title: Rabankyrin-5 interacts with EHD1 and Vps26 to regulate endocytic trafficking and retromer function.
abstract: Rabankyrin-5 (Rank-5) has been implicated as an effector of the small GTPase Rab5 and plays an important role in macropinocytosis. We have now identified Rank-5 as an interaction partner for the recycling regulatory protein, Eps15 homology domain 1 (EHD1). We have demonstrated this interaction by glutathione S-transferase-pulldown, yeast two-hybrid assay, isothermal calorimetry and co-immunoprecipitation, and found that the binding occurs between the EH domain of EHD1 and the NPFED motif of Rank-5. Similar to EHD1, we found that Rank-5 colocalizes and interacts with components of the retromer complex such as vacuolar protein sorting 26 (Vps26), suggesting a role for Rank-5 in retromer-based transport. Indeed, depletion of Rank-5 causes mislocalization of Vps26 and affects both the retrieval of mannose 6-phosphate receptor transport to the Golgi from endosomes and biosynthetic transport. Moreover, Rank-5 is required for normal retromer distribution, as overexpression of a wild-type Rank-5-small interfering RNA-resistant construct rescues retromer mislocalization. Finally, we show that depletion of either Rank-5 or EHD1 impairs secretion of vesicular stomatitis virus glycoprotein. Overall, our data identify a new interaction between Rank-5 and EHD1, and novel endocytic regulatory roles that include retromer-based transport and secretion.
authors.affiliation: Department of Biochemistry and Molecular Biology and Eppley Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198-5870, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.forename: Calliste
authors.initials: C
authors.lastname: Reiling
authors.forename: James B
authors.initials: JB
authors.lastname: Reinecke
authors.forename: Iztok
authors.initials: I
authors.lastname: Prislan
authors.forename: Luis A
authors.initials: LA
authors.lastname: Marky
authors.forename: Paul L
authors.initials: PL
authors.lastname: Sorgen
authors.forename: Naava
authors.initials: N
authors.lastname: Naslavsky
authors.forename: Steve
authors.initials: S
authors.lastname: Caplan
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D001692:Biological Transport; D056649:Cathepsin G / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008954:Models, Biological; D028044:Phosphate-Binding Proteins; D013816:Thermodynamics; D020798:Two-Hybrid System Techniques; D033921:Vesicular Transport Proteins / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism
chemical_list: C118719:ANKFY1 protein, human; C120700:EHD1 protein, human; C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D028044:Phosphate-Binding Proteins; C498296:VPS26A protein, human; D033921:Vesicular Transport Proteins; D014759:Viral Envelope Proteins; D049452:Green Fluorescent Proteins; D056649:Cathepsin G; D020696:rab5 GTP-Binding Proteins