultaneous RNAi depletion of both proteins resulted in a similar phenotype to that observed with Rabenosyn-5-RNAi alone, suggesting that Rabenosyn-5 acts before EHD1 in the regulation of endocytic recycling. Our studies suggest that Rabenosyn-5 and EHD1 act sequentially in the transport of proteins from early endosomes to the endosomal recycling compartment and back to the plasma membrane.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198-5870, USA.
authors.forename: Naava
authors.initials: N
authors.lastname: Naslavsky
authors.forename: Markus
authors.initials: M
authors.lastname: Boehm
authors.forename: Peter S
authors.initials: PS
authors.lastname: Backlund
authors.forename: Steve
authors.initials: S
authors.lastname: Caplan
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism* / Q000502:physiology / Q000648:ultrastructure; D003600:Cytosol / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008856:Microscopy, Fluorescence; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D034622:RNA Interference; D011990:Receptors, Transferrin / Q000235:genetics / Q000502:physiology; D014168:Transferrin / Q000032:analysis / Q000378:metabolism; D022161:Transport Vesicles / Q000502:physiology* / Q000648:ultrastructure; D020798:Two-Hybrid System Techniques; D033921:Vesicular Transport Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology*
chemical_list: C120700:EHD1 protein, human; D008565:Membrane Proteins; C416362:RBSN protein, human; D011990:Receptors, Transferrin; D014168:Transferrin; D033921:Vesicular Transport Proteins