Gpn1 and Gpn3 associate tightly and their protein levels are mutually dependent in mammalian cells

•Gpn1-Flag relocalizes Gpn3-EYFP from the cell nucleus to the cytoplasm.•Gpn3-Flag effectively retains Gpn1-EYFP in the cytoplasm.•Gpn3-EYFP and Gpn1-Flag undergo nucleocytoplasmic shuttling as a complex.•Most transfected and endogenous Gpn1 and Gpn3 copurify in a single protein complex.•Inhibition of Gpn3 expression dramatically decreases Gpn1 levels, and vice versa. Gpn1 and Gpn3 are GTPases individually required for nuclear targeting of RNA polymerase II. Here we show that whereas Gpn3-EYFP distributed between the cytoplasm and cell nucleus, it was mainly cytoplasmic when coexpressed with Gpn1-Flag. Gpn3-Flag retained Gpn1-EYFP in the cytoplasm. However, Gpn3-EYFP/Gpn1-Flag nucleocytoplasmic shuttling was revealed after inhibiting nuclear export with leptomycin B. All Gpn3-EYFP coimmunoprecipitated with Gpn1-Flag, and all Gpn1-EYFP with Gpn3-Flag. Importantly, most endogenous Gpn1 and Gpn3 also associate. Gpn1–Gpn3 interaction was essential to maintain steady-state protein levels of both GTPases. We propose that most Gpn1 and Gpn3 associate, are mobilized, and function as a protein complex. GPN3physically interacts with GPN1 by anti tag coimmunoprecipitation (1, 2) GPN3 and GPN1colocalize by fluorescence microscopy (View interaction)