RanBP1 controls the Ran pathway in mammalian cells through regulation of mitotic RCC1 dynamics.

ABSTRACT

The Ran GTPase plays critical roles in multiple cellular processes including interphase nucleocytoplasmic transport and mitotic spindle assembly. During mitosis in mammalian cells, GTP-bound Ran (Ran-GTP) is concentrated near mitotic chromatin while GDP-bound Ran (Ran-GDP) is more abundant distal to chromosomes. This pattern spatially controls spindle formation because Ran-GTP locally releases spindle assembly factors (SAFs), such as Hepatoma Up-Regulated Protein (HURP), from inhibitory interactions near chromosomes. Regulator of Chromatin Condensation 1 (RCC1) is Ran's chromatin-bound exchange factor, and RanBP1 is a conserved Ran-GTP-binding protein that has been implicated as a mitotic regulator of RCC1 in embryonic systems. Here, we show that RanBP1 controls mitotic RCC1 dynamics in human somatic tissue culture cells. In addition, we observed the re-localization of HURP in metaphase cells after RanBP1 degradation, consistent with the idea that altered RCC1 dynamics functionally modulate SAF activities. Together, our findings reveal an important mitotic role for RanBP1 in human somatic cells, controlling the spatial distribution and magnitude of mitotic Ran-GTP production and thereby ensuring the accurate execution of Ran-dependent mitotic events. ABBREVIATIONS AID Auxin-induced degron; FLIP Fluorescence loss in photobleaching; FRAP Fluorescence recovery after photobleaching; GDP guanosine diphosphate; GTP guanosine triphosphate; HURP Hepatoma Up-Regulated Protein; NE nuclear envelope; NEBD Nuclear Envelope Breakdown; RanBP1 Ran-binding protein 1; RanGAP1 Ran GTPase-Activating Protein 1; RCC1 Regulator of Chromatin Condensation 1; RRR complex RCC1/Ran/RanBP1 heterotrimeric complex; SAF Spindle Assembly Factor; TIR1 Transport Inhibitor Response 1 protein; XEE Xenopus egg extract.